Preterm birth is defined as babies born alive before 37 weeks of pregnancy or fewer than 259 days since the first day of a woman’s last menstrual period. Globally, 14.84 million babies were preterm births. Preterm infants are at risk for specific diseases related to the immaturity of various organ systems. This study aimed to assess the prevalence of preterm birth and associated factors among mothers who gave birth in public hospitals of east Gojjam zone, Ethiopia.An institutional-based cross-sectional study was conducted from April 1 up to June 30, 2021, in public hospitals in the east Gojjam zone. Systematic random sampling was used. Data were collected through structured questionnaires, patient interviews and patient card reviews. We used binary logistic regression analysis with 95% CI and P-value < 0.05 to identify the significant factors with preterm birth.Out of 615 mothers, 13.2% gave a preterm birth. Antenatal care ), educational status of mother ), husband educational status), Average monthly family income),family size), multifetal gestation , having Premature Rupture Of Membrane ), history of chronic illness ), being HIV positive), Ante-Partum Hemorrhage ), pregnancy Induced Hypertension ), mode of delivery ), and onset of labor ) were found to be significantly associated with preterm birth.antenatal care, educational status of the mother, husband’s educational status, family income, family size, multifetal gestation, Premature Rupture of the membrane, history of chronic illness, being HIV positive, Ante-Partum Hemorrhage, pregnancy Induced Hypertension, mode of delivery, and the onset of labor were found to be significantly associated with preterm birth. To minimize the proportion of preterm birth focusing on this important variables, timely identification of obstetric complications, strengthening early screening of HIV and high-risk pregnancies like multiple gestations, PIH and APH were important. Preterm birth (PTB) is defined as babies born alive before 37 weeks of pregnancy or fewer than 259 days since the first day of a woman’s last menstrual period . GloballPreterm infants have a higher risk of developing specific diseases due to the immaturity of various organ systems and the causes of preterm birth. As a result of their prematurity, preterm babies are subjected to serious illnesses or deaths during their neonatal period. In the absence of appropriate treatment, survivors are more likely to suffer a lifelong disability and a compromised quality of life. Prematurity complications are the leading cause of neonatal death and the second leading cause of death among children under 5 years old. . PrematuIn low- and middle-income (LMIC) countries, preterm births account for more than 60% of all births, and the rate has steadily increased. Despite this high preterm birth rate, it is challenging to determine the trend of preterm birth in the majority of low-income economies due to a lack of accurate data . Sub-SahThe complication of infants born at preterm gestational age results in a trivial cost to the health sector, parents, and society. The prediction and prevention of preterm birth is a major health care priority . Global Previous studies conducted in different regions indicated that several risk factors were identified for preterm birth. This includes having a previous preterm birth, having a short cervix, short intervals between pregnancies, and certain pregnancy complications , chronic illness, educational status, Multiple pregnancies, maternal age, residing in rural areas, antenatal care visits, being HIV positive, family number, and income , 18–26.PTB is a major public health problem. However, in most low-income countries, including Ethiopia, little emphasis is given to PTB intervention as a means of reducing infant mortality. Health care providers or other stakeholders who worked in this public health problem need data related to common factors associated with preterm birth. However, in our country, the studies conducted about this problem were minimal. Although few studies have been conducted in some areas of Ethiopia, the magnitude and possible risk factors of PTB vary by area. A number of methodological issues are addressed in our study  that were not considered in previous studies. Studying a large region is essential to designing effective interventions and programs in public health. Most of the previous study focused mainly on the prevalence of PTB, rather than associated factors of PTB. In addition, different cultures and socioeconomic statuses within a society have varied factors of preterm birth. Therefore studying in different and multicenter settings was important. Determining preterm birth prevalence and associated factors greatly guides health professionals and health policymakers to identify indicators for monitoring preterm birth strategy and applying necessary preventive and appropriate measures to decrease preterm birth. Moreover, the study area’s magnitude of preterm births and associated factors were unknown. Therefore, this study was conducted to assess the prevalence of preterm birth and to identify factors associated with preterm birth in public hospitals in the east Gojjam zone.The hospital based cross-sectional study design was conducted using interviewer administered questionnaire from April 1 up to June 30, 2021. Additional information was obtained from medical records of the mothers and babies.The study was conducted in public hospitals in the east Gojjam zone, Amhara National Regional State, Ethiopia. Debre Markos city is a zonal administrative city. It covers 14,010 square kilometers and is divided into 18 administrative districts, further subdivided into 49 urban and 392 rural kebeles, the smallest administrative units . The EasAll mothers who gave birth in randomly selected public hospitals in the east Gojjam zone during the study period were our study population. Mothers who gave birth and had known either LNMP or had early ultrasound (before 24 weeks) diagnosis were included for this study. Mothers who were deaf and comatose, had unknown last normal menstrual period (LNMP) and had no early ultrasound (before 24 weeks) were excluded from this study.A systematic sampling technique was used. The sample was arranged based on the three-month patient flow before the data collection period by referring to the hospital’s delivery registration book/ record. To calculate K, the summation of the three months delivery report for the hospital was 1698.Then K = N/n, 1698/615 = 2.8 ≈ 3. Where k = interval, N = total population, n = sample size. Every third mother’s was interviewed, gestational age of the newborn was calculated based on the mothers LNMP or first-trimester ultrasound result, in estimating gestational week, when there are extra days it was counted to the near lowest gestational age. The next participant was taken when the selected study participant was not eligible for the study.The sample size was determined by using a single population proportion formula by considering the prevalence (p) of preterm birth = 15.5%, which was obtained from the previous study in Ethiopia , 95% conFinally, after taking a 10% non-response rate the total sample size (n) was 615. An average delivery report for a month before the actual data collecting period was computed for each hospital by analyzing the client’s registration book to distribute the sample size proportionally to each hospital. The sample size was then proportionally allocated to each hospital. Finally, the immediate postnatal mother with her baby had been selected every three intervals using a systematic sampling technique.Data were collected and extracted by reviewing medical records of mother’s .The data collectors were four fourth year undergraduate midwives students from Debre Markos University. One staff midwife from each hospital was assigned to supervise the data collection process. The data collection process was supervised by both the principal investigator and the supervisor. Information collected from the mother included Residency, Age, Religion, Education status, Occupation, Marital status, Average monthly Average monthly family income, Antenatal Care (ANC), inter pregnancy interval parity, Educational Status of husband, Ante-Partum Hemorrhage (APH), Premature Rupture Of Membrane (PROM), Pregnancy Induced Hypertension (PIH), multiple pregnancies, polyhydramnios, anemia, cardiac disease, hypertension, HIV status, Modern contraceptive use before current pregnancy, Urinary Tract Infection (UTI) and malaria were the predictor variable for this study. Anemia was defined as an HGB level below 11gm/dl (HCT < 33%). Obstetric complications was defined as challenges or problems that happen during labor or delivery. PIH was defined clinically as a blood pressure of > 140/90 mmHg after 20 weeks of gestation with or without proteinuria and/or edema as diagnosed and documented by the attending clinician. APH was defined as any vaginal bleeding in the mother after 24 weeks of gestation as documented in the records by the attending clinician. UTI was defined as a documented clinical/laboratory diagnosis of UTI any time during the pregnancy and/or a positive history of treatment of burning sensation with micturition as reported by the mother. Birth to pregnancy interval was defined as the time between the start of the index pregnancy and the preceding live birth. Last normal menstrual period was defined as the date of the starting of last normal menstruation the women had to index pregnancy. Preterm birth was defined as a newborn with a gestational age of 28 weeks to less than 37 weeks. To assure the quality of data, the questionnaire was pre-testing on 31 mothers in public hospitals in the east Gojjam zone, and the questionnaire’s fitness was confirmed from pre-testing. One-day practical training on how to collect data was given to the data collectors and the supervisor before data collection. During the data collection period, the collected data were reviewed, checked for completeness, and signed by the supervisor at the end of each day.All questionnaires were checked, coded, and entered into the SPSS version 25 software packages, which were then analyzed using R 4.1.3. The data were presented using frequency tables and graphs. The relevant determinants of preterm birth were identified using binary logistic regression. The researchers used both bivariable and multivariable analyses. In the bivariable analysis, independent variables with a p-value less than 0.25 were chosen for the multivariable analysis. An adjusted odds ratio with a 95% confidence level was used to examine the degree of relationship between independent and dependent variables, and variables with a p-value of 0.05 were considered statistically significant.All participants completed the interview (100% response rate). The majority of the respondents 358 (58.2%) were rural residents. The majority of the study participants 360 (58.5%) were between 25 and 34 years old. The entire respondent belongs to Amhara by ethnicity, and 516 (96.1%) were Orthodox Christians in religion. Regarding the marital status of the respondents, the majority of them 596(96.9%) were married. 