Streptococcus suis in China, including a subset with capsular serotype 2. Short post-antibiotic effects (PAEs) (0.5–2.6 h) and PA-SMEs (2.4–7.7 h) were observed for gamithromycin against S. suis. The serum matrix dramatically facilitated the intracellular uptake of gamithromycin by S. suis strains, thus contributing to the potentiation effect of serum on their susceptibilities, with a Mueller-Hinton broth (MHB)/serum minimum inhibitory concentration (MIC) ratio of 28.86 for S. suis. Dose-response relationship demonstrated the area under the concentration (AUC)/MIC ratio to be the predictive PK/PD index closely linked to activity (R2 > 0.93). For S. suis infections, the net stasis, 1–log10, and 2–log10 kill effects were achieved at serum AUC24h/MIC targets of 17.9, 49.1, and 166 h, respectively. At the current clinical dose of 6.0 mg/kg, gamithromycin PK/PD cutoff value was determined to be 8 mg/L. A PK/PD-based dose assessment demonstrated that the optimum dose regimen of gamithromycin to achieve effective treatments for the observed wild-type MIC distribution of S. suis in China with a probability of target attainment (PTA) ≥ 90% was 2.53 mg/kg in this study. These results will aid in the development of clinical dose-optimization studies and the establishment of clinical breakpoints for gamithromycin in the treatment of swine respiratory infections due to S. suis.Gamithromycin is a long-acting azalide antibiotic that has been developed recently for the treatment of swine respiratory diseases. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) targets, PK/PD cutoff, and optimum dosing regimen of gamithromycin were evaluated in piglets against  Actinobacillus pleuropneumoniae, followed by Bordetella bronchiseptica and Streptococcus suis  due to bacterial agents, the primary pathogens are cus suis . Albeit A. pleuropneumoniae and Pasteurella multocida (Haemophilus parasuis (S. suis infections. While it is recognized that the area under the concentration-time curve/MIC (AUC/MIC) is the predictive PK/PD index that well-describes the efficacy of azalide antibiotics  value for gamithromycin was estimated for S. suis. This would be useful in setting the final clinical susceptibility breakpoints for gamithromycin against Streptococcus spp.The objectives of this study were to characterize the PK/PD relationship and the magnitude of gamithromycin target exposures associated with efficacy against S. suis collected from diseased swine suffering septicemia, pneumonia, and meningitis were used in this study. These isolates were gathered through a 10-years antimicrobial resistance surveillance study for animal original bacteria in China between 2009 and 2018. Two well-described strains (ATCC 43765 and 05ZYH33) of S. suis serotype 2 (SS2) were used for the PK/PD studies, as SS2 is the most virulent and dominant pathogenic serotype for both swine and human  with 5% lysed horse blood and sheep blood agar. Gamithromycin injection  for in vivo studies was purchased from Boehringer Ingelheim Animal Health, Toulouse, France, and gamithromycin powder for in vitro studies was obtained from NMT Biotech Ltd., Suzhou, China.A total of 197 clinical isolates of nd human , 17. S. S. suis isolates were undertaken by the broth microdilution technique in accordance with CLSI guidelines  were consequently removed  uptake assay as previously established . NPN uptake results were calculated as a percentage of pure serum control.The plasma membrane permeability of ablished , 21. BacS. suis in MHB and porcine serum were determined as our previously established  were determined in sub-MIC treated phases (0.1 to 0.3 × MICs) after exposure to gamithromycin at 4 × MICs for 1 h. The calculations of PAEs and PA-SMEs have been described in detail elsewhere , 23.Gamithromycin concentration measurement methods and serum PK in piglets after intramuscular injection at 6.0 mg/kg have been described elsewhere in detail . In brieEx vivo PK/PD studies were performed using the drug-containing serums collected from piglets at different time points after receiving intramuscular dosing of gamithromycin at 6.0 mg/kg. Bacterial cells of ATCC 43,765 and 05ZYH33 were subcultured and inoculated to each serum sample, giving an initial inoculum of ~106 CFU/ml. The mixtures were then serially diluted and plated using a drop-plate technique to measure the log10 change of bacterial count after 24 h of incubation. The PK/PD relationship was determined by measurement of the correlation between ex vivo activity and the PK/PD parameter of AUC24h/MIC. The dose-response curve was modeled according to a Hill-type sigmoid Emax model using the Phoenix WinNonlin software  (E = E0 + Emax × CN/(EC50N + CN), where E0 is the log10 change of bacterial count in the absence of drug, C is the AUC24h/MIC ratio, EC50 is the AUC24h/MIC required to achieve 50% of the maximum effect (Emax), and N is the Hill coefficient representing the slope of the dose-response curve. The coefficient of determination (R2) was used to estimate the variance that might be due to regression with the AUC24h/MIC ratio. The PK/PD index targets in serum necessary for the net static, 1-log10, and 2-log10 kill effects were determined for each strain.The AUC/MIC ratio was chosen as the pharmacodynamic parameter for gamithromycin based on previous studies demonstrating this PK/PD index to be predictive of treatment efficacy for the macrolides , 23–25. S. suis infections, the population dose covering the observed wild-type MIC distribution of S. suis (n = 125) was explored by a 10,000-iterations Monte Carlo simulation (MCS) using the Crystal Ball software . The wild-type distribution was chosen because only the isolates devoid of phenotypically detectable resistance genes that clinically can be successfully treated with gamithromycin and that should be considered to calculate a dose. In addition, macrolides generally act bacteriostatic effect   was considered negligible herein.where Cl//3 days) ; AUC/MICPD), a series of MCSs were performed separately for each possible MIC based on our previously reported PK parameters and the PK/PD targets obtained in this study . Cl/F was the clearance scaled by intramuscular bioavailability that was assumed to be 0.91 ± 0.26 L/kg/h  was used for COPD determination. COPD was defined as the MIC value at which the probability of target attainment (PTA) reached 90%  values were markedly lower in porcine serum than those determined in MHB, with an MHB/serum ratio of 28.86 for MIC and 11.23 for MBC, respectively  after intramuscular injection at 6.0 mg/kg. Increasing the serum concentration of gamithromycin further to the peak concentration resulted in a decrease of 2.87-log10 CFU/ml after 24 h , resulting in PA-SMEs of 2.4–7.7 h  of the best-fitting line based on the sigmoid Emax model are also shown in S. suis strains tested over the entire drug-exposure range.The serum of >0.93 . The PK/ctively, . The meaS. suis infections was 2.53 mg/kg in China  of gamithromycin in piglets  were higher than 86.2%, which is an approximation to the 90% criterion  are taken into account as the proper PK/PD parameters to determine the optimal dosing regimen of macrolides , 14. Con PA-SMEs . Data geS. suis infections, while it is currently not indicated for the treatment of streptococcosis. As a result of its spectrum and potency against SRD-associated pathogens, the clinical efficacy of gamithromycin is currently being investigated in a confirmatory clinical trial with S. suis infections  in Pseudomonas aeruginosa  of South China Agricultural University (SCAU).Y-FZ designed the experiment and conceptualized the model. R-LW, and Y-FZ drafted the manuscript. R-LW, PL, X-FC, and XY carried out the experiments. X-PL revised the Introduction section. Y-HL and JS analyzed the data. All authors read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China (31902318), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (32121004), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02N054), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT_17R39), Guangdong Major Project of Basic and Applied Basic Research (2020B0301030007), and the Innovative Team Project of Guangdong University (2019KCXTD001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Marital status, tobacco use, and fetus sex affected birth weight centile classification according to gestational age. Multivariate statistical comparisons of the extracted metabolomes revealed that asparagine, aspartic acid, deoxyribose, erythritol, glycerophosphocholine, tyrosine, isoleucine, serine, and lactic acid were higher in both SGA and LBW placentas, while taurine, ethanolamine, β-hydroxybutyrate, and glycine were lower in both SGA and LBW. Several metabolic pathways are implicated in fetal growth restriction, including those related to the hypoxia response and amino-acid uptake and metabolism. Inflammatory pathways are also involved, suggesting that fetal growth restriction might share some mechanisms with preeclampsia.Fetal growth restriction is an obstetrical pathological condition that causes high neonatal mortality and morbidity. The mechanisms of its onset are not completely understood. Metabolites were extracted from 493 placentas from non-complicated pregnancies in Hamilton Country, TN (USA), and analyzed by gas chromatography–mass spectrometry (GC–MS). Newborns were classified according to raw fetal weight  and non-low birth weight ), and according to the calculated birth weight centile as it relates to gestational age , large for gestational age (LGA), and adequate for gestational age (AGA)). Mothers of LBW infants had a lower pre-pregnancy weight (66.2 ± 17.9 kg vs. 73.4 ± 21.3 kg,  Low birth weight (LBW) is a condition characterized by a neonatal birth weight of less than 2500 g, occurring either in preterm or in full term. Small for gestational age (SGA) neonates are also LBW, but are more precisely defined according to gestational age. A low birth weight is correlated with many socioeconomic factors and parameters affecting intrauterine growth restriction (IUGR) and preterm birth.LBW has a global incidence of 17% and is an important predictor of infant mortality ,3. BirthMaternal exposure to environmental pollutants occupies a central role in the genesis of LBW ,6. In HaAn innovative approach for examining the complexities associated with LBW is the field of metabolomics, which is the study of the identities and abundances of the entire metabolome, which is the whole set of metabolites present in a biological sample at a given time ,9. MetabAlthough it is a powerful approach, metabolomics has only recently been applied to the investigation of LBW associations. In 2012, Kenny and Baker  earned aRecent applications of metabolomics to investigate a variety of obstetrical complications include a study on polycystic ovarian syndrome  and the The University of Tennessee College of Medicine’s Institutional Review Board (IRB) approved the gathering and utilization of placentae for the present research . The specimens were collected at Baroness Erlanger Campus of the Erlanger Health System, located in Chattanooga, TN, between June 2007 and July 2010. Sample collection was based on the timing of maternal arrival and placental delivery. Sample collection was only performed when investigators of this study could be present to ensure that standard collection procedures were followed. As this timing was somewhat random (subject to personnel availabilities), this reduced any possibility of a “timing-based” bias. Further information on mothers and infants was obtained