The exact mechanism by which pelvic tissues lose their anatomic support and undergo descent through the genital hiatus remains elusive. In this case, large Gartner cyst causes secondary cervix elongatio due to the weight of the cyst. Gartner cyst arise from the remnants of the mesonephric ducts. They are typically located in the anterolateral wall and are small. The rarity of this case is very interesting because of the mass size reaches 15 cm and causes cervical elongation.This article report a case of 37 years old women who complained large mass came out from vagina. Patient was diagnosed with large anterior vaginal cyst and cervical elongatio. Ultrasound finding suggested that the cyst was originated from anterior wall of vagina in accordance with Gartner cyst. Patient underwent cyst excision, there was no connection between the cyst and urethra. Operation was continued with Manchester Forthegill to restore normal anotomy of the cervix.Secondary cervical elongatio in this case was caused by large Gartner cyst, eventhough there are possibility involvement of other factors. Cervical elongatio plays important role in the decision of operation in this case after removal vaginal cyst.Secondary cervix elongation due to Large Gartner cyst is rare condition. Enlarge cyst causes secondary cervix elongatio, and operation technique was also challenging. Pelvic organ prolapse may includes downward descent of the vaginal wall and/or uterus. In general, 40% women with POP have cervical elongation. The extent of cervical elongation is proportionate with the degree of uterine descent. Cervix may elongate either in the supravaginal or infravaginal part. Supravaginal elongation is commonly associated with uterine prolapse, whereas infravaginal elongation is congenital. It is uncommon for the cervix to elongate until 10 cm in length. Cervical elongation plays an important role in the decision between hysterectomy and uterine preservation during genital prolapse surgeries. In recent years, uterine sparing procedures are becoming more common, though hysterectomy has classically played a role in pelvic floor. General risk factors for the development of POP include parity, age, obesity, genetic factors, and surgery, but specific pathways that may be involved in prolapse remain elusive .The prevalence of vaginal cysts is estimated to be 1 in 200 women; however, this number is probably inaccurate, since most cysts are not reported. They are more common in women in their third and fourth decades and are usually detected incidentally. When symptomatic, patients may present with mild vaginal discomfort, vaginal pressure or fullness, vaginal mass or swelling, dyspareunia, vaginal bleeding, or urinary symptoms [Gartner cyst arise from the remnants of the mesonephric ducts. They are typically located in the anterolateral wall and are small (ranging in size from 0.1 to 4 cm), but rarely > 10 cm. The cysts are lined by cuboidal or low columnar nonmucinous epithelium. In most cases, Gartner cyst is asymptomatic, less than 2 cm in size and is accidentally found during routine gynaecological examination. The cyst may enlarge and become symptomatic and/or compress surrounding organs like the bladder, urethra or colon .This case is very interesting because of the Gartner cyst size reaches 15 cm and causes cervical elongation.This case report has been reported in line with the SCARE criteria .2A 37 years old woman complained protrussion mass which came out from vagina since 14 years ago. Initially the mass was small and the mass become enlarge when she was pregnant. As her pregnancy close to her due date the mass become larger. After delivery patient couldn’t insert the mass into vagina. Patient felt the mass become largest since 3 month ago and patient got difficulties to do activities. Micturition and defecation were normal.3Physical examination showed 18 × 11 × 5 cm mass outside hymenal ring, with smooth vaginal mucosa and portio, with uterine sondage 15 cm. Cystic mass size 18 × 11 × 5 cm was palpated in anterior vaginal mucosa, smooth surface, fluctuated, without pressure pain. Uterus and adnexas was normal. Pelvic Organ Prolaps Quantification examination revealed that point Aa and Ba were +2, point C was +6, point Ap and Bp were −3, point D was 0. Genital hiatus was 7, perineal body was 4, and total vaginal length was 4. Rectal examination was within normal limit .Fig. 1Pr4Ultrasonography showed cystic mass with firm border contained coarse echointerna material. Mass with size 150 × 97 mm didn’t contain solid and vascular part. Mass was separated from canalis cervicalis originated from anterior vagina (area around anterior fornics and came out to vagina). Uterus size and shape were normal, and more caudal position (retracted by mass). Miometrium was homogen. Uterine cavity didn’t contain abnormal mass. Regular stratum basale endometrium, with thickness 4 mm. Cervix lengthwise to caudal (possible because retracted by mass). Cervical canal was normal with orificium uteri externum in posterior cystic mass. Both ovaries shape and size were normal. No abnormality was found in both adnexas. Conclusion: Cystic neoplasm of anterior vagina with secondary uterine prolapse . No sign of malignancy .Fig. 2Ul5Excision of Gartner cyst and Manchester Forthegill operation was performed to restore normal anatomy. Uterus preservation considered to be appropriate choice because patient still want to have future fertility .Fig. 3a)6Histopathology result found cyst wall was formed by stratified squamous epithelium keratinized and connective tissue with chronic inflammation lymphocyte an plasma cell not contradicting with Gartner cyst and cervical elongatio and malignancy was not found . Post op7The exact mechanism by which pelvic tissues lose their anatomic support and undergo descent through the genital hiatus remains elusive. The “connective tissue deficiency” hypotheses are plausible since the cervix, vagina, uterosacral ligaments, and periurethral tissues are known to contain collagen, elastin, and smooth muscle. Collagen and elastin are important constituents of the extracellular matrix of the cervix. Collagen predominates, with type 1 collagen being the most abundant form (70%) and type 3 making up 30% of total collagen .Biomechanical studies of the stretch and tension relationships of vaginal tissue have shown increased stretch ability of the vaginal skin with