253 (41.1%) were have no formal education, 217(35.3%) of the respondents were taken education in primary school, and 253(41.1%) of them can only read and write, and the remaining 145 (23.6%) were secondary and above education level. The occupational status of most of the respondents 228(37.1%) were farmers, 144 (23.4%) of the respondents were housewives, 150 (24.4%) of the respondents were employers, and the remaining respondents had other occupations . 264 (42.9%) of the husband were have no formal education. 264(42.9%) of the respondent’s family average monthly income is less than or equal to 3000 Birr and 212(34.5%) of the respondent’s family average monthly income is average greater than 5000 Birr. The majority of the respondents, 415 (67.5%) had between 3 and 5 family members (Table Most of the respondents (96.3%) had ANC follow up and 56.1% of the respondent had at least 4 visits. The majority of the respondents used modern contraceptives (94.8%) before their pregnancy, and the majority of mothers had their pregnancy wanted and planned (99.3%). More than two-thirds of the respondents had birth-to-pregnancy intervals greater than or equal to 36 months (83.5%). Labor spontaneously started in 87.8% of the respondents. Three-quarters of the respondents (74.3%) gave birth by SVD. Out of the total respondents, 595(96.7%) were tested for HIV status, and 36 (5.7%) respondents were HIV positive. (133) 21.6% of women had APH, (142) 23.1%, of women had PIH, (70) 11.4%, of mothers had multiple pregnancies, and women had Polyhydramnios (10) 1.6%. 105(17.1%) of the respondents had a history of chronic illness. 70(11.4%) of mothers had urinary tract infection  ), educational status of mother ), husband educational status), Average monthly family income),family size), multifetal gestation , having PROM), history of chronic illness ), being HIV positive), APH), PIH ), mode of delivery ), and onset of labor ) were found to be statistically significant at p-value of < 0.05 (Table .75),famiThis study was conducted to assess the magnitude of preterm birth and its associated factors in public hospitals in the east Gojjam zone. During the study period, the overall proportion of preterm birth was found to be 13.2%. The finding was greater than the preterm birth rate for the world (9.8%) and North America (9%) . It was The proportion of preterm birth in this study however was higher than another study conducted in our country at Gondar town health institutions, which reported a prevalence rate of 4.4% . This diThe study conducted in Malawi shows that the prevalence of preterm birth was 16.3% . This hiThe current study’s finding was lower than those conducted in Kenya National Hospital and Jemma University Specialized Hospital, which reported the prevalence of preterm birth was 20.2% and 25.9%, respectively , 38. ThiMothers with less than 3000 birr Average monthly family income were 1.95 times more likely to develop preterm than those with greater than 5000 birr family income. This study was in line with the study in SSA , 39,whicMothers from three up to five family members were 0.15 times less likely to develop/give preterm as compared to mothers from greater than five family members. This study was in line with the study in Ethiopia , which sThe education status of mothers were significant factors of preterm. Mothers who had no formal education were 2.79 times more likely to develop/give preterm than mothers who had secondary and above education levels. This study was in line with the studies in Sub-Saharan African and European countries –41, whicMothers who had husbands who had no formal education were 2.11 times more likely to develop/give preterm than mothers who had husbands with secondary and above education levels. This study is in line with the previous study in Iran , which sMothers with APH were 3.62 times more likely to have a preterm birth than mothers without APH. This finding is in line with the study conducted in Kenya, Ethiopia, and East Africa , 44, 45.Mothers with multifetal gestation were 3.30 times more likely to have a preterm birth than mothers without multifetal gestation. This finding is in line with the study conducted in Ethiopia, Korea, Greek –48. It mMothers who delivered with SVD were 7.16 times more likely to develop preterm as compared to mothers delivered with CS or Instrumental delivery. This finding is in line with the study conducted in Ethiopia , which sMothers with spontaneous labor were 0.10 times less likely to have preterm birth as compared to mothers with induced labor. This finding is in line with the study conducted in Ethiopia , which sHistory of chronic illness was significantly associated with the outcome variable, mothers who had a history of chronic illness were 3.94 times more likely to give preterm birth than mothers who had no chronic illness history. This finding is in line with a study conducted in Ethiopia , 44, 49,This study revealed a significant association between pregnancy-induced hypertension and preterm birth. Mothers who had complications PIH were 3.61 times increased risk of having a preterm birth than those mothers without this problem during the index pregnancy. This finding is in line with the study conducted in East Africa, Ethiopia, Nigeria, Iran, Ghana, and Kenya , 51–55, Another significant association was found between mothers who had premature rupture of membranes (PROM) and preterm birth. Mothers with PROM were 6.46 times more likely to have a preterm birth than their counterparts. This finding was in line with the previous findings in Tehran, Iran, Ethiopia, East Africa, Nigeria , 56, shoIn this study, the ANC follow up were significantly associated with the outcome variable preterm birth. Mothers with < 4 times ANC follow-up in the index pregnancy were 2.87 times more likely to have a preterm birth than mothers who had the ANC visits ≥ 4 times. This finding is in line with a study conducted in Ghana, Nigeria teaching hospitals, and western Ethiopia , 54, 57,Being HIV positive was significantly associated with preterm birth. HIV-positive mothers were 6.99 times more likely to give preterm compared to HIV-negative mothers. This finding is in line with a study in Malawi and Ethiopia , 58, 59,In contradiction to the previous studies in our study, age, marital status, RH factor, Pregnancy status, and UTI , 62, 63.Despite efforts to reduce recollection biases by informing local events, there may be a recall bias. When ultrasound facilities are not accessible, the menstrual history and clinical examination are used to confirm gestational age and which may subject to considerable error. Another drawback of our study is using secondary data for some variables.The proportion of preterm birth in public hospitals in the east Gojjam zone is 13.7%. number of ANC, educational status of mother, husband educational status, family income, family size, occupation, multifetal gestation, having PROM, history of chronic illness, being HIV positive, APH, PIH, mode of delivery, and onset of labor were found to be significantly associated with preterm birth. As a result, focusing on these important variables would reduce the number of premature births. Furthermore, it was suggested that educating the community about the importance of ANC service utilization of mothers and preventing preterm birth be strengthened. Moreover, strengthening early screening of HIV and high-risk pregnancies like multiple gestations, PIH and APH were important to prevent preterm.


In this work, MBGNs and PHBV-MBGN microspheres, both loaded with Astaxanthin (ASX), were synthesised and compared in terms of ASX release kinetic, ASX entrapment efficiency, and cell viability. Moreover, the correlation of the release kinetic to phytotherapeutic efficiency and side effects was established. Interestingly, there were significant differences between the ASX release kinetic of the developed systems, and cell viability differed accordingly after 72 h. Both particle carriers effectively delivered ASX, though the composite microspheres exhibited a more prolonged release profile with sustained cytocompatibility. The release behaviour could be fine-tuned by adjusting the MBGN content in the composite particles. Comparatively, the composite particles induced a different release effect, implying their potential for sustained drug delivery applications.In recent years, composite biomaterials have attracted attention for drug delivery applications due to the possibility of combining desired properties of their components. However, some functional characteristics, such as their drug release efficiency and likely side effects, are still unexplored. In this regard, controlled tuning of the drug release kinetic via the precise design of a composite particle system is still of high importance for many biomedical applications. This objective can be properly fulfilled through the combination of different biomaterials with unequal release rates, such as mesoporous bioactive glass nanoparticles (MBGN) and poly(3-hydroxybutyrate- In 85 g/mol ,11.co-3-hydroxyvalerate) (PHBV) are well-studied types of PHAs that have been largely employed for the development of drug