from medical records. The placentae were obtained in a standardized manner from single birth mothers over 18 years old, who were free of HIV and hepatitis. To avoid analytical bias, all of the samples were collected, stored, processed, and analyzed in the exact same way. The whole placentae were stored at −80 °C until they were processed. Prior to the analysis, samples were thawed overnight at room temperature and then the chorionic plate and decidua basalis were removed with ceramic scissors, leaving the villous core tissue to be homogenized. A portion of the homogenized villous core was taken for the metabolomic analysis. Samples were oven-dried at 60 °C to constant weight and then powderized with a mortar and pestle.In this study, two key factors were analyzed: raw birth weight and the calculated birth weight percentile. The obstetrical community considers infants with a weight less than 2500 g as being low birth weight (LBW). The birth weight percentile was calculated for each sample using the gestation related optimal weight (GROW) software [The MetaboPrep GC kit from Theoreo was used for untargeted metabolome extraction, purification, and derivatization. Sample analysis was performed in the period from December 2016 to August 2017. Following the manufacturer’s instructions, 25 ± 1 mg of tissue was added to an Eppendorf microcentrifuge tube along with the extraction mixture, including the internal standard . The sample and extraction mixture were mixed via vortexing at 1250 rpm for 30 min and then subjected to ultrasonic treatment (30 min at 30 °C). The extracted metabolome was centrifuged to separate any solids before the purification process. The purified sample was then freeze-dried overnight.Trimethylsilyl derivatization was carried out by adding the first derivatization mixture and vortexing, followed by the second derivatization mixture. The derivatized metabolomes were transferred to 100 µL inserts for the auto sampler injection. Before injection into the GC–MS, the samples were centrifuged.A sample of the derivatized solution (2 microliters) was injected into a GC–MS system  using split mode. Chromatography was performed with a 30 m × 0.25 mm CP-Sil 8 CB fused silica capillary GC column with a 1.00 µm film thickness from Agilent, using helium as the carrier gas.The oven temperature program consisted of a 0.5-min hold at 100 °C and was then increased to 320 °C at 4 °C/min, resulting in a GC run time of 60 min. Carrier gas flow linear velocity was maintained at 39 cm/s and the split flow was set to 5:1. The mass spectrometer was programmed with a 4.5 min solvent delay and then operated in full scan mode (35–600 m/z) using electron ionization (70 eV) and a scan velocity of 3333 amu/s.The samples were analyzed in groups of 25, with four controls per batch: a blank injection of hexane, a standard mix of 50 molecules, a pool of 2 microliters from 50 randomly selected treated samples, and a repeated injection of one randomly selected sample. Each batch was deemed valid if the blank showed no peaks, the standard peak area was within 10% of what was expected, the ratio of the 100 major peaks in the repeated sample was within 15% of the original, and the pooled sample was within 5% of the model built from previous samples.The chromatograms were deconvoluted using the most intense fragment for metabolites meeting the integration criteria . 2-isopropyl malic acid was used as the internal standard (SIM = 147) and the extracted chromatograms were used to calculate the metabolite area. Relevant metabolites (see below) were annotated according to level 1 of the Metabolomics Standard Initiative . Brieflyp < 0.05). Comparisons of variables reported as percentages were done by means of the Yates correction of the Χ2 test.The clinical and demographic data were not normally distributed according to the Kolmogorov–Smirnov test, so that only non-parametric statistical tests were employed for a comparison of these characteristics. The comparison between groups was made with the Rank Sum Test, according to Mann–Whitney or by means of analysis of variance on rank preformed single way , also using the post-hoc test of Dunn . Chromatographic data pre-treatment included chromatogram alignment, calculation of peak areas normalized to that of the internal standard, followed by mean centering and unit variance scaling , and were conducted using the MetaboPredict software , Italy). Class separation was investigated by PLS-DA, which is a supervised method that uses multivariate regression techniques to extract, via linear combination of the original variables (X), the information that can predict the class membership (Y). Variable importance in projection (VIP) scores were calculated for each component. Features with VIP score ≥ 2 were identified for further biochemical pathway investigation. Lower scores were not considered to be significant contributors to class separation. Classification and cross-validation were performed as well as a permutation test to assess the significance of the class discrimination and the lack of model overfitting.Partial least square discriminant analysis (PLS-DA) was performed using the R statistical platform  scores were calculated for each component. VIP is a weighted sum of the PLS loadings’ squares, considering the amount of Y-variation explained in each dimension. The weights are determined by the reduction in the sums of the squares across the number of PLS components. The average of the feature coefficients was used to indicate the overall coefficient-based importance.The involvement of metabolic pathways was analyzed using the MetPa tool . It is bResults were obtained from 493 placental tissues, 124 from pregnancies with LBW newborns and 369 from pregnancies resulting in birth weight >2500 g. Newborns were determined to be SGA in 118 cases, AGA in 326, and LGA in 49 by normographic data. The demographic and clinical characteristics of all pregnancies are reported in p < 0.0001), a lower body mass index (BMI) , and a shorter gestation time (246.4 ± 24.0 days vs. 267.2 ± 19.4 days p < 0.001) when compared with non-LBW subjects. Marital status, tobacco use, and fetus sex affected the birth weight centile classification according to gestational age. For example, single mothers had an LGA infant more frequently than an AGA infant  and the LGA infant was more frequently male . A higher tobacco use frequency was associated with SGA infants compared with AGA infants .Women who had an LBW newborn also had the following characteristics: lower pre-pregnancy weight   (A well-defined differentiation of the LBW and non-LBW was achieved (R = 0.59) -A1. Disc = 0.71) -B1. VariFive metabolites were selected as being relevant by the volcano plot: hydroquinone and hydroxylamine were lower in LBW, whereas deoxyguanosine, glutathione, and linoleic acid were higher in LBW .p < 0.05 assessed by the ANOVA of the three classes were also evaluated in a heatmap. The consistent selection of relevant metabolites using the different models was synthesized with an UpSet visualization [Metabolites with lization  showing In the present study, we report the placental metabolomic fingerprint of LBW and SGA newborns, a subject of growing interest due to the recent importance given to the concept of “fetal origin of adult disease”. This idea suggests that early life conditions can “program” the fetus for a spectrum of adverse health outcomes in adulthood, such as coronary artery disease, hypertension, obesity, and insulin resistance ,36. IndeAlthough the placenta is the organ mainly involved in fetal nutrition and weight determination, few studies have investigated the placental metabolomic variations associated with newborn weight ,39. The 2 tension.Taurine is an essential nutrient in fetal metabolism, as the fetus and placenta lack the enzyme for taurine synthesis . ReducedTwo possible reasons for increased glycerophosphocholine in LBW/SGA placentas can be suggested. First, the increase may be due to increased cell death . Second,Amino acids are among the main nutrient sources for fetal growth, accounting for 20–40% of fetal energy requirements . Amino a+ gradient. Subsequently, glycine diffuses into the fetal circulation and back to the mother. The strong polarization of System A provides the basis for the net transport of glycine to the fetus. Paolini et al. [The glycine transport system  is an Na-dependent system and is concentrated in the syncytiotrophoblast, energized by the Nai et al.  reports Our observation of increased lactic acid concentration in LBW/SGA placentae strongly indicates hypoxic conditions of the placental cells. In this condition, ketone utilization was increased. In murine models, hypoxic tolerance was increased in those mice treated with 3-hydroxybutyrate . This fiWomen with SGA babies showed higher self-reported tobacco use. One of the main ways in which smoking affects fetal growth is through the reduction of oxygen and nutrients that reach the developing baby. Moreover, nicotine, a component of tobacco smoke, is a vasoconstrictor, which means it narrows the blood vessels and decreases blood flow. This reduction in blood flow can result in a decrease in oxygen and nutrients reaching the placenta, which can lead to SGA babies. Additionally, smoking increases the risk of placental abruption, where the placenta separates from the uterus prematurely, cutting off the baby’s oxygen supply and increasing the risk of stillbirth. Another way in which smoking can affect fetal growth is through the release of harmful chemicals from tobacco smoke. These chemicals can cross the placenta and reach the developing baby, causing damage to the developing cells and organs. For example, carbon monoxide, a component of tobacco smoke, can reduce the amount of oxygen in the baby’s blood, leading to fetal growth restriction. The risks associated with smoking during pregnancy are not limited to women who smoke cigarettes. Secondhand smoke exposure can also have negative effects on fetal growth and development.Nevertheless, the use of tobacco is not associated with the reported differences in terms of metabolomics profiling within our cohort. Indeed, the abundances of VIP-score and Volcano plot selected metabolites did not show significant differences in smoker and non-smoker enrolled subjects (data not shown). The same evaluations were performed for marital status, education, and infant sex; the abundances of statistically significant metabolites were also not correlated with these parameters.The placental metabolic profile of LBW/SGA newborns highlights the role of amino acid metabolism and degradation, and the role of folate, prostaglandin, and leukotriene metabolism. This illustrates the great importance of hypoxic and nutritional placental deficiency, but also suggests a possible role of inflammation, which may be responsible for a subclinical preeclampsia condition.The present study used a novel placental analysis technique and contributes to previous metabolomic studies carried out on cultured placental explants. Indeed, while oven drying is not standard in human metabolomics investigations, it is widely used in plant metabolomics  with no Our method provides a unique tool for understanding placental disease mechanisms in utero, thereby phenotyping diseases based on the whole placental involvement and not on results from cultured cells. Because these kinds of studies require a large cohort to provide accurate and reproducible findings, our strength is that our cohort is one of the biggest ever used in a metabolomics study.