prolapse. It is not clear if these findings were confounded by age-related tissue changes. Worsening prolapse was associated with increased extensibility of the vaginal skin, but this finding was felt to be a result of prolapse rather than a direct cause .Distal mesonephric duct in the female may persist as vestigial remnants in the anterolateral vaginal wall down to hymen as Gartner duct. The cyst developed as a result of secretory activity. Gartner ducts are identified in approximately 25% adult women and 1% evolve into Gartner duct cyst. Classically Gartner cyst was solitary, unilateral, and less than 2 cm in diameter. Large Gartner cyst can cause dyspareunia and other symptoms .In this case, secondary cervical elongatio was caused by large Gartner cyst. The elongation affect vaginal part of the cervix. The large size of Gartner cyst causes cervical elongation in this patient eventhough there are possibility involvement of other factors .Whether or not to preserve the prolapsed uterus is still a matter of debate. Furthermore, in recent years, more emphasis on the uterosacral ligaments as the most prominent structures to prevent uterine or middle compartment descensus has emerged. Therefore, it appeared logical to use techniques that incorporate these ligaments in the restoration of the middle compartment. Cervical amputation is part of the classical Manchester procedure, a surgical procedure for the correction of a prolapsed uterus .Then cyst was excised in the proximal part. After cyst removal, reevaluation was performed to confirm cervical elongatio. Uterine sondage was 13 cm, operation was continued with Manchester Forthegill procedure. Longitudinal incision was perfomed in anterior and posterior vaginal wall. Vaginal wall was separated sharply and bluntly. And then amputation of cervix was performed. Forthegill technique was performed to tied bilateral cardinale ligament into anterior cervix. Stumdorf technique was performed with hegar dilator number 6 as guidance intra cervical canal. With needle pick up anterior lip or the stump remaining after amputation, 0,5 cm to the right or the midline, and bring the needle out through cervical canal. Afterward suture was is carried out through vaginal mucosa. The suture is carefully tightened with each stitch, thus causing the wound to become gradually covered by vaginal mucosa. The suture is tightened as strongly as possible and both ends are tied together tightly. Uterine sondage after operation was 8 cm. Manchester forthegill technique was chosen to preserve uterus and reproductive function.Cervical elongatio plays important role in the decision of operation in this case after removal vaginal cyst. However, cervix may elongate postoperatively after uterine preservation. Preoperative assessment of cervical elongatio in this patient should be confirmed intraoperative after removal of the cyst. Because preoperative assessment can be inaccurate since the large cyst retract the cervix downward. Rare large Gartner cyst and cervical elongatio has aroused the interest to report this case, as Gartner duct cyst commonly present in small size.8Secondary cervix elongation due to Large Gartner cyst is rare condition. Enlarge cyst causes secondary cervix elongatio, and operation technique was also challenging.Source of funding is form the author only. There is no involvement of sponsor.Because this is the case report and it is not a research, ethical approval was not required.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Surahman Hakim: concept, operator, data analysis, final approvalAhmad Kemal Harziq: drafting and revisingYulia Margaretta Sari: data collection, data analysis, writing the paperNA.Surahman Hakim MD.Yulia Margaretta Sari MD.Achmad Kemal Harzif MD.Not commissioned, externally peer-reviewed.This case report do not have any relationship with other people or organisations.


Major birth defects increase the risk of fetal death and pediatric hospitalization, which also impact on healthcare costs. Sociodemographic factors can drastically affect reproductive health and be used to discriminate the exposure to hidden risk factors. Foz do Iguassu is a Brazilian city located in the triple-border region of Brazil / Paraguay / Argentina with high rates of birth defects. However no study aimed to verify factors associated with this incidence or preventive care is reported. The current work investigated the prevalence of major birth defects and its association with maternal sociodemographic factors in Foz do Iguassu.In this population-based cross-sectional study we used data of all live births occurred in Foz do Iguassu from 2012 to 2017. The associated sociodemographic variables such as maternal age, maternal education, maternal race, country of residence, maternal parity and onset of prenatal care were analyzed. Each major birth defect was described according to absolute and relative frequencies, Kruskal-Wallis and logistic regression models were used to evaluate variables associated with selected birth defects.The most prevalent major birth defects were Cleft Lip and/or Palate , gastroschisis , spina bifida , hydrocephalus , hypospadias , Down syndrome , anencephaly , anorectal atresia / stenosis , undetermined sex , esophageal atresia / stenosis with or without fistula  and limb reduction defects . Maternal age was associated with gastroschisis and Down syndrome. Only maternal education up to 7 years was statistically associated with major birth defects considering all other sociodemographic variables.Cleft Lip and/or Palate and Gastroschisis prevalence were higher than those found in the literature. This findings may suggest a distinct epidemiological behavior regarding major birth defects in the region. The work opens new perspectives for birth defects risk factors in the triple-border. Birth defects are structural, functional, or metabolic disorders diagnosed at birth or in the course of life . They arIn Brazil, the staff from the hospital where the delivery occurred collects data regarding pregnancy care, and maternal and newborn characteristics using a paper-based form (Declaration of Live Birth). Then, this form is sent to the Municipal Health Department, which inputs the data from all births into the web-based Information System on Live Births (SINASC). If a neonate presents a birth defect, this information is recorded into the Declaration of Live Birth and