delivery microspheres [In recent years, ASX has drawn extensive attention for dermatological and cosmetic applications due to its outstanding antioxidant activity, which prevails over that of tocopherol, its supportive effect on skin health, and its protective role against UV radiation ,13. Howeospheres ,27,28,29ospheres ,31. Li eospheres , for insospheres ,33,34. IPHBV was purchased from Goodfellow , dichloromethane and Astaxanthin (ASX) from Sigma-Aldrich , and Polyvinyl Alcohol (PVA) from Baxter Healthcare . Tetraethyl orthosilicate , triethyl phosphate , and calcium nitrate were bought from Aldrich . Furthermore, ethyl acetate and cetyl-trimethylammonium bromide (CTAB) were provided by Merck , ammonium hydroxide (28%) was provided by VWR , and distilled water (MilliQ) and absolute ethanol (99.8%) were provided by Alfa Aesar . All the used chemicals were of analytical grade.w/w) and dried for 6 h in an incubator at 60 °C.The mesoporous bioactive glass nanoparticles (MBGN) were produced following a modified Stöber procedure, which was previously reported in . For thew/w) of loading. There was a PHBV/MBGN ratio of (10:1) for all the samples. Subsequently, the emulsion was centrifuged at 6000 rpm, and the supernatant was removed entirely to let the precipitated microspheres dry overnight in an incubator at 60 °C. All the particle synthesis was carried out in the beakers and tubes protected against light.Simultaneously, composite PHBV-MBGN-ASX microspheres were produced using a solid-in-oil-on-water (S/O/W) method, which was previously reported in ,32. BrieThe average particle diameter and zeta potential measurement of the Astaxanthin-loaded MBGN (MBGN/ASX), the PHBV-MGBN-ASX (composite microspheres), blank MBGN, and blank PHBV particles were carried out by using a Zetasizer Nano ZS  in deionized water for MBGN conditions and ethanol for PHBV conditions, similar to previous reports ,36.Micrographs of the surface morphology of the MBGN/ASX and the composite microspheres were obtained via Scanning Electron Microscopy (SEM) using an SEM Auriga . All samples were sputtered with gold in a turbomolecular-pumped coater Q150T Plus  prior to SEM examination.−1 .Absorbance infrared spectra were obtained for the neat components, MBGN/ASX, and composite microspheres by using a Fourier Transform Infrared spectrometer (FTIR) Nicolet 6700 . Measurements were carried out in the middle-infrared (MIR) region from 4000 to 400 cmsup is the ASX mass measured in the supernatant in mg, and Mthe is the initial ASX mass added during the fabrication in μg.The ASX Entrapment Efficiency (EE) was calculated according to the supernatant method previously reported . The ASXThe ASX-release kinetics were calculated by adding 5 mg of each sample to 5 mL of ethanol in triplicate ,37. The 3 cells were taken from 90% confluency flasks and seeded in 24-well plates. Considering the EE of each condition, master solutions of ASX/MBGN, composite microspheres, and free ASX were so prepared to have a similar content through each condition. Afterward, 10-fold serial dilutions of 10 µL, 100 µL, and 1000 µL from each master solution were added to the 24-well plates previously seeded with cells. A similar amount of blank MBGN and blank PHBV microspheres was used as additional controls, and untreated cell samples were used as control. All experiments were carried out in triplicate. The cell viability was determined via WST-8 assay  after 24 and 72 h post-administration according to the provider protocol. Briefly, the supernatant was carefully removed from all well plates and washed with HBSS; later, freshly prepared cell culture medium containing 1% v/v WST-8 solution was added to the 24-well plates and incubated for 3 h. After incubation, 100 µL of supernatant from each well plate was transferred, by duplicates, into a 96-well plate for the absorbance measurement at λ = 450 nm in a PHOmo Autobio , similar to previous reports [NIH-3T3 mouse fibroblasts cells  were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with BCS, sodium pyruvate, and L-glutamine . Afterward, 50 × 10 reports .Cell samples were stained with Life & Dead Hoechst protocol using Calcein AM, Propidium Iodide, and Life & Dead assays  for cell morphology observation after 72 h of incubation. Cell staining was carried out according to the provider’s protocol. After staining, the samples were stored and protected from light prior to observations. Fluorescent micrographs were obtained and processed using a fluorescent optical microscope Primovert with AxioCam ERc 5s .MBGN/ASX and composite microsphere samples were immersed in SBF, produced according to the protocol of Kokubo , and incThe surface morphology of the samples was analysed via SEM according to p < 0.05), using the software Origin 8 , and the data were presented as mean ± standard deviation for each measurement.Experiments were carried out in triplicate. The analysis of statistical significance was conducted by analysis of variance One-Way ANOVA followed by the Tukey test , MBGN/ASX, and composite microspheres recorded in the mid-infrared region (MIR) from 4000 to 400 cmonjugate ,48. Excethe MBGN . The ban of PHBV , which iThe ASX entrapment efficiencies (EE) of the MBGN/ASX and composite microspheres are shown in The cumulative release with respect to the initial theoretical amount of ASX in both drug delivery systems exhibited a characteristic biphasic controlled release curve. Nonetheless, the final concentration in MBGN/ASX was slightly lower than that in the composite microspheres, a behaviour that corresponds to the EE difference between the systems. Additionally, a steeper slope was observed in the ASX-MBGN kinetic, which is related to a higher release ratio during the burst phase and resulted in an apparent complete depletion of the release during the first 40 h. In contrast, the composite release kinetic exhibited a lower slope during the burst phase and an extended controlled phase up to the first 80 h after administration, allowing a prolonged ASX release. This dissimilarity in the release behaviour may be explained based on the morphology and composition features of the systems, as the MBGN exhibited a mesoporous surface that favours the release as soon as the nanoparticles are in contact with the medium. In addition, the surface contact area/volume ratio is higher in the MBGNs because of their nanometric dimensions, promoting a fast drug release.On the contrary, in the composite microspheres, the surface contact area/volume ratio is low. Furthermore, the homogeneous polymer surface acts as an extra phase where the medium needs to permeate through the polymeric barrier to trigger the diffusion, and once the process starts, the diffusion may be delayed in comparison to pure MBGN ,37. The Nevertheless, both systems exhibited a proper potential for the ASX-controlled release applications, and this behaviour may be extended to other phytotherapeutics and other applications in which high initial concentrations must be avoided ,49,50. AAfter 14 days of immersion in Simulated Body Fluid (SBF), the structure and morphology of MBGN/ASX and composite microspheres were further analysed via XRD and SEM observation c,d, wher−1), MBGN/ASX (10−2 and 10−3), and composite microspheres (10−1) presented significant differences with respect to the control. On the other hand, after 72 h of incubation (−1 and 10−2) and MBGN/ASX (10−1) exhibited a significant decrease in cell viability, while the composite microspheres (10−1 and 10−2) sustained an increase in cell viability. At this time, free ASX and blank PHBV presented no significant difference with respect to the control.The cell viability of both MBGN/ASX and the composite microspheres administrated to NIH-3T3 cells was assessed after 24 and 72 h; blank MBGN, blank PHBV particles, and free ASX were used as additional conditions, and cells without treatment were considered as control shown in . The ASXcubation b, some dFluorescent microscopy observations after 72 h c correspThe results mentioned above show the importance of the release kinetics for the effective administration of the model drug by the two studied delivery systems. Another important aspect was the difference in the EE of both systems, indicating that a higher amount of MBGN/ASX is required to release equivalent amounts of ASX for extending the drug’s beneficial effects. Nonetheless, this also will increase the amount of MBGN in contact with cells, which, as discussed before, may impact biocompatibility. Furthermore, it could be interesting to characterise the influence of the MBGN content in the composite release kinetic as an easy alternative for tailoring the release in these kinds of carriers to determine if this behaviour can be extended to other model drugs. In any case, both the MBGN/ASX and composite microspheres exhibited an optimum potential for controlled phytotherapeutic delivery needed for specific applications.FTIR measurements confirmed the successful incorporation of ASX in MBGNs and composite microspheres. The MBGN/ASX and composite microspheres exhibited an optimum ASX entrapment efficiency . The release kinetic curves showed a significant difference between the release behaviour of the MBGN/ASX and composite microspheres, where the latter exhibited prolonged release of ASX up to the first 80 h compared to half (40 h) of the MBGN/ASX.The WST-8 cell viability results and fluorescent cell morphology observations showed that the difference in the release behaviour affected the NIH-3T3 cell viability during the first 72 h of incubation. These results suggested that the release behaviour of the composite microspheres may be tuned by modifying the MBGN content in the composite microspheres. Additionally, the exhibited release kinetics may be extended to other phytotherapeutics similar to ASX.Although the composite microspheres are promising for sustained in vitro drug release applications, additional characterisation on these systems could be interesting, such as the effect of varying the MBGN content on the drug release kinetics and the stability and antioxidant activity of ASX during the particle synthesis process and the release.