Epidermoid cysts are most often benign cystic lesions, with uterine cervical localisation being very unusual. We present the case of a 52-year-old female patient diagnosed with an epidermoid cyst at the level of the uterine cervix. A bioptic and haemostatic uterine curettage was performed, followed by total hysterectomy with bilateral adnexectomy. The histopathologic analysis and immunohistochemical essay of the resection specimens confirmed the cervical epidermoid cyst. The presence of high-risk HPV (human papillomavirus) was only seen in the cervical mucosa. The exact etiopathogenesis is unknown, but postpartum cell implantation of reminiscent embryonic tissue can be involved in the development of these lesions. Epidermoid cysts are most often benign cystic lesions, commonly seen on the face, scalp and trunk, and rarely can occur on the glabrous skin of the palm and sole and genital organs ,2,3,4. TProbably the simplest and most accurate way to describe such lesions was proposed by Ackerman , who speAlthough some of these cysts (particularly those located in the fingers) result from traumatic inclusion of the epidermis—hence the term epidermal inclusion cyst—the majority probably arise from the cystic dilation of the infundibular portion of the hair follicles . A few oHPV (human papillomavirus)  has been found in cases of keratinous cysts of palmoplantar and other locations, suggesting that it may play an etiological role ,6,7,8,9.The other type of keratinous cyst is the pillar or trichilemmal type. It occurs preferentially on the scalp and is microscopically characterised by a trichilemmal type keratinization, which is a sudden keratinization process without the formation of a granular layer and an uneven interphase between the keratinised and non-keratinised cells. The keratin inside the cyst is not lamellated, some of the nuclei are retained and focal calcification is frequent. Ultrastructural and immunohistochemical studies also support a trichilemmal derivation for this lesion . EpidermA particularly rare localisation for an epidermoid cyst is represented by the uterine cervix. A survey of the specialized literature brings to our attention only one other such case, published in 1925 by Bacon . WillsonA female patient, aged 52, comes to the obstetrics–gynaecology clinic for haemorrhagic metropathy and fatigue. A bioptic and haemostatic uterine curettage was performed, and the histopathological analysis shows an endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) with focal atypia. After the prescribed treatment, there were no notable results; as a consequence, it was decided to radicalise the intervention and perform a complete hysterectomy with bilateral adnexectomy. The macroscopic examination of the surgical piece was performed revealing a polypoid grey mass with brown areas in the endometrial cavity, overall thickened myometrium, two ovarian cysts less than 3 cm in diameter with vitrine serous content and smooth inner surface, the same at the fallopian tube level, some brown areas at the surface of the cervix less than 2 mm each in diameter and a conspicuous cyst filled with a fragile grey substance of 1.8 cm in diameter located at relative deeper layer of the exo-cervix. The primary sections were stained with haematoxylin–eosin (HE). An immunohistochemical analysis was performed on sections prepared from formalin-fixed paraffin embedded tissue using an automated immune-stainer . Immunohistochemical assays were performed on a Ventana Benchmark GX automated staining instrument according to the manufacturer’s instructions ,21,22. FThe macroscopic and microscopic analysis of the resection specimens revealed chronic exo-cervicitis with signs of viral infection, chronic micropapillary endo-cervicitis, hyperplasic endometrial polyp, adenomyosis, follicular ovarian cysts, multiple para tubal cysts, and an immunohistochemically confirmed cervical epidermoid cyst.The endo-cervix was lined by a single layer of columnar mucous cells and had an micropapillary architecture, moderate inflammatory infiltrate in the surface chorion, and areas of squamous metaplasia and moderate inflammatory infiltrate in the surface chorion in some other parts could be seen.The exo-cervix was found to be lined by a non-keratinised stratified epithelium (squamous epithelium) showing a distinct basal layer, but the appearance of atypical keratinocytes was noted, with some of them showing koilocytotic atypia with sharply outlined perinuclear vacuoles, dense and irregular staining peripheral cytoplasm and an enlarged, modified nucleus . The p16The exo-cervix shows a cystic structure in the deeper layers that is filled with an amorphic, eosinophilic, lamellar substance . The cysOther areas of the cyst are lined by stratified, flattened epithelium with focally outer fine, but distinct, granulated eosinophilic cytoplasm cells . The cysThe cervical localisation of an epidermal-type keratinocyte cyst is particularly unusual. The immunohistochemical studies of the specimens proved without any doubt that the cystic structure was indeed an epidermal cyst and the etiopathology of an epidermoid cyst with “classical” localisation is probably very well understood. In this case, the etiopathology of such a lesion located in the cervix is an ongoing debate. Some causes of it could be: local irritating conditions such as trauma, chronic inflammation, infection with HPV, squamous metaplasia, growth originating from vestigial structures and heterotrophic intrapartum implants. The exact etiopathology is unknown, although animal research studies suggested that reminiscent vestigial structures are likely candidates to induce such a lesion . The uteDespite the fact that epidermoid cysts are commonly seen localised in hair-bearing skin, diagnosing them at mucosa sites, such as in our case, is extremely rare. The association between HPV infection and the epidermoid cyst presence has been debated in many studies ,26. HighIn our study, we also tried to assess whether there was any HPV expression in the samples of the cervix and/or epidermoid cyst that we had diagnosed. The positive p16 nuclear and cytoplasmic expression was detected, in our case, at the level of the exo-cervix, but it was revealed to be totally negative in the epidermoid cyst. The negative result only excluded high-risk HPV strains and thus left enough space for other HPV subtypes to be evaluated.Moreover, p16 plays an important role in the cell cycle as a cyclin-dependent kinase inhibitor; p16 acts like an indirect marker for the HPV infection and the overexpression is induced by viral E7 protein . The malAccording to Stolnicu et al. , very feIn our case, we also tried to find other cysts whose presence could be explained on the basis of a genetically determined background, but in the end, there was actually no evidence of multiple epidermoid cysts, so this cyst most probably had a sporadic nature.The exact etiopathology of the epidermoid cyst is unknown, but reminiscent vestigial structures are a likely candidate to induce such lesions. Further studies should be performed in order to assess the recurrence rate or even malignancy transformation potential, since an increasing number of cases are being reported at unusual sites such as the genital areas. The association between any HPV subtypes and epidermoid cysts is still a debated issue because its pathology is extremely rarely encountered.


Due to the increasing demand for electrification in the automotive sector, the interest in the manufacturing and processing of pure Copper  is also increasing. Laser-based technologies have proven to be challenging due to Cu’s high optical reflectivity. Processing pure Cu with Electron Beam Melting (EBM) is a promising manufacturing route, allowing for high design freedom. The highest priority is to achieve outstanding thermal and electric conductivity in manufactured Cu components. Chemical contamination or manufacturing defects, such as porosity, significantly reduce the thermal and electric conductivity. The literature on post-processing  of additively manufactured Cu is scarce. Therefore, this study discusses the correlation between as built and heat treated microstructure, as well as surface roughness on the EBM electric conductivity. EBSD analysis is performed to analyze the effect of microstructure on electric conductivity. The effect of sandblasting and vibratory finishing on surface roughness and electric conductivity is investigated. Additionally, the samples are mechanically tested in terms of hardness. Due to the distinctive combination of physical and mechanical properties, copper and copper alloys are interesting for a wide range of applications ,2,3. ThePowder bed fusion (PBF) systems are commonly used to print 3D models by using either a laser beam (L-PBF) or an electron beam  as an energy source ,7. FundaWhile pure copper reflects up to 98% of infrared laser radiation commonly used in L-PBF ,11. The Hardness values of 46–48 HV and tensile strengths of up to 231.6 MPa with an elongation at fracture of 59.3% were achieved with EBM ,15. The Guschlbauer et al.  studied Research work on typical post-processing methods for EBM parts, to further tailor the microstructure of pure copper, is limited. Studies with conventionally manufactured copper alloys show great improvements in mechanical and physical properties with conventional two-step heat treatments. Since an in situ heat treatment is incorporated during the EBM process , thermalA) around 5 J/mm2 achieved the highest electric conductivities; >99.6–100% International Annealed Copper Standard (IACS). Electric conductivities of 94%IACS were achieved with an EA of 11 J/mm2  is present. This can be attributed to grains growing along the thermal gradient, parallel to the build direction. EBSD analysis was carried out for four samples manufactured with E2 and 7.7 J/mm2, the differences in microstructure are considered to be negligible. Therefore, the EBSD analysis of 7.7 J/mm2 is rotated by 90° to identify changes in grain orientation, parallel to the build direction. While more grain orientations are recorded, columnar grains can still be seen. The epitaxial grain growth can clearly be identified in the 7.7 J/mm2 EBSD analysis, since some grain lengths exceed 3 mm, corresponding to more than 60 layers. The grains with unfavorable grain orientation are overgrown by grains oriented in the build direction.For the samples manufactured with energy densities 7.57 J/mmA 8.64 J/mm2, the grain size is 55.3 µm. The heat input, combined with the elevated preheat temperature throughout the build , leads to grain coarsening. The effect of in situ heat treatment during EBM has previously been reported [2 sample does not follow the increasing grain size with energy density trend. This is due to the orientation of the EBSD scan. Along the build direction, grains with unfavorable grain orientation are overgrown and are hence smaller compared to those with favorable grain orientation. The average grain size of the 7.7 J/mm2 sample is, therefore, not representable, since smaller overgrown grains are also considered. The grain orientation is not affected by the energy density in the investigated energy density range. The average grain sizes increase with the energy densities, as shown in reported ,27. The 2 (relative density 99.99%). Five EDX spectra were taken, and since all spectra are similar to each other, one representative spectrum was chosen. As can be seen from A 4.5 J/mm2, 7.57 J/mm2, 7.7 J/mm2 and 8.64 J/mm2.A 4.5 J/mm2 (relative density > 99.5 %) is shown in 2O, which was measured at a finer scale than used in this study. The sub-structure shown in 2O. The sub-structure was also found for higher energy densities.An SEM-image for a cube manufactured with EA 8.64 J/mm2 is 39.8 MSm−1−1. This is equivalent to 66.66%IACS, which is significantly lower than expected. Based on the compilation of Jiang et al. , it can A 8.64 J/mm2 sample. The black lines indicate angles greater than 10° , and red lines show grain boundary angles below 10° . −1) is attributed to the amount and curvature of LAGBs. The possible presence of Cu2O precipitates in the sub-structure further reduces the electric conductivity. The reduced electric conductivity measured  is reduced, compared to the as built condition, by up to 35%. The area surface roughness (Sa) shows similar results. Sand blasting and vibratory finishing remove sintered and partially molten particles from the sample surface, reducing the surface roughness. Compared to sand blasting, vibratory finishing improved the surface roughness by 7%. This can be attributed to the more aggressive collision of the polymer granulates on the surface, as well as the high frequency applied during vibratory finishing. The duration of the vibratory finishing process is also three times longer compared to sand blasting. However, all cubes were post-processed simultaneously during vibratory finishing, whereas each sample was individually sandblasted. The time efficiency of vibratory finishing should also be considered. −1 (as built condition) to 44.72 MSm−1 −1 (as built and abrasively post-processed). This corresponds to an increase of 12% compared to the as built condition. A decrease in surface roughness led to an improved electric conductivity, as seen in the measured electric conductivity after sand blasting in this study. The electric conductivity increased from 38.9 MSmWith an increased surface roughness, the overall thickness of the sample does not remain constant. The electric conductivity is, therefore, hindered at the edges of the samples increasing the electric impedance. Reducing the surface roughness ensures a more constant sample thickness and that electric conductivity can flow through the sample with reduced impedance. 