the birth defect is coded according to Chapter XVII of the International Classification of Diseases Review 10 (ICD-10). The aggregated data is available on the Brazilian government website for epidemiological assessment. Foz do Iguassu is a Brazilian city located in the triple-border region of Brazil / Paraguay / Argentina, according to government records, in the period from 2012 to 2017, Foz do Iguassu presented a birth defect rate of 11.78 / 1000 live births, roughly 50% more than that observed in the state of Paraná and Brazil (7 / 1000 live births and 8.07 / 1000 live births respectively) , birth dDespite the high rates of birth defects in this region, the risk factors associated with its occurrence have not been studied yet. The present work aims to describe the prevalence of major birth defects and its association with maternal sociodemographic factors in Foz do Iguassu. The results may contribute to the understanding of how sociodemographic factor influence the occurrence of major birth defects in Brazil’s borderlines.This is a population-based cross-sectional study.birth defect identification field were excluded.Data regarding births in Foz do Iguassu between 2012 and 2017 was obtained from the Municipal Health Department. All live births in this period were eligible. Declaration of Live Birth forms lacking The 25 most severe major birth defects were selected to the study , 11. TheBirth defects was expressed as both absolute and relative frequency . The association between maternal age and birth defect was analyzed for each of the most prevalent major birth defects . We also pooled all birth defects to analyze the association between the other independent variables and the occurrence of birth defect.Shapiro-Wilk test was applied to verify data distribution in each birth defect and Kruskal-Wallis test was used to verify differences between the mean maternal age of newborns with and without major defects.Maternal education, maternal race, country of residence, maternal parity and the onset of prenatal care were described according to absolute and relative frequencies considering the distribution of newborns with and without major birth defects. To investigate the maternal sociodemographic factors associated with birth defects, a logistic regression models were proposed. Unadjusted and adjusted models were performed. The strength of association between dependent and independent variables was estimated by the Odds Ratio (OR) with 95% Confidence Interval (95% CI). Statistical analyses were conducted in Epi Info 7® and BioEstat 5.3®.A total 30,761 births were registered in Foz do Iguassu from 2012 to 2017. 32  cases were excluded due to no filled birth defect identification field. Among 305 (0.99%) cases of birth defects, 140 (46%) corresponded to selected major birth defects included in this study.N = 3), palate (N = 15), lip and palate (N = 11)) with mean maternal age of 26.4 ± 6.8 years. Among CLP cases, 41% (N = 12) presented more than one birth defect. Gastroschisis was the second most prevalent birth defect with 6.83 / 10,000 live births (N = 21) and a mean maternal age of 21.5 ± 4.4 years. Birth defects of nervous system  accounted for 43 cases with a mean maternal age of 25.6 ± 9.2 years, 25.2 ± 6.5 and 24.5 ± 5.1 years, respectively. Down syndrome had 4.23 / 10,000 live births (N = 13) and the highest mean maternal age of 33.5 ± 7.3 years; including 3 cases with cardiovascular defect as well. Holoprosencephaly, Transposition of the great arteries, Tetralogy of Fallot, Coarctation of the aorta, Omphalocele, Patau syndrome and Edwards syndrome was no registered  ; white race (65.7% vs 62.9%); resident in other countries (7.9% vs. 5.5%); primiparous 42% vs 39%); onset prenatal care after the first trimester (20% vs 16%). In the logistic regression analysis, maternal education up to 7 years was the only variable associated with the major birth defects in both unadjusted and the adjusted analyzes  of total cases and have been reported as the most common outcome. This fact may reflect the complex interactions between genes and poorly understood environmental factors . LiteratCompared to other studies, a low number of cardiovascular birth defects  were observed in the present study , 18, 19.The association between Down syndrome and advanced maternal age is well described in the literature –22. HoweGastroschisis is a full-thickness defect in the abdominal wall, usually in the right side of normal umbilical cord insertion . The preAbout 60% of birth defects have unknown etiology. Genetic defects such as chromosomal disorders are more often investigated when compared to environmental factors . The expWe also hypothesized the high incidence of some major birth defects may be related to the indiscriminated use of pesticides in the region with mutagenic and endocrine disruption properties . The expAnother potential risk factor considered is the residence proximity to the Electromagnetic Fields (EMF) from one of the world largest producers of energy power lines in this area. Studies have suggested an association between exposure to EMF and leukemia, abortion, and birth defects –44. We eIn our study, we analyzed only records of live births based on data collected at delivery. Considering that birth defects are an important cause of abortion, the prevalence of birth defects presented in our study may be underestimated, so comparisons with other settings should be done with caution. It is important to note that the Brazilian Mortality Information System (SIM) only records fetal death that occurred after 19 weeks of gestation; thus, severe congenital defects associated with abortions within the first 19 weeks of gestation would never get registered. Also, some defects are not easily diagnosed on a physical examination of the newborn, such as cardiovascular defects, which may be underreported.The most prevalent cases of birth defects in Foz do Iguassu were CLP and gastroschisis. Moreover, the prevalence of birth defects in the region is higher than other regions. Regarding sociodemographic factors, maternal age is associated with gastroschisis and Down syndrome and education up to 7 years is determinant to the occurrence of major birth defects. Health information systems are important tools for epidemiology and data acquisition procedure should be better addressed to assure accurate diagnosis since the early stages of life.