Metabolomic analysis methods were employed to determine biomarkers for various chronic kidney diseases (CKDs). Modern analytical methods were developed and applied successfully to find a specific metabolomic profile in urine samples from CKD and Balkan endemic nephropathy (BEN) patients. The aim was to explore a specific metabolomic profile defined by feasible/easy-to-identify molecular markers. Urine samples were collected from patients with CKDs and BEN, and from healthy subjects from endemic and nonendemic areas in Romania. Metabolomic analysis of urine samples, extracted by the liquid-liquid extraction (LLE) method, was performed by gas chromatography-mass spectrometry (GC-MS). The statistical exploration of the results was performed through a principal component analysis (PCA) evaluation. Urine samples were statistically analyzed using a classification based on six types of metabolites. Most urinary metabolites are distributed in the center of a loading plot, meaning that these compounds do not represent significant markers for BEN. One of the most frequent and higher-concentration urinary metabolites in BEN patients was p-Cresol, a phenolic compound that implies a severe injury of the renal filtration function. The presence of p-Cresol was associated with protein-bound uremic toxins, which have specific functional groups such as indole and phenyl. In prospective studies for future investigation, prevention, and disease treatment, we suggest a larger sample size, sample extraction using other methods, and analysis using other chromatography techniques coupled with mass spectrometry, which can generate a more significant data set for statistical analysis. Chronic kidney diseases (CKDs) affect almost 15% of the world population and are considered an emerging global health problem . Many stUrine-based metabolomics has been applied in research on early detection, renal toxicity, diagnosis, biomarker identification, and pathogenic pathways of kidney disorders and kidney cancer . One ana2-microglobulin, creatinine, urea cycle metabolites, and uric acid, were determined by nuclear magnetic resonance (NMR) , and aristolochic acid I . These were purchased from Sigma-Aldrich .A general overview of the study population is presented in Morning midstream urine samples were collected from informed volunteers, and written consent was obtained before sample collection. The collected samples (~10 mL) were mixed with a solution of 1% sodium azide prepared in sterile deionized water to inhibit bacterial growth, and stored at 4 °C for 2 to 4 h (until extraction). The urine samples were then extracted (as described in (1)n = 35; code names 23, 25, 28–36, 38–61) who are family members of BEN patients (n = 8) or live in BEN areas (“endemic controls”) (n = 27); these were self-selected by availability. We ensured that only people without any documented history of kidney disease and not currently taking prescribed medication participated in our study. Urine samples  collected during the first part of the day served as controls for the samples collected from the BEN patients.Healthy control volunteers ((2)n = 5; code names C1, C2, C3, C4, and C5) registered at the Clinical County Hospital, Timisoara, Romania, served as disease controls .Patients with nephrological diseases other than BEN ((3)n = 25; code names 1–22, 24, 26, and 27) under hemodialysis at Renamed Centre, Drobeta-Turnu Severin, Mehedinti County. Patients  were residents of Erghevita, Bistrita and Valea Izvorului villages in Mehedinti County. The subjects were self-selected during visits for hemodialysis sessions performed at the clinic, and none was suspected to have urinary tract tumors.Patients diagnosed with BEN  protocol: 2 mL urine was extracted with 4 mL DCM. This mixture was left to a continuous extraction process in 10 mL tubes on a horizontal shaker for 24 h at room temperature. As a result of the separatory process, two liquid phases were formed: the upper phase contained the aqueous urine phase, and the lower phase contained the organic DCM (solvent) phase. After the complete separation of the two phases, the lower one was transferred to another tube and evaporated to dryness under inert nitrogen gas, and the dry residue was kept at −20 °C until analysis.Prior to injection into the GC-MS system, the final residue obtained in the previous stage was restored in 50 µL of PRA grade DCM after reaching room temperature. Then 2 µL of the extract was injected manually in splitless mode into the inlet, which was heated at a temperature of 180 °C. Three different concentrations of urine samples spiked with aristolochic acid I were prepared in DCM, yielding final molar concentrations of 1.03 µM, 0.20 µM and 0.02 µM. The GC-MS conditions were established as follows: the injected sample was passed through a non-polar capillary column  carried by a helium flow rate of 1 mL/min. The temperature program of the oven was set to increase from 50 to 250 °C at a heating rate of 6 °C/min and a final hold of 5 min. The conditions of the mass spectrometer system were: source temperature set at 230 °C, quadrupole detector temperature set at 150 °C, and electron ionization (EI) mode of 70 eV. The mass range of detected compounds was from 50 to 550 amu, registered after 3 min of solvent delay. Peaks were integrated using the MS software Chemstation Data Analysis  and AMDIS , and mass fragments specific for aristolochic acid I were identified in urine samples using a spiked urine sample as a standard. All chromatograms were processed with Enhanced MSD ChemStation software ver.D.02.00/2005  and AMDIS GC/MS Analysis software version 2.70. Deconvoluted spectra obtained by the specific data processing protocol were introduced into the Agilent ChemStation and AMDIS GC/MS Analysis search software, and an automatic search of compound information from the NIST MS Search 98 and 2.0 libraries  was performed. Search results with match similarity above 90% were accepted as candidate compounds, and their mass spectra were compared to those obtained from the MS libraries. The relative concentrations calculated with Chemstation Data Analysis software were used for statistical evaluations.1 is obtained as the direction in the original property space, which provides maximum covariance of the projected data. PC2 is obtained in the same manner, but with the restriction of orthogonality to the PC1, and so on. As a consequence, these PCs are linear combinations of the original variables. In terms of linear algebra, PCA is an approximation of the dataset, defining the X matrix as a product of two matrices with reduced dimensionality, T and P. The first matrix provides a score plot by plotting its columns, while the second matrix is obtained by plotting its rows, resulting in the loading plot. The score plot provides information about the grouping of cases, represented by the BEN patients, patients with other types of CKDs, and control volunteers. The loading plot provides information about the influence of variables on this grouping, represented by the urinary metabolites. The first PCs will be sufficient to explain the main variance of the data. In order to reduce bias, the original data are centered and normalized [PCA is a powerful multivariate analysis technique able to extract useful information from a large dataset. This technique provides a lower dimensional principal subspace that consists of orthogonal principal components . PCrmalized ,19. In om/z) ratio and polarity. The classes of compounds are: aliphatic and aromatic acids, fatty acids, amines, amides, sterols, alkanes, alkenes, alcohols, phenols, esters, aldehydes, ketones, hydroxides, indoles, pyrazines, pyridines, xanthines, carbohydrates, phenyl compounds, and carboxylic acids  of our method.The 65 subjects included in the present study were males and females aged between 58 and 75 and living in villages in Mehedinti County, Romania. A total of 262 compounds were identified in urine samples in several main classes; most of them were miscellaneous, having more than one reactive group. Compound appearance in a chromatogram is based on mass-to-charge  and all variables (urine metabolites) included. The distribution pattern of cases  is similTo obtain a specific distribution of cases and variables, six groups (Groups 1 to 6) were generated. PC1 versus PC2 score plots  and loadIn Group 1, urine samples and 12 metabolites resulting from the drug metabolism were considered. The following drugs were found in urine samples: brallobarbital, cyclobarbital, diazepam, amobarbital, phenobarbital, 3- salicylic acid, antipyrine, and flufenamic acid. Some of the resulting compounds represent drug metabolites: ampyrone, phenylhydrazone, 4-(diethylamino) salicylaldehyde, 6H-1,4-diazepin-6-one, 2,3-dihydro-5,7-dimethyl-, and metacetamol. Ampyrone and flufenamic acid were the most frequent drugs found in 39 urine samples, 13 of them being present in BEN patients. The statistical model did not reveal any significant results when BEN patients were compared with CKD patients and the control group. BEN patients, marked with red dots, cluster in the middle and the right side of the score plot , meaningn = 20) of BEN patients versus 66% (n = 23) of the controls, because of the small number of samples, the statistical model did not reveal any significance. Only one subject (C3)  , represen = 18) were included for statistical evaluation, these being present in 74% of urine samples . Generally, this is one of the most common phenolic compounds present in urine samples, typically found at a high percentage in subjects with affected kidneys .In Group 6, all metabolites of urine samples from family members of patients were considered. Ten families were identified as living in BEN areas, some of the members being BEN patients and others being healthy controls  and the number of the individual . The following families have one member with BEN , while other families include two members with BEN . There are also two families with healthy controls . A statistical model without significance was obtained when urine samples of BEN patients and controls were compared. As can be observed, BEN patients appear at the right upper and lower parts of the score plot .In the research field of Balkan endemic nephropathy, this study is the first involving human urine samples collected from patients with BEN, volunteer controls, and patients with other types of CKDs. Upon extraction, the samples were analyzed using the simple and sensitive GC-MS technique. In BEN, as in AKI and CKD, normalization to urinary creatinine (UCr) concentration is not necessary when comparing across individuals with impaired renal function. The UCr concentration changes rapidly, leading to different creatinine generation rates . AccordiThe GC-MS technique was chosen as the method of analysis because even when limited to the detection of volatile organic compounds (VOCs), it is a non-invasive screening technique, and volatile metabolites from urine analysis result from different classes of compounds that could reveal specific disease patterns . The mecThe first classification for the statistical evaluation was made with drug metabolites resulting from self-medication, although none of the patients and volunteers disclosed specific treatments they were following at the time of sampling. Most of the main molecular ions were specific for the non-steroidal anti-inflammatory drug class. It is worth mentioning that the distribution pattern of the score plot from A second classification was made for metabolites that contained phenolic groups. Phenolic compounds are considered typical urine biomarkers. The activity of gut microbiota through dietary polyphenols and aromatic amino acids fermentation leads to the presence of phenols in urine samples. Phenol derivatives result from the metabolism of tyrosine and phenylalanine through the intestinal microbiome activity. One of the main phenolic compounds is p-Cresol, which was found in 80% of BEN patients. This compound is slowly cleared by dialysis, and is accumulated in the kidneys, leading to inflammation and fibrosis . VariatiAnother major class of compounds present in urine samples was hormones, which have endogenous and exogenous sources. Most hormone presence is due to hormonal medications. Most of the subjects were older than 45 (70%), and some of the female subjects were receiving estrogen replacement therapies at the time of sample collection. The most common hormonal metabolites detected in urine samples are found in the center of the loading plot .Aristolochiaceae plants [The major aristolochic acid ions were searched based on the mass spectrum of the aristolochic acid I standard, but none of these molecular ions could be detected, probably because the subjects were not exposed to aristolochic acids around the time of urine sample collection, or because the concentrations were below the detection limit . One of e plants . In a ree plants . In a ste plants .Other environmental toxins to which BEN patients are exposed, and which are considered cofactors in disease causation, are polycyclic aromatic hydrocarbons (PAHs), which contaminate groundwater supplies and have anthropogenic or geogenic sources such as Pliocene lignite deposits found in the vicinity of the endemic villages . AlthougThis study has some limitations because it presents preliminary results obtained from a limited number of patients. Because of the limited number of tested subjects, the PCA statistical method was chosen to reveal specific urinary biomarkers. There is a need for control samples from the population living in BEN areas, as well as urine samples taken from BEN patients at different timepoints. Furthermore, a continuous study over a longer period, and a complex epidemiological study associated with other biological biomarkers , could provide better knowledge for BEN prevention. The chosen liquid-liquid extraction method without derivatization resulted in a limited number of volatile compounds, usually thermostable ones. Other limitations of this study include the limited incidence of BEN cases in Romania (only in the southwestern part of the country). Differences in urine sample concentration, using creatinine levels as a proxy, were absent. Other extraction techniques and analytical methods should be used to identify other types of urinary metabolites beside those obtained by the GC-MS method in order to generate a large dataset of compounds from all chemical compound classes. The generated results should be analyzed using univariate and multivariate statistical methods combined with artificial intelligence techniques, which could lead to robust models necessary to obtain specific and discriminative biomarkers . An inteTo the best of our knowledge, this study is the first to use urine metabolomics to discriminate among specific biomarkers in a limited number of individuals, including BEN patients, patients suffering from other types of CKD, and healthy individuals living in BEN areas. The liquid-liquid-extraction method was employed to extract a large panel of metabolites. The GC-MS analysis resulted in a panel of 262 urinary compounds, which seemed to be appropriate before statistical evaluation using the PCA technique. Although the statistical models generated using PCA did not reveal any significant results because of the limited number of samples of studied groups, it is worth mentioning that some key metabolites were found in a higher percentage and in a higher number of BEN samples than those found in the control group. The increased metabolites in urine BEN patients are phenolic compounds, especially p-Cresol, which implies an impairment of the renal filtration function. The discovery of p-Cresol as a marker involved in BEN pathology development could be used for early renal impairment diagnosis before the disease is clinically manifested. The excretion of dicarboxylic acids, cholesterol, and phenolic acids in the urine suggests severe oxidative stress processes. Other specific compounds for BEN urine samples that came to light after the PCA statistical evaluation were protein-bound uremic toxins with specific functional groups: indole, phenyl, phenol. These urinary metabolites provide a particular metabolomic signature when compared to normal urine samples, and could be used in a screening procedure to diagnose early forms of BEN in the population in endemic villages. Because of the limited number of CKD patients, no specific urinary metabolites were identified compared to BEN patients. So far, no studies have been performed using urinary analysis in search of specific biomarkers for early BEN diagnosis. All these methods, including extraction, chromatography, and statistics (LLE—GC-MS—PCA), could serve as primary noninvasive screening tools for generating preliminary data, leading to prompt decision making, which is necessary for further investigation of BEN patients.