2, and have a relative density of 99.99%. The surface roughness was not affected by heat treatment. The microstructures of the heat treated samples are shown in −1 (as built and sandblasted) to 47.46 MSm−1 −1 , the electric conductivity rises from 44.72 MSmrefer to , which iEven though an increase in electric conductivity was found, with a decrease in surface roughness and heat treatment see  in this Electric conductivity is heavily dependent on grain boundary angles, chemical composition and surface finish. An increase in electric conductivity is achieved by increasing grain size. Further research into achieving a single crystal with EBM should be considered to study the effect on electric conductivity.Straight grain boundaries improve electric conductivity. This can be achieved by increasing the overall build temperature. There is trade-off between hardness and electric conductivity: For high electric conductivity, large grains are required. For high hardness, a small grain size is needed.Chemical impurities, at smaller scales than EDX or EBSD, lead to a sub-structure within grains and significantly reduce electric conductivity.In this study, the correlation between as built and heat treated microstructure, as well as surface roughness on the EBM electric conductivity of pure copper, is investigated. The following conclusions can be drawn:


Numerous studies have suggested significant associations between prenatal exposure to heavy metals and newborn anthropometric measures. However, little is known about the effect of various heavy metal mixtures at relatively low concentrations. Hence, this study aimed to investigate associations between prenatal exposures to a wide range of individual heavy metals and heavy metal mixtures with anthropometric measures of newborns.We recruited 975 mother–term infant pairs from two major hospitals in Israel. Associations between eight heavy metals  detected in maternal urine samples on the day of delivery with weight, length, and head circumference at birth were estimated using linear and Bayesian kernel machine regression (BKMR) models.Most heavy metals examined in our study were observed in lower concentrations than in other studies, except for selenium. In the linear as well as the BKMR models, birth weight and length were negatively associated with levels of chromium. Birth weight was found to be negatively associated with thallium and positively associated with nickel.By using a large sample size and advanced statistical models, we could examine the association between prenatal exposure to metals in relatively low concentrations and anthropometric measures of newborns. Chromium was suggested to be the most influential metal in the mixture, and its associations with birth weight and length were found negative. Head circumference was neither associated with any of the metals, yet the levels of metals detected in our sample were relatively low. The suggested associations should be further investigated and could shed light on complex biochemical processes involved in intrauterine fetal development.The online version contains supplementary material available at 10.1186/s12940-022-00950-z. Heavy metals are naturally occurring elements with a high atomic weight and density at least five times greater than water. Some of these heavy metals are essential nutrients in the body, and a deficiency in one of them can result in diseases . On the While the mechanisms underlying the effect of over-exposure to heavy metals on the development of newborns remain the subject of ongoing studies , 10, somIn recent years, many epidemiologic studies have examined the associations between heavy metals measured in maternal urine and various adverse health outcomes among newborns, including low birth weight , low birIn the current study, we examine the association between prenatal exposure to a mixture of heavy metals  and newborn anthropometric measures. We investigated the concentrations of eight heavy metals  in maternal urine samples and examined their association with anthropometric measures, both individually and by using a modeling approach that accounts for possible non-linear associations, as well as any interactions between the metals .Beginning in 2016, pregnant women and their newborns were recruited in delivery rooms of two hospitals in Israel: (1) Rambam Medical Center – the largest hospital in the Northern District of Israel, which accounts for around 5500 births annually, and (2) Shamir Medical Center – located in the Central region of Israel and which accounts for around 8000 deliveries annually. Women were considered eligible if they were Hebrew-speaking, aged 18 years or older, and pregnant with a singleton. Exclusion criteria included: (1) preterm birth ; (2) pregnancies considered by the medical staff to have a high risk of complications  ; (3) minEach participant was asked to provide a single urine sample. The samples were frozen at − 80 °C immediately after receiving them and then transported at − 20 °C for further analysis at the Central Public Health Laboratory of the Israeli Ministry of Health (Abu-Kabir). We measured levels of As, Cd, Cr, Hg, Ni, Pb, Se, and Tl using inductively coupled plasma mass spectrometry (ICP-MS), on an Agilent 7800 × ICP-MS instrument equipped with an Integrated Sample Introducing System (ISIS) and High Matrix Introducing mode (HMI). The procedure involved acid dilution of urine and direct injection into the ICP-MS instrument, followed by helium dilution in the HMI instrument. The method followed standard quality assurance and quality control procedures. Urinary metal concentrations were quantified using internal standard calibration procedures and certified analytical standards. Quality control was performed by analyzing aliquots of control material in each series (every ten samples), and accuracy was validated by the successful annual participation in the international proficiency test (G-EQUAS) for all parameters. Urine creatinine was measured using a well-established colorimetric method at the Central Teratology Laboratory at the Shamir Medical Center. It was used to standardize the metal concentration detected in the urine samples by a simple adjustment and a covariate in statistical models, as previously suggested by O'Brien et al. , 38.As part of a routine physical examination by trained neonatologists performed upon all infants following birth, birth weight, length, and head circumference were measured. The data were documented under an anonymized number for each mother–child pair. A total of 975 weight and head circumference measurements were conducted, as well as 887 length measurements. Each measurement was repeated three times for reliability, and mean values were computed. All results were documented in the newborns' medical records.Using the comprehensive data collected from each mother via the questionnaires and data gathered from maternal medical registries, we were able to adjust our final models to account for possible confounders, including maternal age , newborn’s gender, parity (nulliparous vs. multiparous), tobacco exposure during pregnancy (yes vs. no), socioeconomic status (SES) (standardized score), geographic area and creatinine concentration as measured in maternal urine. The maternal standardized SES index was individually calculated by matching maternally reported home address zip codes and the geographical distribution of SES as reported yearly by the Central Bureau of Statistics , using aAs gestational age could function as a mediator affecting the pathway between exposure and outcome , and theInformation on cigarette, cigar, or pipe smoking and the degree to which women were exposed to environmental tobacco smoke during pregnancy was self-reported by participants. Women were considered smoke-exposed if they reported either being an active smoker or were exposed to environmental tobacco smoke for 1 h or more per week during at least one-half of their pregnancy.Distributional plots and descriptive statistics were examined for all variables by the recruitment center (Rambam and Shamir). Mean values and standard deviations (SDs) were used to describe continuous variables, and independent t-tests were used to compare differences between groups. Median values, interquartile ranges (IQRs), and Mann–Whitney U tests helped describe and compare maternal urinary metal concentrations between groups. We used frequencies and chi-square tests to present and compare categorical variables between groups. All metal concentrations were modeled as natural log-transformed and standardized for IQR to achieve a standard scale and account for the positive skewness detected. The mean values of repeated anthropometric measurements were calculated and then standardized to the mean and SD of the study population.p < 0.05. All statistical processes were performed using R  and the data.table, ggplot2, dplyr, lubridate, and bkmr packages.For further analysis, statistical significance was two-sided and set at First, we evaluated the associations between exposure to individual metals during pregnancy and standardized anthropometric measures using multivariate linear regression models adjusted for maternal age, parity, newborn’s gender, tobacco exposure, SES, geographic area and creatinine concentration as measured in maternal urine. The standardized birth weight of newborns was also included as an independent variable in models that examined the association between exposure with birth length and head circumference. First, models were adjusted for covariates without considering interactions among the metals. Then, two- and three-way interactions of metal concentrations were included in the models. The results are presented as mean differences in SD of anthropometric measures  per IQR change in the log-transformed urine metal concentrations.i, an independent exposure–response function, h, as well as covariates (Xi) and their corresponding coefficients (β).Alongside the single pollution models, possible effects of joint exposures were examined. To examine potential interactions between metals on and their associations with the standardized birth weight, length, and head circumference, Bayesian kernel machine regression (BKMR) models were run. This novel non-parametric method enables a Bayesian variable selection framework to conduct analyses of mixtures without any prior assumption of linearity of the associations  and has In our study, BKMR models were fit using the Markov chain Monte Carlo algorithm, with 25,000 iterations using the Gaussian kernel . All metth, 50th, and 75th percentiles. To further examine the possible bivariate metal–response associations, we visualized the anthropometric measures as functions of two exposures while concentrations of one metal change and the second were fixed at their 10th, 50th, and 90th percentiles.For PIP, a minimal threshold of 0.50 was previously suggested  to deterAmong 975 mother–newborn pairs recruited for the study Table , the meaP = 0.004) in birth weight, as well as an average decrease in birth length of 0.133 SD . A 1-IQR increase