Aim: To analyze short term outcomes of very low birth weight infants (VLBWI) born preterm after maternal preeclampsia and HELLP syndrome within the German Neonatal Network.Methods: The German Neonatal Network is a large population-based cohort study enrolling VLBWI since 2009. Two thousand six hundred and fifty two infants below 32 weeks of gestation born after maternal preeclampsia or HELLP syndrome and 13,383 infants born prematurely for other causes between 2009 and 2018 were included in our analysis. Descriptive statistics and multinomial regression models including preeclampsia and HELLP syndrome were performed for short-term outcome measures such as intracerebral hemorrhage, necrotizing enterocolitis requiring surgery, bronchopulmonary dysplasia, retinopathy of prematurity, periventricular leukomalacia, persistent ductus arteriosus requiring surgery, blood culture positive sepsis and death.Results: After adjustment for confounding variables, preterm birth due to preeclampsia or HELLP syndrome was associated with a reduced risk for intracerebral hemorrhage , necrotizing enterocolitis requiring surgery (OR 0.35 95% CI 0.15–0.82), periventricular leukomalacia (OR 0.61 95% CI 0.40–0.92), and death (OR 0.72 95% CI 0.55–0.96) as compared to other causes of preterm birth.Conclusions: The indication for preterm birth has an impact on neonatal outcome in preterm infants born below 32 weeks. This notion should be included when counseling the families. Globally, about 11% of all infants are born preterm, but the rates significantly vary between different countries and continents . Very loHypertensive disorders are one of the world's leading causes of maternal and perinatal mortality . PreeclaAn interdisciplinary perinatal collaboration is crucial to discuss all the risk factors of threatening preterm birth in order to guide counseling and medical decision-making for an optimal timing of delivery. There are many studies showing that indications for delivery like preterm premature rupture of membranes (PPROM) with chorioamnionitis  or IUGR In our analysis we included data of VLBWI who were born in 62 GNN centers between January 1st, 2009, and December 31st, 2018. Infants with lethal abnormalities were excluded. After written informed consent was obtained from the parents or legal guardians, data was collected prospectively by neonatologists or trained study personnel. A clinical data set including pre-, peri- and post-natal treatment and outcome data was recorded by according data sheets. The cause of preterm birth was determined by the attending obstetrician, multiple causes were possible. All case record forms were sent to the study center at the University of Luebeck. Data quality was evaluated by annual on-site monitoring by a study nurse or a pediatrician experienced in neonatology.For statistical analysis, we included VLBWI with at least one known reason for preterm birth. Recorded indications for delivery were clinical chorioamnionitis, preterm labor, pathological cardiotocography, IUGR, preeclampsia and HELLP syndrome, abruption of placenta, PPROM as well as “other reasons.” In this analysis, infants born due to preeclampsia or HELLP syndrome were analyzed against all other infants.U-test were used for calculating statistical significance. The type I error level was set to 0.05. To test associations between cause of preterm birth (preeclampsia and HELLP syndrome) and different outcome variables we performed a multinomial regression model to calculate odds ratios (OR) and corresponding 95% confidence intervals (CI). Confounding variables included: gestational age, application of antenatal steroids, mode of delivery, gender, birth weight, multiple birth and IUGR. The following outcome variables were tested: intracerebral hemorrhage (ICH), necrotizing enterocolitis (NEC) requiring surgery, bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP), periventricular leukomalacia (PVL), persistent ductus arteriosus (PDA) requiring surgery, blood culture positive sepsis and death. Missing data were not imputed. All statistical analyses were performed with SPSS 25.0 software .Descriptive statistics using percentages for peri- and post-natal parameters and corresponding indications for preterm birth were carried out. For categorical variables Pearson's-Chi-square test and for continuous variables Mann-Whitney-The analyzed group “preeclampsia and HELLP syndrome” comprises all infants who were delivered because of maternal HELLP syndrome, preeclampsia or eclampsia as documented by the attending obstetrician. IUGR is defined as intrauterine growth <10th percentile for gestational age in combination with pathologic arterial and venous Doppler ultrasound examination in pregnancy. The diagnosis was recorded by the attending neonatologist as documented in the maternal medical record by the obstetrician. The term is used distinct from small for gestational age (SGA), which is defined as birth weight <10th percentile according to gestational age . IUGR feBPD is diagnosed when needing supplemental oxygen or ventilation support at 36 weeks of post-menstrual age . ClinicaBetween 2009 and 2018, 17,213 VLBWI below 37 weeks of gestation were enrolled in the GNN. After exclusion of VLBWI born > 32 weeks, datasets of 16,035 VLBWI were available for analysis. Of these infants, 2,652 were born after maternal preeclampsia or HELLP syndrome see .p < 0.001), LOS , blood culture positive sepsis , ICH , PVL , NEC requiring surgery , PDA requiring surgery , and death  as compared to infants born due to other reasons , blood culture proven sepsis , BPD , NEC requiring surgery , and death .VLBWI born after preeclampsia and HELLP syndrome with IUGR showed increased frequencies for complications compared to infants born without IUGR , in partn = 3,428 in total), the neonates who were additionally exposed to maternal preeclampsia and HELLP syndrome had higher frequencies for clinical sepsis , BPD , and NEC requiring surgery  compared to VLBWI with IUGR but without preeclampsia and HELLP syndrome , NEC requiring surgery , PVL , and neonatal death  and the accurate phenotypic characterization of the infants. This is accomplished by regular monitoring of data quality within the GNN. There also are some limitations which need to be discussed. One limitation is the fact that it was common in our cohort to have more than one prenatal cause for preterm birth per infant-mother pair. For example, 56 of 2,652 VLBWI (2.1%) were exposed to preeclampsia and HELLP syndrome in combination with preterm labor  found that preeclampsia was associated with an improved neonatal outcome compared with fetal and obstetric indications for delivery by using a composite neonatal outcome consisting of death, cord blood pH <7 or base excess < -12, 5-min Apgar ≤ 3, cardiopulmonary resuscitation during resuscitation, culture-proven sepsis, intraventricular hemorrhage, and necrotizing enterocolitis. We found a lower risk for ICH and PVL in VLBWI born after maternal preeclampsia or HELLP syndrome in our cohort. This is in line with the retrospective cohort study of Morsing et al.  which found that infants born after maternal preeclampsia showed lower odds of ICH . Furthermore, an analysis from the EPIPAGE cohort noted a lower risk for PVL and ICH for infants born after hypertensive disorders than for infants born after preterm labor or PPROM  did not show a higher risk for neonatal BPD after preeclampsia  from Sweden reported similar results of a lower NEC risk for infants born after preeclampsia , Luebeck, Germany, and by the local ethics committees of all participating centers. Written informed consent to participate in this study was provided by the participants' legal guardian.AH and VB: study concept and design. All GNN sites: acquisition of data. AH and TR: statistical analyses. All authors analysis and interpretation of data. VB and AH: drafting of the manuscript. All authors critical revision of the manuscript for important intellectual content. WG: obtained funding and study supervision. All authors contributed to manuscript revision and approved the final version.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.