This paper reports a rare case of adrenocortical carcinoma (ACC) diagnosed during pregnancy presenting with gestational hypertension. Hypertensive disorders in pregnancy should receive enough attention to identify and exclude the possibility of adrenal diseases, thereby making a timely diagnosis and active treatment. Gestational hypertension, a common complication of pregnancy, remains one of the leading causes of maternal and perinatal fetal and neonatal mortality, which seriously compromises the health of mothers and infants , 2. The A 26-year-old, gravida 2, para 1 (G2P1) woman at the 31st week of gestation was admitted to the hospital with a chief complaint of elevated blood pressure for 3 months. The woman occasionally suffered from dizziness and amaurosis when changing her posture during pregnancy. Regular antenatal examinations were performed, and there was no sweating or other special discomfort during pregnancy. The oral glucose tolerance test (OGTT) revealed 5.46-8.76-8.42mmol/L, and the patient was diagnosed with gestational diabetes mellitus and given diet and exercise interventions, but no regular blood glucose monitoring. The blood pressure was 106/78 mmHg at the 7th week of gestation, 127/94 mmHg at the 16th week of gestation, and 149/100 mmHg at the 25th week of gestation. However, the patient did not pay attention enough attention to it and also did not receive further diagnosis and treatment. Palpitation and chest tightness occurred once at 31 weeks + 2 days of gestation. The next day, the patient felt headache and dizziness, without blurred vision, and her blood pressure was 161/112 mmHg. Therefore, the patient was transferred to the local hospital. The presence of a solid mass in the right-side adrenal region was identified during the admission obstetric ultrasound evaluation. The Fig. Two months after delivery, the patient underwent surgery of “excision of the right adrenal gland and space-occupying lesion + partial resection and repair of vena cava + stripping of right renal capsule” in our hospital. During the operation, a tumor about 10 cm in size was seen in the right retroperitoneum, with a fixed, position and hard texture, which was closely related to the right liver, kidney, and retrohepatic inferior vena cava. The tumor tissue was sent for pathological examination after the operation, and the results indicated an adrenal space-occupying grayish red nodular mass 10*7.5*7.5 cm), and the cut surface was grayish red and grayish yellow with medium texture, accompanied by hemorrhage and necrosis. A histopathologic description of cells is arranged in an acinar shape. Fig. . Immunoh*7.5 cm, Considering the patient’s condition, obstetrics and urology department recommended chemotherapy. After 3 months of follow-up, she has completed chemotherapy at another hospital. During the follow-up, she survived with no clinical evidence of disease recurrence.ACC is a rare endocrine malignancy, and the etiology of most sporadic cases is unknown . ACC typAdrenal disorders are relatively rare in pregnancy, and in most cases, the diagnosis can only be confirmed at autopsy. The pathophysiologic repercussions of adrenal disorders are enormous to both mother and fetus, leading to significant maternal and fetal morbidity. Hence, timely diagnosis and appropriate treatment are critical to the prognosis of the mother and fetus –9.Pregnancy-associated ACC is even less seen. The reported clinical symptoms of the vast majority of ACC patients in pregnancy are atypical, mainly including hypertension, diabetes, fatigue, depression, acne, etc. What is worse is that ACC in pregnancy is usually found at the advanced stage, resulting in a larger tumor than that observed in non-pregnant patients .A retrospective study that compared 12 women diagnosed with ACC during pregnancy or immediately after delivery with non-pregnancydiagnosed ACC patients, and found that patients diagnosed during pregnancy or postpartum had larger tumor volumes. However, the survival rate of patients diagnosed during pregnancy was 50% at one year and only 13% at five years . SeveralSTAT3, and destroy insulin receptor substrate-1 (IRS-1) and IRS-2, eventually resulting in insufficient glucose uptake and hyperglycemia [It has been pointed out that more than 17% of tumor patients are accompanied by diabetes and abnormal elevation of blood glucose . The impglycemia . On the Numerous studies have proven the functional alternations of the hypothalamic-pituitary-adrenal (HPA) axis in patients with type 2 diabetes, especially the high secretion of cortisol. Some scholars have proposed that there is a reciprocal causation relationship between hyperglycemia and high cortisol secretion. Glucocorticoids can not only promote glycogenolysis, inhibit liver glycogen synthesis, and stimulate gluconeogenesis, but also inhibit insulin secretion and thus lead to elevated blood glucose , 17. AdrAccording to reports, a woman affected by GDM was accidentally diagnosed with pancreatic cancer during the growth scan in the third trimester of pregnancy, so the growth scan in the third trimester of pregnancy is also very important in diagnosing the maternal abdominal mass .This study reported a rare case of ACC in pregnancy, that presented with gestational hypertension. Due to an accurate abdominal ultrasound evaluation the suspicion of an adrenal mass occurred.Surgical treatment is the first choice for patients during an early second trimester of pregnancy who have an apparent mass on the adrenal gland, medication is usually ineffective. The best time for surgical intervention during pregnancy is the second trimester.In our opinion, after explaining the relative risks and surgical expertise involved in each case, individualized treatment should be carried out according to the preferences of patients and surgeons.This study suggests that hypertensive diseases occurring in pregnancy should receive enough attention, and the possibility of adrenal diseases should be differentiated and excluded, so as to make a timely diagnosis and active treatment.