in log Tl concentration [μg/g creatinine] was also associated with an average decrease in birth weight, of 0.081 SD . Head circumference was not significantly associated with any of the exposures. Neither two-way nor three-way significant interactions among the metals were detected for birth weight, length, and head circumference.The linear regression results are shown in Fig. th, 50th, and 75th percentiles  . In theiSimilar to Cr, increasing levels of Tl were significantly associated with lower birth weight, as shown in the linear models and supported by the BKMR models. These results are consistent with the findings of several studies –61, wherAs shown in Table S2 [see Additional File Previous studies that examined the association of Ni with fetal growth have been inconclusive; however, several studies , 72 haveThe association of maternal urine Hg concentrations with anthropometric measures of newborns has been investigated , 66. WhiAs Pb and Cd levels exceeded the limit of detection (LOD) in less than 70% of participants in our study and had a prominently lower range and mean compared with other studies –80, it iPrevious literature on the association between As exposure and anthropometric measures of the newborn is relatively limited, and reports have had mixed findings: while some failed to reject the null hypothesis , 82, othNone of the metals was found to be significantly associated with head circumference in any of the models run. Using the BKMR models for head circumference, the PIPs detected were less than 0.50 for all metals, and single exposure associations appeared insignificant. Various associations between metals and head circumference were previously shown by Rahman et al. (2021) , who exaThe current study had several strengths: the large sample size, the examination of multiple metals, the use of classic as well as advanced mixture modeling analysis, and the heterogeneous population recruited from two different geographical areas and hospitals. However, there were also several limitations. Since maternal education and income data was not collected, individual zip code-based SES was used. As our study included only term newborns, any association between prenatal maternal exposure to metals and preterm deliveries could not be examined , 78, 87.Using a large sample size and multi-metal mixture data, we delineated a potential association between prenatal maternal exposure to heavy metals and newborns’ weight, length, and head circumference. Our findings suggested that Cr was the most influential metal in predicting weight and length, as it was also negatively associated with both. An inverse association between Tl and birth length was detected, while a positive association between Ni and birth weight was suggested. Although some findings were not consistent with those of other studies, the levels of heavy metals observed in our study were relatively low, with low variances. Hence some associations detected might be spurious and should be further investigated in future epidemiologic studies as well as in vitro and in vivo biochemical studies.Additional file 1: Table S1. Urinary Metal Concentrations (n=975) corrected for creatinine levels (μg/g) Stratified by recruitment center: Shamir and Rambam. Figure S1. Bivariate Exposure-Response Functions for z-standardized weight model (n = 975). Figure S2. Bivariate Exposure-Response Functions for z-standardized length model (n = 887). Figure S3. Bivariate Exposure-Response Functions for z-standardized head circumference model (n = 975). Table S2. BKMR Posterior Inclusion Probabilities (PIP) obtained for each metal from models of anthropometric measures including gestational age as an independent variable.


Electrochemotherapy (ECT) and irreversible electroporation (IRE) are being investigated for treatment of hepatic tumours. The liver is a highly heterogeneous organ, permeated with a network of macro- and microvasculature, biliary tracts and connective tissue. The success of ECT and IRE depends on sufficient electric field established in whole target tissue; therefore, tissue heterogeneity may affect the treatment outcome. In this study, we investigate electroporation in the liver using a numerical mesoscale tissue model. We numerically reconstructed four ECT experiments in healthy porcine liver and computed the electric field distribution using our treatment planning framework. We compared the computed results with histopathological changes identified on microscopic images after treatment. The mean electric field threshold that best fitted the zone of coagulation necrosis was 1225 V/cm, while the mean threshold that best fitted the zone of partially damaged liver parenchyma attributed to IRE was 805 V/cm. We evaluated how the liver macro- and microstructures affect the electric field distribution. Our results show that the liver microstructure does not significantly affect the electric field distribution on the level needed for treatment planning. However, major hepatic vessels and portal spaces significantly affect the electric field distribution, and should be considered when planning treatments. ECT is already an established treatment for cutaneous and subcutaneous tumours , due to its high effectiveness, relatively simple application and good cosmetic results6. Based on its effectiveness for superficial lesions, ECT is now also being investigated for treatment of various deep-seated tumours8. A significant part of recent studies is focused on treatment in the liver; results indicate that ECT is a feasible and effective treatment option for primary11 and secondary liver tumours15.Electrochemotherapy (ECT) is a localized tumour treatment that combines the use of chemotherapeutic agents with the application of short high-voltage electric pulses to tissue. The application of pulses causes a transient increase in cell membrane permeability—reversible electroporation—that facilitates the transport of ions and molecules to which the membrane is otherwise impermeable or poorly permeable. Reversible electroporation significantly enhances the local cytotoxic effect of agents with intracellular targets, such as bleomycin and cisplatin19, to models of densely packed cells23, models of single cells, and models of cell membrane electroporation25. The liver is a highly heterogeneous organ, permeated with a network of blood vessels and biliary tracts. Several studies have already shown the importance of considering liver macrostructures (large blood vessels and bile ducts), when constructing models for electroporation-based treatments in the liver28. Moreover, liver parenchyma has a distinct microstructure consisting of functional units called hepatic lobules, each containing a centrilobular vein (CV). Hepatic lobules relate to a network of connective tissue, blood vessels and bile ducts , called the interlobular septa. For modelling purposes, the lobules are usually represented as prisms with hexagonal cross sections of 1000–2000 μm diameter with CVs in the center (80–187 μm diameter), and separated by gaps (~ 50 μm) representing the interlobular septa30. The electric field distribution depends on the electrical properties of the medium. This is especially important when treating target volumes that contain tissues with significantly different conductivities, as the majority of voltage drop, and consequently electric field strength, occurs in tissues with low conductivity32. The natural heterogeneity of the liver structure may have an impact on the electric field distribution and consequently on the outcome of electroporation-based treatments in liver.A prerequisite for a successful electroporation-based treatment is complete coverage of target tissue volume with sufficiently high electric field. For this purpose, numerical models of various scales are being developed for accurate prediction of electroporation in target tissue; from bulk tissue models used for treatment planning33, we examined whether ECT with bleomycin causes clinically significant damage to normal liver tissue with respect to large blood vessels and bile ducts. In our present study, we numerically reconstructed four of the experiments performed in that study33, and computed the electric field distribution in tissue with our previously developed treatment planning framework36. The aim of this study was to compare the computed electric field distribution with the histopathological changes observed in tissue after treatment, and to evaluate how the liver structures affect the electric field distribution. We determined the electric field thresholds that best correspond to the changes present in tissue after ECT, and evaluated the temperature increase and probability of thermal damage to tissue, with special focus on sensitive anatomical structures such as vessels and bile ducts. We investigated whether the liver microstructure  and the variability in its’ electrical conductivity affect the electric field distribution in any extent relevant for comparison with histopathological findings.In this study, we investigate ECT in the liver using a mesoscale tissue model that is comparable to findings from microscopic images after treatment. In a recent translational animal model study33 and computed the electric field distribution and heating in treated tissue. We fitted the computed electric field to the microscopic images and determined the thresholds that best fit the two zones of histopathological changes observed in the samples—Zone A, immediately surrounding the electrode insertion site that exhibits coagulation necrosis with complete loss of liver microstructure, and Zone B of partially damaged liver parenchyma. The electric field thresholds corresponding to the two zones for each reconstructed case are presented in Table We reconstructed four experiments of liver ECT from the study reported previouslyThere was no significant heating observed in tissue. With linear geometry electrodes, the temperature did not exceed 41 °C anywhere in the tissue, while in case of hexagonal electrodes the temperature reached 47 °C only at the electrode surface. Probability of thermal damage was determined by integrating the Arrhenius equation over the time of treatment and was < 1% everywhere in tissue in all four cases; therefore, the tissue necrosis observed in Zone A is not considered to be a direct consequence of elevated temperature in tissue. Nevertheless, a map of maximum temperature distribution for all four cases is shown in Supplementary Fig. We evaluated how the liver microstructure—hepatic lobules, septa and centrilobular veins (CV)—affects the electric field distribution. We compared the electric field distribution computed with numerical models incorporating a varying degree of geometrical detail of the liver parenchyma: a heterogeneous model , a semi-homogenous model (homogeneous hepatic tissue with CVs) and a fully homogenous model (only hepatic tissue). Detailed description of the models is provided in the Methods sub-section “Liver microstructure and parametric study of tissue properties”.37. In this example the mean relative error between electric field computed with a heterogeneous model and a fully homogenous model is 7% with a standard deviation of 12%, while the median relative error is 3%. The mean relative error between the electric field computed with a semi-homogenous and a fully homogenous model is 3% with a standard deviation of 10%, while the median relative error is 0.5%.Figure Using the heterogeneous model, we performed a parametric study, to evaluate how the differences in conductivity of the septa affect the electric field distribution and determined thresholds for Zones A and B. Electric field was computed for all combinations of parameter values of the septa conductivity function  with a higher base electrical conductivity than the surrounding liver parenchyma (0.26 S/m compared with 0.091 S/m38) and is therefore exposed to a lower electric field strength than its surroundings..The four studied samples are different from the ones presented in our previous study; special attention was given to select samples where several major hepatic vessels were involved in the treatment area. In three cases at least one electrode was inserted directly into the vessel, while in the fourth case sample S1) the electrodes were positioned around one of the hepatic veins. The presence of these hepatic vessels affects the gross electric field distribution. In Figs.  the