Most of the women who smoke before pregnancy continue smoking during pregnancy, and some start to quit smoking after being pregnant, although existing guidelines for pregnancy recommend that women who smoke should quit smoking before pregnancy. Findings about the timing and intensity of maternal smoking, especially low-intensity smoking (1–9 cigarettes per day), and preterm birth are still inconsistent and ambiguous. This study aimed to examine the association of the timing of smoking and doses of smoking before pregnancy and during the first or second trimester of pregnancy with preterm birth in a large-scale population-based retrospective cohort study.n = 2,378,398). We found that maternal smoking during pregnancy, even at a very low level of intensity, was associated with an increased risk of preterm delivery. The adjusted ORs (95% CI) of preterm birth for mothers who smoked 1–2, 3–5, 6–9, 10–19, and ≥20 cigarettes per day during the first trimester compared with mothers who did not smoke were 1.31 (1.29–1.33), 1.31 (1.30–1.32), 1.33 (1.31–1.35), 1.44 (1.43–1.45), and 1.53 (1.52–1.55), respectively , whereas for those who smoked during the second trimester, the corresponding ORs were 1.37 (1.35–1.39), 1.36 (1.35–1.38), 1.36 (1.34–1.38), 1.48 (1.47–1.49), and 1.59 (1.58–1.61), respectively . Furthermore, smokers who quit before pregnancy, regardless of smoking intensity, had a comparable risk of preterm birth with nonsmokers, although this was not the case when cessation occurred in the first or second trimester of pregnancy. The major limitation of this study is the self-reported information about smoking, which may be subject to information bias. In addition, we cannot rule out the possibility of residual confounding caused by unmeasured factors in an observational research design.We used nationwide birth certificate data from singleton mother–infant pairs in the United States National Vital Statistics System, 2011–2018. All adult women with live singleton births, without preexisting hypertension or diabetes, and with complete data on smoking and gestational age at delivery were included. Participants reported their smoking status (yes or no) and daily number of cigarettes consumed before and during each trimester of pregnancy. The outcome of interest was preterm birth, defined as a birth before 37 weeks of gestation. Logistic regression models were used to estimate the odds ratio (OR) with 95% confidence intervals (CIs) of preterm birth associated with smoking status and the number of cigarettes consumed, adjusting for maternal age, race/ethnicity, parity, education levels, prepregnancy BMI, previous history of preterm birth, marital status, infant sex, and initiation of prenatal care. This study included 25,623,479 women, with a mean age of 29 years (range 20–50 years); 13,742,486 (53.6%) participants were of non-Hispanic white ancestry, 5,971,598 (23.3%) of Hispanic ancestry, and 3,417,456 (13.34%) of non-Hispanic black ancestry. The prevalence of preterm birth was 9.3% , and preterm birth are unclear.In a large-scale population-based retrospective cohort study consisting of 25,623,479 mothers, we examined the association of the timing of smoking and doses of smoking before pregnancy and during the first or second trimester of pregnancy with preterm birth.Maternal cigarette smoking during either the first or the second trimester of pregnancy, even as low as 1–2 cigarettes per day, was associated with increased risks of preterm birth.Smokers who quit before pregnancy, regardless of smoking intensity, had a comparable risk of preterm birth with nonsmokers, although this was not the case when cessation occurred in the first or second trimester of pregnancy.Our findings suggest that no safe level or safe trimester exists for maternal smoking during pregnancy.Public health efforts should be made to help women who smoke stop smoking completely before pregnancy to prevent preterm delivery. Preterm birth is not only the leading cause of mortality in children under 5 years old worldwide  but alsoMaternal cigarette smoking during pregnancy is known as one of the most important modifiable causes of poor maternal and fetal outcomes . AlthougGiven the high prevalence and life-threating impact of preterm birth and inconsistent findings from previous studies, a thorough and quantitative understanding of the potential effects of maternal smoking before and during pregnancy is urgently needed. Therefore, this study examined the association between maternal cigarette use before pregnancy and during the first and second trimesters of pregnancy and the risk of preterm birth by taking advantage of a nationwide large cohort in the US.https://www.cdc.gov/nchs/nvss/births.htm).We used nationwide birth certificate data from the US National Vital Statistics System (NVSS). In the US, state laws require birth certificates to be completed for all births, and federal law mandates national collection and publication of births and other vital statistics data. The NVSS, as the federal compilation of this data, is the result of the cooperation between the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention (CDC) and all US states to provide access to statistical information from birth certificates. Standard forms for the collection of the data and model procedures for the uniform registration of the events are developed and recommended for state use through cooperative activities of the states and NCHS. To collect data from the best sources, two worksheets, including the mother’s worksheet and the facility worksheet, were developed for the 2003 revision of the US Standard Certificate of Live Birth. In the mother’s worksheet, data  are directly obtained from the mother; in the facility worksheet, data  are obtained directly from the medical records. The NVSS data have been widely used in annual vital statistics reports by the CDC , other rn = 26,259,852) documented in NVSS from 2011 to 2018 if the mothers were aged 20 years or older and have complete data on smoking before and during pregnancy and gestational age. Mothers with preexisting hypertension or diabetes were excluded  because these diseases are strong risk factors for adverse birth outcomes, including preterm birth. The final sample consisted of 25,623,479 live singleton births. A participant flow chart was provided .In the present study, we used NVSS data from 2011–2018 because some important information, such as maternal prepregnancy BMI, was collected since 2011. We included all live singleton births  for cohort study . BecauseIn this study, our exposure variables of interest were maternal cigarette smoking 3 months before pregnancy and during early pregnancy (first or second trimester). We did not use data on maternal cigarette smoking during the third trimester of pregnancy, because most preterm deliveries occur during the third trimester, which complicates the exposure duration of maternal smoking during this period .Fig 1.The information about smoking was collected using the mother’s worksheet from the 2003 revised birth certificate in the hospital around the time of delivery . MothersGestational age was calculated using the date of the last menstrual period (LMP) and the date of delivery. LMP was mainly obtained from hospital records or the physician. The mother would be asked for this