Objective: To identify the determinants and risks associated with developing hypertension and metabolic syndrome in the first year postpartum in women who experienced preeclampsia. Methods: A cohort study was conducted, involving women who had experienced preeclampsia (PE) recently. The control group was women with the same characteristics but a healthy pregnancy. The variables analyzed were somatometry, disease history, pre-pregnancy body mass index (Pre-BMI), and Third Adult Treatment Panel updated (ATP III) metabolic syndrome (MS) data . These variables were measured at 3, 6, and 12 months postpartum. Results: Women with a history of PE exhibited higher systolic and diastolic blood pressure than women without PE. The risk of developing isolated diastolic arterial hypertension at 3 and 12 months of follow-up was two to eight times greater in women with a history of PE. Factors associated with having higher blood pressure levels were preeclampsia, insulin resistance, age, and BMI. Neither the pre-BMI index nor gestational weight gain (GWG) had any effect on blood pressure in any of the three assessments. Women with preeclampsia had a 5- to 8-fold increased risk of developing MS . However, PE was not identified as a risk factor at the six-month evaluation and was only explained by pre-pregnancy obesity and overweight. Conclusions: Obesity and overweight, as well as preeclampsia, were strongly associated with the development of hypertension and metabolic syndrome during the first year following childbirth. Preeclampsia (PE) is a hypertensive disorder presenting after the 20th week of pregnancy, may or may not be accompanied by proteinuria and a low platelet count, with the involvement of other organs, and affects 2–8% of first pregnancies ,2,3,4. IConversely, prior to pregnancy, women with metabolic syndrome (MS) may face a higher risk of developing preeclampsia and diabetes during pregnancy ,11. AddiThis study aimed to identify the determinants and risks associated with developing hypertension and metabolic syndrome in the first year after childbirth in women with a recent history of preeclampsia, compared to women without this antecedent (controls).To recruit patients, we consulted the medical records of women who gave birth within the last 45 days, using a consecutive approach. This non-probabilistic study was conducted at the National Institute of Perinatology in Mexico City, a tertiary government institution that caters to the uninsured population (from October 2014 to April 2017) in a cohort of women between 18 and 45 years of age with preeclampsia in the last pregnancy. All patients had to comply with the selection criteria . Additionally, participants should not have taken medications during and after the pregnancy that interfere with the variables of interest . Elimination criteria were: women who were detected—during the follow-up—with pathologies such as hyperthyroidism, hypothyroidism, kidney damage, liver failure, or who became pregnant again.The controls were recruited consecutively, at the same time as the cases, matching by age and by the (aforementioned) inclusion and exclusion criteria, among patients diagnosed with normal-evolutionary pregnancy (without pathologies). All eligible participants still hospitalized were personally invited to participate, while those who had been discharged were contacted by telephone. A total of 261 participants met the research criteria and provided informed consent. Among them, 148 had preeclampsia (52 severe and 96 mild), while 113 controls without preeclampsia were recruited.Proteinuria .Other maternal organ dysfunctions include:The diagnosis of preeclampsia was made during pregnancy by senior obstetrics and gynecology specialists in the clinic, commonly based on the criteria of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy . The FIGAcute kidney injury , liver involvement  with or without right upper quadrant or epigastric abdominal pain, neurological complications , or hematological complications .3.Uteroplacental dysfunction  .Although the International Society for the Study of Hypertension in Pregnancy (ISSHP) now does not recommend classifying preeclampsia as moderate or severe , at the General demographic characteristics, pathological and obstetric history, as well as pre-pregnancy weight and height, were collected. Pre-pregnancy BMI  and gestThe research adhered to the principles of the Declaration of Helsinki, always ensuring the confidentiality of the study subjects. It received approval from the ethics, research, and biological sample handling committees (Protocol code 3210-21001-02-14 on 12 December 2019).The variables analyzed were somatometry and clinical characteristics of ATP III metabolic syndrome (MS) . These variables were measured at 3, 6, and 12 months postpartum.(a)Somatometry: This included measurements of weight, height, waist circumference, and hip circumference. Body weight was obtained under normal conditions using a TANITA digital scale . Height was assessed using a SECA stadiometer . Anthropometric measurements were taken following the Centers for Disease Control and Prevention guidelines (CDC) .(b)Blood pressure. Blood pressure measurements were performed using the auscultatory method in triplicate, simultaneously by a doctor and a nurse, both standardized with the Habicht method .(c)Blood pressure was determined after at least 10 min of rest using a sphygmomanometer and a cuff appropriate for the participant’s complexion. The measurements were taken in the morning by triplication with an interval of 2 to 3 min; the participant was seated, leaning on the chair’s backrest, with feet on the floor, and extended left arm. Simultaneous determinations were made by the doctor and nurse using a stethoscope with a double earpiece and a single capsule, allowing them to listen to the same patient at the same time. Systolic and diastolic blood pressure (SBP and DBP) were recorded, utilizing Korotkoff phases I and V as references, respectively. If there was a difference of more than 2 mm/Hg, the procedure was repeated after a 5-min rest. The final value was obtained by averaging the latter two readings. The cut-off points for diagnosing arterial hypertension in this study were based on the criteria established by the American College of Obstetrics and Gynecology, the European Society of Cardiology of 2018 criteria, and the WHO ,22,23.(d)Each time, participants were questioned about their medical history, including gynecology, obstetrics, and pathology.(e)Blood samples were taken by venipuncture to determine glucose, cholesterol, triglycerides, and HDL quantities in serum or plasma. Quantitative in vitro determinations were performed using photometric equipment from System Kits DiaSys, Diagnostic Systems GmbH . Insulin levels were measured by ELISA using the chemiluminescence detection method. Equipment from the Beckman Coulter brand Access  was used. Insulin resistance was calculated by homeostasis model assessment (HOMA-IR). HOMA-IR = fasting glucose levels [mmol/L] × fasting insulin levels [μU/mL]/22.5.(f)Somatometry of newborns was performed within the first 12 hours of life. A calibrated scale (SECA 374) and an infantometer (SECA 416) were used. Weight was classified according to the intergrowth criteria as small for gestational age (SGA) (percentile <10), adequate for gestational age (AGA) (10th to 90th percentile), and large for gestational age (LGA) (>90th percentile) .(g)2, which is why it is used instead of waist circumference ), reduced high-density lipoprotein cholesterol (<1.29 mmol/L [<50 mg/dL]), raised blood pressure (BP) , or raised plasma glucose (≥5.5 mmol/L). Since almost all women experience changes in waist circumference due to a larger gap between the rectus abdominal muscles, especially in the first year after delivery, the WHO and ATP III criteria were modified using a BMI equal to or greater than 30 kg/mmference ,26.The participants’ follow-up was conducted with three evaluations at 3, 6, and 12 months after the birth of their children. Each evaluation consisted of the following:2 to compare groups. As a risk, we calculated the odds ratio and prevalence ratios. We employed probabilistic models, treating PE as the main exposure variable. A univariate general linear model was performed to analyze the association of some maternal factors and covariates  in order to adjust for possible confounding factors and find the exposure variables that could explain the main outcomes (blood hypertension and MS) at 3, 6, and 12 months post-childbirth. To identify factors influencing arterial hypertension after pregnancy in participants with severe, mild, or without preeclampsia, a general linear model was used. Independent variables included age, pre-pregnancy body mass index, weight gain during pregnancy, body mass index, homeostatic model assessment (HOMA), and history of previous preeclampsia at each evaluation. A logistic regression model was used to calculate the factors that could explain MS after pregnancy. The statistical analyses were performed using the standard statistical software package SPSS 25.0 (IBM SPSS Statistics).Our outcome variables were blood hypertension and metabolic syndrome at 3, 6, and 12 months post-childbirth. We performed a Pearson Xp = 0.012). Compared with multiparous women, the risk of PE in primiparous women was determined to be 2.05 (95% CI 1.16–3.63).In our sample, 74 participants (28.3%) were primiparous, of whom 51 (69%) suffered from PE. Among 187 multiparous women, 97 (52%) had PE . Similarly, this risk was higher for mild PE  compared to severe PE , both of them compared with the control group that only had nine participants with a history of previous preeclampsia .The initial sample consisted of 261 participants. Among them, 38 cases were lost between the first and second evaluations , and 29 cases were lost between the second and third evaluations  .p = 0.74), gestational weight gain (p = 0.70), PE severity grade, personal history of hypertension, or gestational diabetes between participants with and without full follow-up.In the 67 cases lost, no significant differences were found in age, pre-gestational body mass index, gestational age (2), while the more significant number of obese or overweight women had PE.Controls, who did not have preeclampsia, were predominantly thin  had mild PE, and 9 (25%) had severe PE. Only 3 of the 113 controls had a history of high blood pressure. Throughout the three evaluations, the blood pressure values of the patients in both groups (with and without preeclampsia) were similar between those with a personal history of arterial hypertension and those without.Women with PE had a higher incidence of premature births (71%), and 87.5% of preeclamptic women had children with low birth weights.Age, gestational diabetes, and pre-pregnancy diabetes mellitus did not differ between groups at the beginning of the study. All our cases of preeclampsia were late-onset.Sixty-nine participants had a personal history of previous preeclampsia; nine (13%) belonged to the control group, and the vast majority (65.2%) belonged to the mild preeclampsia group.When considering the cutoff points for arterial hypertension (systolic hypertension ≥ 140 mm Hg and diastolic hypertension ≥ 90 mm Hg) ,19,20,21IDH was the predominant phenotype of hypertension found in this sample. At three months postpartum, IDH was present in 92.8% of women with a history of PE (13 out of 14 women), while only one out of 14 women without PE (7.2%) had IDH. At six months postpartum, twelve women had IDH, with nine women (75%) being from the preeclampsia group and three (25%) from the control group. Finally, after one year, all seven women with IDH had a history of preeclampsia in their last pregnancy (100%).According to the general linear model, it seems that the history of PE, whether it was severe or mild, cannot fully explain the systolic and diastolic blood pressure figures up to 12 months after pregnancy. According to Our study focused on using the recent ATP III criteria for diagnosing MS and examining the link between PE and the further development of MS. Our analysis of risks revealed inconsistent findings, with a significantly higher risk of MS observed in women who had experienced PE in their last pregnancy during the 3-month and 12-month post-childbirth evaluations. The odds ratios were 5.2 (95% CI 1.9–14) and 8.2 (95% CI 1.0–65), respectively. However, no association was observed between PE and MS during the 6-month evaluation, with an odds ratio of 1.6 (95% CI 0.6–4.1).Additionally, we employed a logistic regression model to identify the factors that could explain the development of MS after pregnancy with PE. Maternal age, pre-pregnancy BMI, gestational weight gain, exclusive breastfeeding, a history of gestational diabetes, a history of previous preeclampsia, and blood levels of LDL-C were used as exposure variables.2 0.410). However, in the 6-month evaluation, only pregestational overweight and obesity explained the MS (R2 0.295). Finally, at one year of follow-up, the MS was only associated with obesity but not with a history of preeclampsia (R2 0.272)  were the variables that explained the MS (R2 0.272) .We sought to determine whether overweight and obesity, in addition to preeclampsia, played a significant role in the development of hypertension and metabolic syndrome in the first year postpartum. We also investigated the factors that could explain overweight and obesity in these women.p = 0.13). In contrast, at 3 and 6 months of follow-up, the mean BMI between these two groups differed with statistical significance . Then we applied an analysis with a general linear model adjusted for age, breastfeeding practice, use of hormonal contraceptives, gestational weight gain, pre-pregnancy BMI, and having or not having PE during the last gestation. 