elecNo significant tissue heating was observed in our models. This comes at no surprise, since only a small number of pulses is used in ECT. The highest computed temperature reached 47 °C in sample S1 with hexagonal electrodes, but only immediately at the surface of the electrode that was positioned in the hepatic vein. In case of the linear geometry electrodes, the temperature did not exceed 41 °C, since a significantly lower number of pulses is cumulatively applied and a slower repetition rate is used. Probability of thermal damage according to Arrhenius kinetics equation was < 1% everywhere in tissue for all four cases.33, we investigated whether ECT with bleomycin causes clinically significant damage to normal liver tissue. Upon histological examination of explanted liver samples, acute changes with clear zonation were observed in the tissue. The area immediately surrounding the electrode insertion site exhibited coagulation necrosis with complete loss of liver microstructure . Surrounding this area was a zone of partially damaged liver parenchyma, which was attributed to irreversible electroporation (IRE) of tissue . No histological changes were observed in areas exposed to reversible electroporation. Furthermore, the addition of bleomycin (electrochemotherapy) did not cause any difference compared with samples only treated with electric pulses. In our current study, we fitted the computed electric field to the microscopic images of the treated area to determine the threshold values that best fit the appearance of the Zones A and B.In our previous study44. The pH change is attributed to the ion transport, which results in a strong acidification at the site of the anode and alcalinization at the site of the cathode, which result in necrotic regions near the electrode insertion site. The necrosis in Zone A observed in our samples may be related to the pH changes around the electrodes, however, further research is needed to verify this speculation. The appearance of Zone B is most likely caused by IRE of tissue. Even though lower pulse amplitudes and pulse number are used in ECT, an area of irreversibly electroporated tissue is also present around the electrodes. Our computations show the best fit with Zone B is achieved with electric field strength of 810 V/cm, which is sufficient to cause IRE of hepatic tissue with reversible electroporation pulse protocols45.The mean electric field threshold that best fits the appearance of Zone A is 1225 V/cm. Although histological examination of Zone A revealed changes characteristic of coagulation necrosis, in Zmuc et al. we postulated that it is not likely these changes were caused by tissue heating during pulse delivery, which was now also confirmed by our computations. In recent years the role of pH changes in cell death mechanisms is being investigated in electroporation-based treatments33, the estimated thresholds for Zone A were 1500 V/cm for linear and 1200 V/cm for hexagonal electrodes. The electroporated volume and electric field threshold depend on the number and duration of applied electric pulses (i. e. exposure time). With one pair of linear electrodes significantly less pulses are cumulatively delivered to tissue compared to hexagonal electrodes (8 pulses vs. 96 pulses respectively), therefore, a higher threshold for linear geometry electrodes is expected24. In present study, however, we found no significant difference between the thresholds for linear and hexagonal electrodes for both zones. We achieved good agreement with the original study for the hexagonal electrodes, however, for linear electrodes our threshold was approximately 300 V/cm lower. The discrepancy between the results could be due to sample selection bias. Although the data was obtained from the same experiments, the thresholds in the present study were determined on samples located at the sites of major hepatic vessels and bile ducts, whereas in the previous study33 the thresholds were determined on samples located exclusively in the liver parenchyma and obtained from a different animal. Biological variability and different methods of threshold determination are among the main reasons for the different threshold values reported in literature48. Lastly, in Zmuc et al., the thresholds were estimated by matching the radius of Zone A to the distance obtained from the image of field distribution. For electric fields of such high intensity (> 800 V/cm), the transition zone is very short and even an error of 1 mm in the estimated zone radius can result in a difference of 150 V/cm in the electric field threshold. However, in the present study, we used a refined image-based fitting method for threshold determination with a step of 10 V/cm.In Zmuc et al.In the second part of the study, we investigated whether the microscopic structure of the liver  and the differences in electrical conductivity of these microstructures affect the electric field distribution during ECT on a level relevant for comparison with microscopic images. The result of our study show, that the inclusion of the liver microstructures does not significantly affect the computed electric field. There is a localized drop in field strength observed in the domain of the septa and CVs Fig. , however33, Zmuc et al. observed that the damage in Zone B was not distributed equally, as it was more pronounced in the centri- and midlobular areas. This observation might be explained by taking into consideration the vascularization of the hepatic lobules. At the center of each lobule there is a centrilobular vein surrounded by sinusoids (capillaries), while the “wall” of the lobule consists of the vascularized septa and portal tracts. Blood flows through the lobule walls, traverses the sinusoids, and flows into the centrilobular vein29. In Fig. In the previous studyOur study was mainly limited by its retrospective nature. While in case of hexagonal electrodes, the positions of the electrodes were easily discernible in the histological samples, the soft liver tissue was somewhat deformed during electrode insertion, therefore the inter-electrode distances in the samples were no longer 7.3 mm in all locations within the samples. Already a 1–2 mm difference in inter-electrode distance resulted in significant difference in the determined electric field thresholds. We applied scaling and shearing deformations to the 2D models, however, we were unable to completely eliminate the deformation, therefore small differences in inter-electrode distance remained. On the other hand, in case of linear geometry electrodes, we were able to fix the inter-electrode distance in the model to 2 cm. However, only one electrode site was visible in the sample and the position of the counter electrode had to be determined to the best of our ability, based on the shape of the Zones A and B.27.In conclusion, we confirmed that the liver microstructure  does not significantly affect the electric field distribution at a mesoscale. The use of a fully homogeneous model of the liver parenchyma is suitable for the numerical computations of electric field in the liver organ, needed for planning electroporation-based treatments. However, major hepatic vessels and portal spaces should be included in the model, as these macrostructures significantly affect the electric field distribution, as already suggested before33, which is reported in accordance with ARRIVE guidelines for reporting of research involving animals. All experiments were performed in accordance with the relevant guidelines and regulations. Regulatory approval for this study was obtained from the National Ethics Committee at The Administration of the Republic of Slovenia for Food Safety, Veterinary, and Plant Protection . Experiments were performed on healthy pig liver; six pigs were treated with ECT with bleomycin and two pigs received pulses only, serving as control. The pulses were delivered with the Cliniporator pulse generator  using either fixed hexagonal geometry electrodes or two linear geometry electrodes with 2 cm spacing. The following ECT protocols were used: for linear geometry electrodes 8 × 100 μs, 2000 V electric pulses were delivered with a 1/s repetition rate; for fixed hexagonal geometry electrodes 96 × 100 μs, 730 V electric pulses were delivered with a 5000/s repetition rate. The applied voltage in both electrode geometries corresponded to a 1000 V/cm voltage-to-distance ratio. Two days after the procedure the liver was explanted, cut and fixed in formaldehyde. After 24 h, the specimens were cut into 2–3 μm thick samples, stained with H&E and microscopically examined and photographed. Further details regarding the experiments, treatment protocols and histological analysis are described in33.The animal experiments were performed in the scope of a recent in vivo animal model study by Zmuc et al.33. A pathologist manually outlined the two zones in microscopic images. The outlined images were imported to Adobe Illustrator CS4 where major haptic vessels, vessel walls, portal spaces and bile ducts were manually outlined as well, and electrode insertion trajectories were determined. The outlines were saved in vector format . The heat dissipation process is much slower than electroporation phenomenon, therefore the base conductivity for the following electrode pair computations is increased due to increased temperature. For a more detailed explanation of the modeling approach, see Supplementary materials Section "Numerical model and computation".COMSOL Multiphysics was used for computation of electric field and temperature distribution during the procedures. The computation process is described in detail in51. DSC calculates the similarity between two binary images (masks) and takes a value between 0 and 1. A similarity of 1 means that the masks match perfectly. We compared the masks of Zones A and B with masks representing isocontours of the electric field at selected thresholds (field masks). The process is shown in Fig. The computed electric field and temperature distributions were imported into MATLAB for comparison with the segmented microscopic images. The 2D model was already identical to the microscopic image of the sample Fig. a, while In order to evaluate, how the liver microstructures and the variability in electrical properties of these structures affect the computed electric field distribution we constructed a 2D model incorporating the hexagonal liver architecture. A hexagonal structure representing the hepatic lobules and interlobular septa was constructed using Adobe Illustrator CS4. The modelled lobule diameter was 1950 μm, CV diameter was 150 μm and septum thickness was 50 μm Fig.  a. PositThree models with a varying degree of geometrical details were computed; a heterogeneous model incorporating the whole liver microstructure , a semi-homogeneous model consisting of homogeneous hepatic tissue with CVs, and a fully homogeneous model consisting only of hepatic tissue. In order to eliminate error due to meshing, identical geometry (whole liver microstructure) was used in all three models; the homogeneous effect was achieved by matching the conductivity of the septa and CVs to the conductivity of the lobules.0), factor of conductivity increase, center (EC) and size (EW) of the transition zone. The parameters of the electrodes and all tissues except for the septa were taken from previous works36, and are listed in Supplementary Table C and EW were taken from conductivity functions of hepatic lobules and vessel wall. All studied parameter values are listed in Supplementary Table The change in electrical conductivity due to electroporation of each modelled structure is approximated in COMSOL as a smoothed step function, with the following parameters: base electrical conductivity  and for the semi-homogenous and homogeneous models. Then we calculated the similarities between the computed electric fields and segmented Zones A and B of sample S1 see Fig. a and comThe animal experiments were performed in the scope of our previously published animal study by Zmuc et al. , which is reported in accordance with ARRIVE guidelines for reporting of research involving animals. All experiments were performed in accordance with the relevant guidelines and regulations. Regulatory approval for the study was obtained from the National Ethics Committee at The Administration of the Republic of Slovenia for Food Safety, Veterinary, and Plant Protection .Supplementary Information.