information if data were unavailable from these resources. Imputation is done by the NCHS if the date of LMP is missing but the month and year are valid . PretermInformation about maternal demographics, parity, education, marital status, prepregnancy weight and height, prenatal care, and infant sex was collected using the maternal worksheet. Data on the previous history of preterm birth were obtained directly from the medical records using the facility worksheet.2), and timing of initiation of prenatal care . A missing category for covariates was added when necessary.In the analyses, we categorized the covariates as follows: maternal age , race/ethnicity , parity , maternal education , previous history of preterm birth , marital status (yes versus no), infant sex (boys versus girls), prepregnancy BMI  with 95% confidence intervals (CIs) of preterm birth according to maternal cigarette smoking status. First, we estimated the association between the timing of maternal cigarette smoking and preterm birth with the mothers who never smoked before and during pregnancy as the reference group. A variety of potential confounders, including maternal age, race/ethnicity, parity, education levels, previous history of preterm birth, marital status, prepregnancy BMI, infant sex, and timing of initiation of prenatal care, were adjusted in the models. Second, we estimated the ORs of preterm birth according to the numbers of cigarettes consumed  before and during pregnancy. We were especially interested in the association of low-intensity cigarette smoking with preterm birth. In addition, we repeated all the analyses for a finer categorization of preterm birth .Moreover, we examined the association of smoking cessation at various periods during pregnancy with the risk of preterm birth. Cessation before pregnancy was defined as having smoked before pregnancy, but no smoking during the first trimester and the second trimester; cessation during the first trimester was defined as having smoked before pregnancy and during the first trimester, but no smoking during the second trimester; smoking throughout the second trimester was defined as having smoked 3 months before pregnancy and during the first and second trimesters of pregnancy; never smoking was defined as no smoking before or during pregnancy.P values for heterogeneity were derived from the multiplicative interaction term coefficients (exposure variable × effect modifier variable) added to the main effects multivariable model. A sensitivity analysis was conducted using data from NVSS 2014–2018, in which the obstetric estimate of gestation at delivery was used to identify gestation age [To evaluate potential effect modification, we conducted interaction and stratified analyses according to age (20–34 or ≥ 35 years), race/ethnicity , and educational levels , an indicator of socioeconomic status, to explore the potential disparities of maternal age, race/ethnicity, and socioeconomic group in the association between maternal cigarette consumption and preterm birth. tion age . To furttion age .P < 0.05 was considered statistically significant.All statistical analyses were performed with SAS software . Two-sided n = 2,378,398). Among them, 9.9% of mothers smoked before pregnancy, 7.4% smoked during the first trimester, and 6.4% smoked during the second trimester. Mothers who smoked before or during pregnancy were more likely to be younger, non-Hispanic white, less educated, unmarried, obese, and with higher parity (Table 1). Of the births, 2,378,398 (9.3%) were preterm birth. The prevalence of preterm birth according to population characteristics is shown in S1 Table.This study included 25,623,479 mothers with live singleton births, with a mean age of 29 years (range 20–50 years); 13,742,486 (53.6%) participants were of non-Hispanic white ancestry, 5,971,598 (23.3%) of Hispanic ancestry, and 3,417,456 (13.34%) of non-Hispanic black ancestry. The prevalence of preterm birth was 9.3% . Positive and significant associations were observed for mothers who smoked in multiple trimesters, as well . Similar results were observed for a finer categorization of preterm birth .Maternal smoking during either the first or second trimester of pregnancy was associated with an increased risk of preterm birth. After adjustment for maternal age, race/ethnicity, parity, education levels, previous history of preterm birth, marital status, prepregnancy BMI, infant sex, and timing of initiation of prenatal care, the OR of preterm birth compared with nonsmokers was 1.00 (95% CI 1.00–1.01) for mothers who only smoked before pregnancy , 1.26 (1.19–1.33) for those who only smoked during the first trimester , and 1.31 (1.21–1.42) for those who only smoked during the second trimester . For mothers who quit smoking during the first trimester  and those who smoked through the second trimester , the corresponding ORs were 1.17 (1.16–1.19) and 1.45 (1.45–1.46), respectively . The adjusted ORs of preterm delivery for mothers who smoked 0, 1–2, 3–5, 6–9, 10–19, and ≥20 cigarettes per day before pregnancy were 1.00 (reference group), 1.22 (1.20–1.24), 1.23 (1.22–1.24), 1.21 (1.19–1.23), 1.32 (1.31–1.33), and 1.36 (1.35–1.37), respectively . For those who smoked during the first trimester, the corresponding ORs were 1.00 (reference group), 1.31 (1.29–1.33), 1.31 (1.30–1.32), 1.33 (1.31–1.35), 1.44 (1.43–1.45), and 1.53 (1.52–1.55), respectively . For those who smoked during the second trimester, the corresponding ORs were 1.00 (reference group), 1.37 (1.35–1.39), 1.36 (1.35–1.38), 1.36 (1.34–1.38), 1.48 (1.47–1.49), and 1.59 (1.58–1.61), respectively . Significant associations were observed for a finer categorization of preterm birth (Table 2).In terms of smoking intensity, significant associations of preterm birth were observed for smoking cigarettes, even at low levels (1–2 cigarettes per day), at either 3 months before pregnancy or the first or second trimester (Table 3). Smokers who quit before pregnancy, regardless of smoking intensity, had a comparable risk of preterm birth with nonsmokers. However, women who quit during pregnancy showed a significant increased risk of preterm birth, even for those who smoked only 1–2 cigarettes per day.It is worth noting that 74.4% of women who smoked before pregnancy continued to smoke during the first trimester, among which 85.4% smoked during the second trimester. Therefore, we examined the association of smoking cessation at various periods during pregnancy with preterm birth to further understand the effects of smoking intensity at various periods . Although maternal smoking during pregnancy was associated with preterm birth in each age group, race/ethnicity group, and education levels (S4 Table), the associations were more prominent among women 35 years or older, non-Hispanic whites, and those with higher education levels. For the intensity of cigarette consumption, a significant association between cigarette consumption, even only 1–2 cigarettes per day, during pregnancy and preterm birth was observed consistently in age, race/ethnicity, and education groups (S7 Table), and the associations were more prominent among women ≥ 35 years old, non-Hispanic whites, and those with higher education levels.The association of smoking status and daily smoking amount with preterm birth was modified by