2 adjusted) with only two variables: the pre-pregnancy body mass index and the gestational weight gain. The Waist Height Index variability was dependent on the pregestational BMI and the GWG, between 52% and 65%, in the three scrutinies. Interestingly, breastfeeding is added as a predictor factor at 12 months postpartum. Finally, the waist hip index at 6 and 12 months postpartum is barely explained, between 13% and 19%, by the same variables, adding the covariate age.The body mass index one year postpartum was similar between women with PE and without PE  showed that those who had a pregnancy with preeclampsia had significantly higher mean systolic and diastolic blood pressure values than controls. In the international literature, evidence of remodeling and structural and functional alterations of the heart has been observed in pregnancies with preeclampsia .Moreover, strong evidence, including a meta-analysis of >2.3 million women, indicates that preeclampsia is associated with an increased risk of future cardiovascular disease. The relative risk for developing cardiovascular disease, including cerebrovascular disease, peripheral arterial disease, and cardiovascular mortality, was found to be 2.00 for mild preeclampsia, 2.99 for moderate preeclampsia, and 5.36 for severe preeclampsia . They alThe literature shows that the recognized risk of PE among primiparous women is between 1.4 and 5.5 times higher than in multiparous women ,30, as op < 0.001). A possible explanation for this high recurrence may be that the institution that makes this report specializes in high-risk pregnancies and is a national reference hospital; therefore, the cases are concentrated.In relation to the above, our case material showed a high number of cases of recurrence of preeclampsia (women with more than one pregnancy). The frequency of presentation of PE among multiparous women with a previous pregnancy with PE was 87% (60/69), while preeclampsia among multiparous women without a history of PE was 13.0% (9/69), . Being overweight or obese is strongly associated with this hypertension phenotype, as observed in the current study . OverweiIsolated diastolic hypertension (IDH) has consistently been associated with adverse cardiovascular events, particularly in young individuals, where it is more prevalent. Therefore, treatment is recommended for individuals under the age of 50 with IDH, as it has been shown to significantly reduce cardiovascular complications. It is worth noting that IDH in elderly subjects is less prevalent and is usually not associated with cardiovascular complications ,39,40. IInterestingly, our results differ from those reported in the literature since the model showed pregestational BMI and GWG did not influence the development of arterial hypertension in the first year post-childbirth; many authors point out that maternal overweight and obesity and excess gestational weight gain influence the subsequent development of cardiovascular and metabolic diseases postpartum ,43. AlthThere is documented evidence suggesting that if PE occurs at term and with a child with adequate birth weight, cardiovascular risk is doubled later in life, but if the pregnancy is full-term with an SGA newborn, the CV risk is tripled. When the birth is preterm, this risk increases to 5, and if preeclampsia occurs before week 34 of gestation, the CV risk will be tenfold times higher . NeverthThe association between insulin resistance (IR) and blood pressure has been extensively studied in patients with diabetes mellitus. It is believed that RI plays a significant role in the development of hypertension. Proposed mechanisms to explain this include enhanced tissue angiotensin II activity and aldosterone, insulin oxidative stress, and increased sympathetic nervous system activity . In the Using the ATP III criteria for MS diagnosis, a high risk of developing MS during the first year postpartum was observed in women who had experienced preeclampsia.2 values ranging from 0.410 to 0.247). Similar to our study, Cho et al. also demonstrated an increased risk of developing metabolic syndrome postpartum in women without pre-pregnancy metabolic syndrome, with BMI showing a direct association with its development [Metabolic syndrome is considered a potential complication following preeclampsia. In a systematic analysis conducted by Udenze, the prevalence of post-preeclampsia MS ranged from 10.9% to 27.3%, with a reported risk for its development ranging from 1.23 to 3.60 . The reselopment .Another study reported a three-fold increased risk of metabolic syndrome associated with preeclampsia and gestational hypertension. Metabolic syndrome is a basic component of diabetes mellitus. Almost 10 years ago, a retrospective, population-based cohort study found that the risk of developing diabetes mellitus doubled after 16.5 years of pregnancy follow-up with pre-eclampsia or gestational hypertension, in the absence of prior diabetes . PreviouIn international literature, there are several studies that demonstrate the protective influence of exclusive breastfeeding in preventing the onset of metabolic syndrome and diabetes mellitus, which seems to be more effective at younger ages. The beneficial effect of breastfeeding has even been observed in women with gestational diabetes ,51.Our study on the potential impact of exclusive breastfeeding on the development of metabolic syndrome (MS) yielded conflicting results. While the 3-month evaluation found an unexpected positive correlation between metabolic syndrome and breastfeeding, we faced a limitation in the low number of women who exclusively breastfed their children for 6 and 12 months, which prevented us from obtaining sufficient statistical power for drawing reliable conclusions.In this study, various measurements of adiposity, including BMI, waist-hip ratio, and waist-height ratio, were calculated during each assessment in the postpartum period. The results indicate that pre-pregnancy BMI explained a significant proportion (greater than 0.60) of the variability in BMI at 3, 6, and 12 months postpartum. On the other hand, gestational weight gain (GWG) accounted for a smaller proportion (between 0.10 and 0.25) of the variability, and preeclampsia (PE) explained between 0% and 2% of the model’s variability. This suggests that, in this particular study, PE did not significantly influence the development of postpartum overweight and obesity. However, both overweight/obesity and PE were strongly associated with high blood pressure and metabolic syndrome up to one year after pregnancy. These findings align with international literature, including a meta-analysis of prospective cohort studies that found an increased risk of preeclampsia in overweight or obese nulliparous and multiparous pregnant women, highlighting maternal adiposity as an independent risk factor for PE ,53.In a 2020 report by Lopes et al., it was observed that women with hypertensive disorders during pregnancy, particularly those with a pre-pregnancy BMI ≥ 35, experienced a slower resolution of their arterial hypertension postpartum . SimilarThe study confirmed that women with a history of preeclampsia have an increased risk of developing arterial hypertension, particularly isolated diastolic hypertension, and metabolic syndrome. Likewise, with a similar or greater risk, overweight and obesity were associated with high blood pressure and metabolic syndrome in the first year postpartum. Furthermore, there was a strong and direct association between overweight/obesity and pre-pregnancy BMI, with pre-pregnancy obesity significantly influencing the development of metabolic syndrome.This study found that being overweight or obese may have a similar effect as preeclampsia on the progression to cardiovascular and metabolic complications. Although there are still no ways to prevent or treat preeclampsia, we believe that overweight and obesity are preventable and treatable risk factors, and control of these would help reduce progression to MS and hypertension.The follow-up of our patients was limited to three evaluations, which covered 12 months after the obstetric event. This could be considered a short follow-up since in the literature there are some reports that at a 2-year follow-up, MS has been found in 25% of women with a history of PE . In this


UC was most commonly classified as left-sided UC (E2) , while the most common location of CD was the ileocolon (L3) . Non-stricturing and non-penetrating CD was the most common behavior (B1) . Approximately one-third of the patients with Crohn’s disease developed perianal disease , with fistulizing Crohn’s being the most prevalent manifestation , followed by abscess formation  and fissures . The most common extraintestinal manifestation was arthropathy . Significant p-values were detected for UC and CD (p = 0.036). Conclusions: This study provides crucial insights into the demographics, patterns, and manifestations of IBD in Saudi Arabia. The findings highlight the need for improved diagnosis, management, and treatment strategies for IBD in this population. The establishment of a multi-center database will help to facilitate future research and improve patient care in Saudi Arabia.Background: Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is becoming increasingly prevalent in Saudi Arabia. However, there is limited data on the characteristics and manifestations of IBD in this population. This study aimed to establish a multi-center database of patients with IBD in the Qassim region of Saudi Arabia to better understand the demographics, prevalence, and manifestations of IBD in this population. Methods: This retrospective study included patients diagnosed with IBD at three healthcare facilities in the Qassim region of Saudi Arabia. The patient’s demographic and clinical characteristics, disease location and behavior, age at diagnosis, medication use, related surgeries, and extraintestinal manifestations were recorded based on the Montreal classification. A total of 257 patients with IBD were included in the study, of which 126 had UC and 131 had CD. Results: Of the 257 patients with IBD, 134 (52.2%) were male and 123 (47.8%) were female. The mean age of patients with CD and UC were 28.74 (range 15–67) and 38.79 (range 15–75) years, respectively. There was a significant difference between all age groups ( Crohn’s disease (CD) and ulcerative colitis (UC), collectively referred to as inflammatory bowel diseases (IBD), are chronic immune-mediated gastrointestinal tract disorders . The etiThe prevalence of IBD is currently the highest in Europe  and North America  . In 2010Data regarding the exact prevalence and incidence of IBD in Saudi Arabia are limited, as there are few published studies in this regard. However, a retrospective analysis of patients within two decades 1983–2022) at King Saud University Hospital in Riyadh, published by Al-Ghamdi et al., demonstrated an annual incidence of CD of 0.94 per 100,000 population per year, while another study published by El-Mouzan et al. showed an annual incidence of 0.5 per 100,000 population per year, with a