Enigmatic phenomena have sparked the imagination of people around the globe into creating folkloric creatures. One prime example is Zana of Abkhazia (South Caucasus), a well‐documented 19th century female who was captured living wild in the forest. Zana's appearance was sufficiently unusual, that she was referred to by locals as an Almasty—the analog of Bigfoot in the Caucasus. Although the exact location of Zana's burial site was unknown, the grave of her son, Khwit, was identified in 1971. The genomes of Khwit and the alleged Zana skeleton were sequenced to an average depth of ca. 3× using ancient DNA techniques. The identical mtDNA and parent‐offspring relationship between the two indicated that the unknown woman was indeed Zana. Population genomic analyses demonstrated that Zana's immediate genetic ancestry can likely be traced to present‐day East‐African populations. We speculate that Zana might have had a genetic disorder such as congenital generalized hypertrichosis which could partially explain her strange behavior, lack of speech, and long body hair. Our findings elucidate Zana's unfortunate story and provide a clear example of how prejudices of the time led to notions of cryptic hominids that are still held and transmitted by some today. The displaced origins of a misconstrued person. Face reconstruction from Tkhina‐75 skull. Bottom right: artistic reconstruction after consideration of contemporary witness accounts. The locals described her as being “part human and part animal,” 2 m tall and dark‐skinned, covered with thick hair, who was able to lift a 50 kg sack of flour with one hand, and outrun a horse in a race.To overcome limitations of a previous DNA analysis and the possible ambiguities of craniometric studies, we sequenced the genomes of both the unknown female and Khwit, to 3.1‐ and 3.3‐fold coverage, respectively. We performed genome‐wide analysis to explore their genetic ancestry and kinship relations, which allowed us to shed light on Zana's story based on objective genome‐wide data.2See the Supplementary Methods for a more in‐depth description of the materials and methods used in this study.2.1All ancient DNA (aDNA) laboratory work was carried out in dedicated clean laboratory facilities at the GLOBE Institute, University of Copenhagen, according to aDNA standards described elsewhere.We used teeth and petrous bones to extract ancient DNA from both individuals and build double‐stranded BGISeq libraries following the BEST protocol, using adapters compatible with BGI sequencing according to Mak et al. 2017.We used the BAM workflow implemented in the PALEOMIX pipelineWe used mapDamage v2.0 to get the read length distribution and approximate Bayesian estimates of damage parameters.2.2https://github.com/ruidlpm/pathPhynder).We used haplogrep2A phylogenetic network analysis of complete mtDNA sequences including that of “Zana”, Khwit and other L2 haplogroup sequenceshttp://www.popgen.dk/software/index.php/IBSrelate) which was followed by estimation of IBS sharing matrix based on the two‐dimensional site frequency spectrum (2d‐SFS) from real‐SFS implemented in ANGSD.The kinship coefficients were calculated by first generating a site allele frequency likelihood file (saf) in ANGSD  was performed using plink v1.9 with the ancient genomes projected onto the modern variation. The first 30 eigenvectors of PCA were used as input for the uniform manifold approximation and projection (UMAP) analysis using the “umap” R package.D‐statistics estimates were calculated using the ADMIXTOOLS3A total of 1 219 599 801 BGISeq sequences were generated from the teeth and petrous bones for both individuals (see Mapping Statistics in The contamination estimates based on mtDNA (for both) and X chromosome (only for Khwit) were less than 1%, and the chromosomal sexes of the individuals matched their anthropological descriptions: Khwit as male and “Zana” as female.3.1The mtDNA sequences were identical for both individuals, consistent with the hypothetical mother‐son relationship, and could be assigned to haplogroup L2b1b, the parental haplogroup of which (L2b clade) is widely distributed in western Africa,Khwit's Y‐chromosomal lineage belongs to the haplogroup R1b1a1b1b which clearly reflects his non‐African paternal heritage. This is a sub‐haplogroup of a major haplogroup R1b1a1b defined by M269 mutation, which is encountered in high frequencies in Europe and western Asia.Although an earlier study that analyzed Khwit's mtDNA sequence revealed "Zana's" maternal origin as African,Silva et al. have estimated that mitochondrial clade L2b likely originated ca. 24 kya,3.2We next further tested whether the two individuals were directly related using PC‐relate as implemented in PCAngsd3.3To further assess the genetic relationship between Zana, Khwit and various worldwide populations using nuclear genome variation, we ran a PCA based on the Human Origins (HO) panel. To aid visualisation, we reduced the total number of reference populations in the panel to represent the major genetic lineages of the world. However, given its geographic relevance, we included relatively more populations from the Caucasus, and given previous hypotheses that Zana may have had some archaic hominid ancestry, we also included genome‐wide data from three archaic humans, and used the chimpanzee as an outgroup. Additionally, we included the two Mesolithic hunter‐gatherers (SATP and KK1) from the South Caucasus for comparison.The results clearly show that Zana is neither genetically close to archaic humans nor the chimpanzee, but clusters closely with modern human populations Figure . As expeUnsupervised clustering analysis using ADMIXTURE also clearly rejects any hypothesis that Zana was of “nonhuman” origin, for example as suggested by various sources.To estimate Zana's ancestry proportion more accurately, we conducted the admixture analysis in “supervised” mode based on 13 African populations Figure  represenTo formally test the possible Neanderthal introgression into Zana's genome we conducted 𝐷‐statistics in the form of  Figure . The resIn summary, our results based on genome‐wide analysis reveal that Zana's genome had a sub‐Saharan African genetic origin, consistent with the results of previous (unpublished) craniometric and mtDNA analyses. This suggests that her presence in the region may have been linked to the Ottoman Empire slave trade to Istanbul, that was one of the main hubs for the slave trade in the region in the 19th century. Moreover, Zana's largely eastern African ancestry is consistent with the historical records indicating that most of the African slaves in the Ottoman Empire originated around areas of the African Great Lakes and present‐day Sudan.The contemporary reports and subsequent tales of Zana's wildness were at least partially based on some of her unusual physical characteristics such as the lack of speech, intellectual disability and long hair covering her whole body to name a few. With the genomic data clearly rejecting all nonhuman hypotheses, we speculate that if these descriptions of her physical characteristics are accurate, she may have had a rare human genetic disorder such as congenital generalised hypertrichosis: a syndrome with dismorphic facial features, intellectual disability, and hypertrichosis.4Our results prove that the unknown female buried in the Genaba family cemetery was Zana herself. In contrast to the speculations that she might have been a female Almasty, we provide definitive genome‐wide data to put an end to the accounts of her as anything but a human woman.Zana was likely of eastern African descent, although we cannot rule out partial western African ancestry. We hypothesise that her lineage could have arrived in the territory of present‐day Abkhazia (South Caucasus) as a result of the slave‐trade practiced between the 16 to 19th centuries CE by the Ottoman Empire. Lastly, we speculate that it was simply her unfamiliar individual physical characteristics  and the subsequent rumors over generations that fueled the myth of a non‐human origin.4.1Following her capture in the forest, Zana was deprived of her basic human rights, and treated as a slave: she was kept in captivity, likely forced to have sexual relations with local men, and worked in forced labor conditions. After she passed away, the accounts on her mythical figure attracted several scientists to unearth her story and her son's bones were exhumed. Our study intends, both to reveal the true human nature of Zana and grant her and her descendants' remains the dignity they deserve.All permissions for excavations in the 1960–1970s and for aDNA analyses were provided by the relevant authorities.Margaryan, Ashot: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Validation; Visualization; Writing‐original draft; Writing‐review & editing. Mikkel‐Holger Sinding: Data curation; methodology; validation; writing‐review & editing. Christian Carøe: Data curation; investigation; methodology; validation; writing‐review & editing. Vladimir Yamshchikov: Conceptualization; data curation; methodology; validation; writing‐review & editing. Igor Burtsev: Conceptualization; investigation; methodology; validation; writing‐review & editing. M. Thomas Gilbert: Conceptualization; funding acquisition; investigation; project administration; supervision; writing‐original draft; writing‐review & editing.The authors declare no competing interests.https://publons.com/publon/10.1002/ggn2.10051 and in The peer review history for this article is available at AppendixS1: Supplementary NoteSupplementary Figure S1.1 Zana's descendantsClick here for additional data file.Supplementary Table 1Click here for additional data file.Supplementary TPR FileClick here for additional data file.