age, race/ethnicity, and education levels ; meanwhile, smoking, even at low levels (1–2 cigarettes per day), before or during pregnancy was associated with a higher risk of preterm birth compared with nonsmoking (S8 Table). These were consistent with our main findings. In addition, similar results were observed in a propensity score analysis (S11 Table).In the sensitivity analysis using data from NVSS 2014–2018, we found that maternal smoking before pregnancy or during either the first or second trimester of pregnancy was associated with an increased risk of preterm birth  with preterm birth among women who quit at various periods, respectively, we found that women who quit before pregnancy had comparable risk of preterm birth with nonsmokers (women who never smoked before or during pregnancy). However, smokers who quit during pregnancy, even during the first trimester, had significantly increased risks of preterm birth. This may be because women who quit later were exposed to higher accumulated levels of tobacco.The association between maternal cigarette smoking and preterm birth is biologically plausible. First, maternal smoking could result in fetal hypoxia, which is a proposed mechanism of preterm birth, although the specific process is unclear . Second,The main strength of this study is the large sample size, which provided high statistical power to thoroughly analyze the association of the timing and the intensity of maternal smoking with preterm birth. In addition, we used a finer category of light smoking to examine the dose–response relationship between very low-intensity smoking (1–2 cigarettes per day) and preterm birth during specific periods. This study has several limitations. First, maternal smoking before and during pregnancy was self-reported, which may be subject to information bias. A previous study showed a high correlation (r = 0.70) between self-reported number of cigarettes and objectively measured biomarker  at any given time point across women, indicating that self-reported variations in smoking during pregnancy may reasonably reflect fetal exposure in epidemiological studies . NonetheOur findings are of considerable clinical and public health implications by highlighting that no level is safe for cigarette smoking during pregnancy. In contrast to some recent studies showing that smoking during early pregnancy (first or second trimester) ,17 or atIn this large-scale population-based study, we found that maternal smoking, even only 1–2 cigarettes per day, during either the first or the second trimester was associated with increased risk of preterm birth. Our findings support the current recommendation for smoking cessation before pregnancy by highlighting that there is no safe level or safe period for maternal smoking during pregnancy. In the future, increased efforts should be made to help women of reproductive age who smoke stop smoking completely before pregnancy to prevent preterm delivery.S1 STROBE Checklist(DOC)Click here for additional data file.S1 FigNVSS, National Vital Statistics System.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 TableNVSS, National Vital Statistics System.(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.


The aim of this study is to investigate the association between maternal gestational weight gain (GWG) and preterm birth according to pre-pregnancy body mass index (BMI) and maternal age. We did a cohort, hospital-based study in Quzhou, South China, from 1 Jan 2018 to 30 June 2019. We selected 4274 singleton live births in our analysis, 315 (7.4%) of which were preterm births. In the overall population, excess GWG was significantly associated with a decreased risk of preterm birth compared with adequate GWG (adjusted OR 0.81 [95% CI 0.72–0.91]), and the risk varied by increasing maternal age and pre-pregnancy BMI. Interestingly, underweight women who older than 35 years with excess GWG had significantly increased odds of preterm birth compared with adequate GWG in underweight women aged 20–29 years (2.26 [1.06–4.85]) and normal weight women older than 35 years (2.23 [1.13–4.39]). Additionally, low GWG was positively and significantly associated with preterm birth overall (1.92 [1.47–2.50]). Among normal weight women category, compared with adequate GWG women aged 20–29 years did, those older than 20 years with low GWG, had significantly higher odds of preterm birth, which increased with maternal age . In conclusion, maternal GWG was significantly associated with the risk of preterm birth, but the risk varied by pre-pregnancy BMI and maternal age. It is reported that the incidence of preterm birth in China increased significantly recently, from 5 to 10%, and 1.5 million preterm infants are born every year. As the well‐known risk factor of perinatal and neonatal morbidity, preterm birth is also considered as the cause leading to long-term adverse outcomes in children2. Therefore, it is imperative to identify risk factors for preterm birth and explore preventive measures to reduce it.Preterm birth is one of common adverse perinatal outcomes that affects approximately 11% of births worldwide4. With excess GWG, pregnant women are more likely to experience gestational diabetes mellitus and macrosomia, which can contribute to preterm birth6. Meanwhile low GWG increases the risk of small for gestational age, low birth weight, as well as preterm birth7. As early as 1990, the Institute of Medicine (IOM) was the first to recommend guidelines for GWG. With the growing knowledge, the IOM revised its recommendations for GWG in single pregnancy women in 2009. However, they do not apply to areas in the world where women are much shorter or thinner than American women or have insufficient obstetric services8.Gestational weight gain (GWG), whether too much or too little, is associated with adverse maternal and infant outcomes11. In 2018, China recommended guidelines for GWG in single pregnancy women, which were in line with IOM guidelines. However, the association between GWG and preterm birth is still controversial and inconclusive. Although low GWG was a risk factor for preterm birth in some studies, in others positive or inverse associations between excess GWG and preterm birth were noted10. Otherwise, pre-pregnancy BMI might play an important role in the relationship between GWG and preterm birth13. Similarly, the function of maternal age, too, cannot be overlooked14. But almost all of those studies were carried out in industrialized countries. Only a few studies have evaluated this issue in non-industrialized countries, including China.Many studies have used the IOM recommendations as reference to investigate the relationship between GWG and pregnancy outcomesIn this cohort study, we aimed to examine the association between GWG and preterm birth according to pre-pregnancy BMI and maternal age in China. Promoting appropriate weight gain advice and prenatal care to mothers before and during pregnancy might reduce the burden of preterm birth.Our study finally enrolled 4274 women of live singleton births with complete data Fig. , of whicIn the overall population, mothers who with low GWG had a significantly increased risk of preterm birth compared with adequate GWG (adjusted OR 1.92 [95% CI 1·47–2.50]), while excess GWG was negatively and significantly associated with preterm birth (adjusted OR 0.81 [95% CI 0.72–0.91]). In the subsequent stratified analyses, pre-pregnancy BMI or maternal age modified the associations between GWG and preterm birth on the basis of the IOM recommendations. It was indicated that the positive relationship between low GWG and preterm birth consistently across maternal age groups and pre-pregnancy BMI groups, in which the adjusted ORs of preterm birth varied by increasing maternal age and pre-pregnancy BMI. Similarly, the significant inverse association between excess GWG and preterm birth also could be partly found in different subgroups. Interestingly, women who were older than 35 years or underweight tended to have positive relationships between excess GWG and preterm birth, although it was not statistically significant.  