prevalence of 5 per 100,000 population per year in the pediatric age group 83–2022 a,13,14,15We aimed to establish a comprehensive understanding of IBD trends in the Qassim region by compiling data from multiple centers. This data would include information about the clinical diagnostic features of CD and UC, baseline characteristics, natural history, location, and behavior of IBD in the region. The study aimed to compare this data with international statistics to identify patterns and trends in IBD occurrence and management.This retrospective study included patients diagnosed with inflammatory bowel disease at King Fahad Specialist Hospital-Burydah (KFSH-B), King Saud Hospital Onaizah (KSU), and Buraidah Central Hospital (BCH) from 22 April 2014 to 30 November 2018. We collected data on patients’ characteristics, disease location, behavior, age at diagnosis , anti-TNF use, surgical complications related to IBD, and extraintestinal manifestations.The study included adult Saudi patients with documented endoscopic or radiological (CT enterography) features along with histopathological findings of either CD or UC. All samples underwent an assessment for the typical histopathological features used in the diagnosis of UC and CD, which include crypt architecture distortion, granulomas, identification of histologic changes in areas of normal endoscopy, and a search for dysplasia. In addition, a virology workup was performed during any admission. Patients who did not meet these criteria were excluded from the study based on specific exclusion criteria, which included patients younger than 14 years of age, those with inconclusive or insufficient diagnostic data for IBD, and non-Saudi patients. These exclusion criteria helped to ensure that the study’s findings were representative of the clinical characteristics of CD and UC in the target patient population .Data were collected from the medical records of patients with IBD into a checklist fulfilling the following variables: age, diagnosis, time from presentation to diagnosis, sex, body mass index (BMI) at diagnosis, and family history of IBD. We also collected data on comorbidities, baseline laboratory investigations at the time of diagnosis (hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), liver function tests (LFTs)), and Montreal classification of the extent of UC and CD. Furthermore, CT enterography and MR enterography (MRE) techniques were utilized, when available, as part of the methodology for evaluating the characteristics of Crohn’s disease and ulcerative colitis, in order to further characterize the tissue.The study included a total of 257 patients, with 190 patients from KSFH-B, 37 patients from KSU, and 30 patients from BCH. These patients were selected based on specific inclusion and exclusion criteria, and their files and data were reviewed by two residents in the Department of Gastroenterology. Duplicate files were identified and removed from the study. The files and data of the selected patients underwent independent review by two residents from the gastroenterology department to ensure the accuracy and reliability of the data.The research team implemented quality control measures to minimize bias in the study’s design, conduct, and analysis. Frequent meetings were held to critically appraise each step of the study and identify potential sources of bias. Two independent reviewers evaluated each component of the study, including the data collection, analysis, and interpretation of results. Any discrepancies or disagreements were resolved through open discussions and consensus-building among the research team. These measures ensured the validity and reliability of the study’s findings, providing confidence in its conclusions and increasing the credibility and generalizability of the results.χ2) test and Fisher’s exact test, while quantitative variables were analyzed using the appropriate test. Appropriate statistical methods were applied to compare variables such as age, extraintestinal manifestations, anti-tumor necrosis factor (anti-TNF) use, and surgical rates between Crohn’s disease (CD) and ulcerative colitis (UC). These methods helped determine the significance of the observed differences between the two groups and contributed to the overall analysis of the study’s findings.The data collected for the study were entered into Microsoft Excel and then transferred to SPSS version 23.0  for analysis. Numerical variables were analyzed by calculating the mean, median, and standard deviation (SD), while categorical variables were presented as numbers and percentages. Categorical variables were compared using the Chi-square  had UC and the other half  had CD  age group, >40 years , and the <17 years  age groups  age group, followed by >40 and <17 years age groups , respectively, as shown in p < 0.005), with more patients aged >40 years being diagnosed with UC. A total of 134 (52.2%) patients were male and 123 (47.8%) were female. Regarding disease distribution, according to gender in UC patients, 66 (52.4%) patients were male and 60 (47.6%) patients were female. In the CD group, 68 (52%) patients were male and 63 (48%) patients were female. No significant difference between the sexes was detected (p = 0.939).A total of 257 patients with IBD were included in the study from 22 April 2014 to 30 November 2018. Almost half of the patients  had CD . The meae groups . On the n = 70; 27.2%) (p < 0.005). Among the CD group, fistulizing Crohn’s was the most prevalent among perianal disease , followed by abscess formation  and fissures . In the UC group, a low incidence of perianal disease, including fistula and fissures, was detected . Significant p-values were detected across all variables, as presented in p = 0.418).Approximately one-third of the patients developed perianal disease . The specifications of the extraintestinal manifestations are shown in n = 15; 5.8%). Among the 15 patients with IBD, arthropathy was more common in the CD group than in the UC group. Six patients developed peripheral arthritis , but only two of these patients had CD . Significant p-values were detected for UC and CD (p = 0.036). More cases of sacroiliitis were noted in patients with CD  than in those with UC. Regarding primary sclerosing cholangitis , most of the patients with these conditions had UC   see .n = 4; 1.5%) were highest in the UC group  than in the CD group . Overall, the incidence of osteoporosis was highest in patients with CD , and no patient with UC had osteoporosis  who developed eye manifestations, a total of three patients  in both groups were noted with insignificant differences. Skin manifestation in patients with IBD  who developed symptoms was highest in patients with CD , with a significant difference between the two groups of IBD.Regarding eye diseases in patients with IBD , while less than half of patients with UC received steroids . Most patients with CD received anti-TNF , while only a few patients  with UC were on anti-TNF (p < 0.005). A significant difference was seen in patients who underwent abdominal surgery for IBD, especially CD , compared with those for UC .A total of 100 (38.9%) patients with IBD were taking either budesonide or prednisone, which are commonly used as steroids in our hospitals. Most patients with CD received steroids , followed by the <16 years age groups (A1) , and lastly the >40 years (A3)  age group Regarding the classification of CD based on the Montreal classification, for the age groups at diagnosis, most cases were in the 17–40 years age group (A2)  ), followed by the colonic region (L2) , and isolated upper disease . For CD behavior, the non-stricturing and non-penetrating (B1) type was the most common , followed by the stricturing (B2) type , and lastly is the penetrating disease (B3) .Regarding CD location, the most common was the ileocolonic region ((L3)  was the most common type of UC according to the Montreal classification, followed by ulcerative proctitis (E1) , and the least common was extensive UC (pancolitis) (E3)    .n = 8; 80%)  and ), respectively  . Most ofectively .p-values of 0.012 and 0.004, respectively. The most common behavior was the penetrating behavior (B3) in patients with CD, presenting with more abscesses , fistulas , and fissures   .n = 16; 51.6%) and penetrating (B3)  Crohn’s disease had previous surgery with a significant p-value of 0.007 (Approximately 50% of patients presenting with stricturing (B2)  was considered rare in the Middle East, especially in Saudi Arabia . Howeverp = 0.939). These data are different from those in North America and Europe, which show a female predominance in CD and a male predominance in UC [Regarding sex differences in IBD, the data in our study showed that there was a male predominance in both CD and UC, which is consistent with previously reported data in Saudi Arabia . Howeverce in UC ,25.The reported median age at presentation of CD in Saudi Arabia varies among centers. In one study it was 23.8 years , while aOur data show that in 17.5% of patients with IBD that developed extraintestinal manifestations, arthropathy involvement was the most common, followed by skin manifestations and eye manifestations, similar data were reported in our country and North America ,27,28,29n = 4) in IBD, while a rate of 0.8% (n = 1) was observed in patients with CD, and 2.3% (n = 4) in patients with UC. In our study, we observed a lower prevalence of thromboembolism than what was reported in the literature  [Thromboembolism occurred at a rate of 1.5% (a 1.22%) ,29,30.n = 76, 58%), with only 1.5% (n = 2) of cases being confined to the colon. In patients with CD, the non-stricturing and non-penetrating (B1) behavior was the most common , and this is similar to the report by Aljebreen et al. However, our data is different from those of some Western countries in terms of the behavior of CD according to the course of the disease [According to the Montreal classification, most of our patients had ileocolonic involvement , followed by proctitis , but pancolitis being the least, while it is the most common type in the literature [Data regarding UC involvement in this study differ from data published in our country and in North America, as our findings show that the main type of UC is the left-sided type , with an overall incidence of 38.1%  ,33,34,35of 38.1% . Among pThis study showed that there was a significant increase in the use of anti-TNF for both UC and CD compared to the previously reported data, which could be attributed to advances in pharmacotherapy and changes in recent guidelines . FurtherIn this study, the related rate of surgery was significant for both UC and CD , which is similar to the data reported in our country and in North America, but the surgery rates for UC were lower ,28,36,37The study had several limitations that may affect the generalizability and interpretability of its findings. Firstly, the sample size was relatively small, with only 257 patients with IBD included in the analysis. This limited sample size may not be representative of the broader population of patients with IBD and may also affect the statistical power of the study. Consequently, the findings of the study should be interpreted with caution, and further research with a larger sample size may be necessary to confirm the study’s results. Secondly, the study was retrospective, which may have led to incomplete or inaccurate data collection. Retrospective studies rely on data collected from medical records, which may be incomplete, inconsistent, or missing information. This limitation may have affected the validity and reliability of the data used in the study, potentially leading to biased or inaccurate results.Finally, the study did not investigate the impact of treatment on disease outcomes. Although the study aimed to examine the demographic and clinical factors associated with IBD, the lack of information on treatment efficacy may limit the practical implications of the study’s findings. Future studies may need to explore the impact of different treatments on IBD outcomes to provide a more comprehensive understanding of the disease and its management.IBD is commonly seen in this region, with CD and UC predominantly affecting younger men. Our study identified differences in disease characteristics, including location and behavior based on the Montreal classification, when compared to international data. Furthermore, we observed a notable rise in the use of anti-TNF medications for both CD and UC.