A pseudoaneurysm or false aneurysm is the consequence of a persistent blood leak caused generally by iatrogenic rupture of a vessel wall. The blood leak creates a new cavity delimited by surrounding tissues and allows blood flow to remain in continuity between this cavity and the vessel. In hemodialysis fistula, pseudoaneurysm generally results from repeated puncturing of the vein at the same site, leading to a bulging anatomical defect in the vein. Over the past few years, interventional radiological treatment has evolved and taken the place of surgery, with different kinds of percutaneous and endovascular treatment methods in pseudoaneurysm management. We reported a case report of successful treatment of arteriovenous fistula pseudoaneurysm with no-measurable neck. We performed ultrasound-guided percutaneous direct thrombin injection while an inflated balloon transiently obstructed flow out of the pseudoaneurysm, in order prevent non-target embolization. A 79-year-old man with end-stage renal disease undergoing hemodialysis with a radio-cephalic arteriovenous fistula (AVF). Nine months after fistula creation, the patient arrived to our attention because of the presence of a post-anastomotic hemodynamic venous stenosis.Under local anesthesia, an ultrasound-guided puncture of AVF was performed and a 5F sheath was positioned in the draining vein. The diagnostic angiography confirmed a focal post-anastomotic stenosis that was treated with a 5 mm × 40 mm balloon with good angiographic result .Fig. 1ThA month after, as a result of this procedure, the patient presented just after her hemodialysis session, a pulsatile and painful mass localized in cannulation site, so a new diagnostic angiography was performed and it was confirmed the presence of wide-necked pseudoaneurysm in the draining vein, at the level of previous puncturing site.A 6 × 60 mm balloon was advanced over the wire and positioned and inflated across the pseudoaneurysm basis in order to exclude the cavity from the circulation then, under ultrasound guidance, the pseudoaneurysm was punctured with a 21-gauge needle and 2 mL of thrombin was real-time injected under ultrasound guidance, until to complete filling the pseudoaneurysm . The balFlebography demonstrated, also, a juxta-anastomotic stenosis, that was treated with a 6 mm × 60 mm balloon with good angiographic result.At the final check, there was no residual stenosis, while the pseudoaneurysm was completely excluded from the circulation.Depending on size and location, it is reported in literature that some pseudoaneurysms may undergo spontaneous thrombosis, as the majority of small post-catheterization pseudoaneurysms ,2. BasedMany kinds of endovascular or percutaneous methods are described for pseudoaneurysm treatment, such as ultrasound-guided compression, covered stent, detachable balloon, coil embolization, or percutaneous thrombin injection However, while for wide-necked pseudoaneurysm treatment, it also reported thrombin injection under ultrasound guidance with the use of covered stent deployed across the neck of the pseudoaneurysm In this case, ultrasound-guided local thrombin injection has the advantage of being a low cost, to guarantee the effectiveness of treatment, not needing sedation, less traumatic, technically simple procedure in experienced hands, paying highly attention to avoid non-target embolization Finally, the absence of a covered stent across puncturing site guaranteed AVF long-term patency: after 1 month from the treatment, the patient continued to dialyze successfully with no further complications.Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.Our institution does not require ethical approval for reporting individual cases or case series. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


A gestational age of 22 to 23 weeks is the lower threshold for selective active intervention. Few infants delivered at a gestational age of 22 to 23 weeks survive if resuscitated. Among those who survive, most develop severe complications, especially in cases of multiple births at the limit of viability.We report the intact survival of extremely preterm twins, a girl (Twin A) and a boy (Twin B), weighing 504g and 475g, respectively, born at the edge of viability at 22 2/7 weeks gestation without significant morbidity.extremely preterm twins born at the edge of viability at 22 2/7 weeks.Twin A required 6 weeks of mechanical ventilation. She received conventional and high-frequency oscillation ventilation. She was extubated to noninvasive positive airway pressure ventilation at 28 weeks and 2 days post conception. Twin B required longer duration of invasive ventilation lasting 11 weeks. Moreover, he had several episodes of feeding intolerance and abdominal distension. However, his serial abdominal radiographs showed nonspecific findings. The gastric tubes were eventually removed from both twins. Full oral feeding was successful on discharge.Both infants are presently in good condition.They were discharged home with a full oral feeding, and without any respiratory support. Now they are 18-month-old with unimpaired development.This report would support healthcare providers in decision-making. It highlights the importance of perinatal and neonatal management optimization to improve survival rates and clinical outcomes of periviable birth. In addition it emphasize the individuality of each case and the need to consider the parents’ wishes in the management decision. Consequently, the survival rates of significantly premature babies have improved; yet, their morbidity rate has increased.,5There are several reported cases of singleton infants delivered before 23 weeks of gestation who survived without significant disabilities and achieved appropriate neurodevelopment.,7 The intact survival of twins born before 23 weeks are rare.Recent data have shown continuous improvements in general health and neurodevelopmental outcomes and an increasing tendency to consider active interventions for infants at the edge of viability.9/L, an absolute neutrophil count of 23 × 109/L, a C-reactive protein level of 105 mg/L, and a negative test result for procalcitonin. Both fetuses exhibited good cardiac activities and active movements. The mother had conceived following in vitro fertilization and embryonal transfer. She was also the mother of healthy 3-year-old twins, who were also conceived by in vitro fertilization. The medical team offered the parents comfort and counseling regarding proactive care, intensive care, and predicted outcomes. The parents requested that all available life-support measures be initiated, despite their complete understanding of the risks and long-term expected problems.A 30-year-old woman who was pregnant with diamniotic dichorionic twins at 22 2/7 weeks of gestation was admitted to the delivery room for preterm labor. She experienced bleeding, and her membranes had ruptured 23 hours before admission. Her condition was complicated by chorioamnionitis, diagnosed due to fever, tachycardia, and elevated inflammatory markers. She had a white blood cell count of 24 × 10The parents’ decision to initiate full resuscitation was unique to our unit. A complete plan, with some flexibility under our policy, was adopted in consultation with the obstetricians and parents. After completing the prenatal steroid course, a live female infant (Twin A) weighing 504 g with Apgar scores of 4, 5, and 7 at 1, 5, and 10 minutes, respectively, was spontaneously delivered. Subsequently, a live male infant (Twin B), weighing 475 g, with breech presentation and Apgar scores of 3, 5, and 7 at 1, 5, and 10 minutes, respectively, was delivered. Both newborns unexpectedly were born in good condition. Each of them was resuscitated, intubated, and administered 1 dose of surfactant.Twin A required 6 weeks of mechanical ventilation. She initially received conventional respiratory support; however, within a few days, she required high-frequency oscillation. Thereafter, she was extubated to noninvasive positive airway pressure ventilation at 28 weeks and 2 days post-conception and, eventually, to nasal high-flow oxygen. Two weeks later, Twin B was extubated to biphasic positive airway pressure. However, after a few days, he required reintubation and invasive ventilation. His second intubation lasted for 3 weeks because of bilateral irreducible inguinal hernias that required urgent herniotomy. He was finally successfully extubated after 11 weeks of invasive ventilation. He then received continuous positive airway pressure on day 92 . The patient successfully weaned to nasal high-flow oxygen after 3 weeks.For both twins, parenteral nutrition was started on the first day of life via an umbilical venous catheter and continued via a peripheral venous catheter. The mother’s milk was administered on their second day of life by trophic feeding through nasogastric tubes and was continued with a gradual, incremental increase in volume until full feeding was tolerated. For Twin B, oral feeding was suspended several times because of intolerance and abdominal distension. However, his serial abdominal radiographs showed nonspecific findings. The gastric tubes were eventually removed from both twins, and full oral feeding was successful upon discharge.Their serial cranial ultrasound scans were normal. On echocardiography, Twin B had a patent ductus arteriosus with a maximum size of 2.5 mm. This was conservatively managed with fluid restriction. Follow-up echocardiography revealed a closed ductus arteriosus. His neonatal course was more complicated than his sister. He also had a right femur fracture due to osteopenia of prematurity, bilateral retinopathy of prematurity, which was treated with ranibizumab injections , and bilateral inguinal hernias, which were treated surgically.Four months after birth, respiratory support was weaned at the postconceptional age of 40 weeks and 5 days for Twin A and 43 weeks and 2 days for Twin B. Both twins are in excellent condition. They have returned home without respiratory or feeding support Table .At discharge, Twin A weighed 3708 g (47.35 centile), her length was 50 cm 16.27 centile), and her head circumference was 33.5 cm (5.21 centile). Twin B was discharged with a weight of 4120 g (3.52 centile), length of 49 cm (<1st centile), and head circumference of 34.8 cm (<1st centile). Neurological examination at the chronological age of 16 months, which corresponded to a corrected age of 12 months, demonstrated no focal deficit. Moreover, the age-appropriate neurodevelopmental-behavioral test did not exhibit any major developmental delay based on their corrected age in any of the 5 domains–motor, language, cognitive, social-emotional, and adaptive for either infant. Their growth is being monitored, and their parents are satisfied with their progress (Table 7 centile,9This report aimed to document the uniqueness of each patient in a premature birth, even in twins, and to emphasize that the decision to resuscitate infants born at the lower end of the viability range should be shared between clinicians and parents. Patient care should be individualized based on the risk factors and the parents’ wishes and beliefs. Nevertheless, advances in intensive care and a great willingness by obstetric and neonatal care providers to provide active care has led to a steady increase in the survival rates of extremely preterm newborns during the last decade.Infants born at periviability at 22 to 25 weeks of gestation could potentially survive with active support. However, their risk of mortality and morbidity is high.,12 Patients born at 22 to 23 weeks of gestation have a higher risk of severe disability, although the data for babies born at 22 weeks are based on a small number of patients.–4The risk of severe disability rises with increasingly extremely preterm births. Among actively managed infants, the rate of severe disability is approximately 1 in 7 at 24 weeks of gestation, 1 in 4 at 23 weeks of gestation, and 1 in 3 at 22 weeks of gestation.,13Absolute survival and survival without severe impairment have been associated with protective factors. These include, but are not limited to, exposure to prenatal corticosteroids, female sex, singleton birth, higher birth weight, and an advanced clinical setting with experienced staff. However, the decision to administer intensive care at the lower end of viability between 22 to 23 weeks gestational age is challenging for clinicians and parents because of variable and complex ethical components and institutional practices and policies.Risk assessment and effective parental counseling, as well as an explanation of the risks and aiding in decision-making, should be undertaken. The family needs support to help them understand the expected outcomes, bearing in mind that each case is individualized. Moreover, the parents’ wishes should be considered in the decision to resuscitate.We are thankful to the patients and their family members for participating in this research study. The project was not funded by any organization.Formal analysis: Ashraf Abuobayda, Nabil Elmansoury.Writing – original draft: Wafaa AlRawi.Writing – review & editing: Wafaa AlRawi,TaisserAtrak, Ashraf Abuobayda, Nabil Elmansoury, Omar El Nakeib, Abhijeet Lonikar.