also had a significantly decreased risk, and who with low GWG had increased risk (adjusted OR 2.10 [95% CI 1·09–4.05]). Moreover, due to small sample size of extremely preterm birth, we didn’t have too much results in stratified analysis. However, it was also explored that the risk of low GWG to preterm birth increased with its severity, especially in women with normal weight. The opposite results were found in women with excess GWG.To further explore the relations between GWG and preterm birth, we examined joint effects of maternal age with GWG on the risk of preterm birth Table . Among nWe also examined joint effects of pre-pregnancy BMI with GWG on the risk of preterm birth Table . In wome9. In those studies, women with lower GWG were at higher risk of preterm birth than higher GWG. However, a review including 12 studies showed that the risk of preterm birth increased consistently in both the highest and lowest GWG categories15. A potential positive association between excess GWG and preterm birth was also reported in recent study10. It is interesting to note that these studies used different definitions of high and low GWG, as well as different analytic methods to characterize the association between GWG and preterm birth, which might lead to the controversial results.The increasing frequency of preterm birth worldwide makes it a critical endpoint to consider in relationship between GWG and preterm birth, which was inconsistent and inconclusive in previous studies. In the 1990s, quite limited literatures pointed out low GWG increased the risk of preterm birth. Some other previous studies showed an inverse association between excess GWG and preterm birth17, we also found decreased risk of preterm birth in women with excess GWG, however risk increased once those women were older than 35 years and had lower pre-pregnancy BMI, which indicated the association between GWG and preterm birth varied by maternal age and pre-pregnancy BMI. Our findings provide a comprehensive review of the association between GWG and preterm birth for women in South China for the first time and suggest that the differing maternal age and pre-pregnancy BMI might contributed to the inconsistent findings.In our study of 4274 mother–child pairs, we noted that women with low GWG have a significantly increased risk of preterm birth compared to women with adequate GWG. Additionally, we found women aged 30–34 and underweight women had highest risk of preterm birth if they didn't gain enough weight during pregnancy. Partially inconsistent with previous studies18. In these women, excess GWG could lead to pregnancy complications more likely, such as gestational diabetes mellitus and gestational hypertension, which is why the risk of preterm birth is increased5. Additionally, excess GWG also was associated with alterations in maternal metabolism and placental microenvironment, which was proposed as a possible pathogenesis in preterm birth19. Unfortunately, there were no significantly results among women younger than 20 years old in our study due to small sample size, but we cannot ignore this group. Adolescent  pregnancy was also showed to be associated with increased risk of preterm birth20. And Howie et al. reported that younger adolescents have an increased likelihood for greater GWG compared to older women in a retrospective review21. However, the relationship between GWG and preterm birth in adolescents is unclear. Only several studies showed an inverse association between pre-pregnancy obesity and preterm birth among them24. Therefore, further research is necessary among adolescents to establish the specific GWG guidelines and explore the underlying mechanisms.The effect of maternal age on the association between GWG and preterm birth suggests that age may play an important role in the potential mechanism of GWG on preterm birth. In this study, we found excess GWG could decrease the incidence of preterm birth in general, but opposite result among women aged 35 years or older. Actually, women with advanced maternal age was considered to have higher risk of preterm birth25. The association between GWG and preterm birth was modified by pre-pregnancy BMI in 5 studies in the review including 12 studies mentioned above15. The findings of these studies consistently showed a stronger effect of low GWG on preterm birth among underweight women. Similarly, our results also showed the magnitude of increased risk associated with low GWG diminished as pre-pregnancy BMI increased. And the risk of low GWG to preterm birth increased with maternal age, especially in underweight populations. By contrast with previous studies that showed excess GWG tended to decrease risk of preterm birth in underweight women4, our results indicated that the negative association between excess GWG and preterm birth was more significant among normal weight populations. Additionally, we also found that the protective effect of excess GWG to preterm birth was appeared in more age groups among normal weight women than among other pre-pregnancy BMI. Yet, we did not advocate excess GWG as a preventive method against preterm birth, as other adverse effects on perinatal and neonatal outcomes that excess GWG should not be ignored26. The tailored recommendations for pregnancy according to different pre-pregnancy BMI groups are needed to stratify the risk of preterm birth accurately.The association between GWG and preterm birth was inconsistent in different BMI categories, although some previous studies considered GWG as a mediator in the association between pre-pregnancy BMI and preterm birth, rather than a confounder27, as well as increased oxidative stress and/or altered immune functions28, important interactions remain unanswered. However, understanding the disparities in the effects of GWG on preterm birth among women at different ages and in different pre-pregnancy BMI groups could help to identify novel pathways in the pathogenesis of preterm birth.Although not clearly elucidated, at least five possible primary pathogenic mechanisms have been postulated for preterm birth, including: activation of the maternal or fetal hypothalamic–pituitary–adrenal (HPA) axis, uteroplacental thrombosis and intrauterine vascular lesions, amniochorionic-decidual or systemic inflammation, cervical insufficiency and pathologic distention of the myometrium. There was not enough evidence so far to show a causal relationship between GWG and preterm birth, because no studies showed direct link of GWG to pathways mentioned above. Although there are intriguing data linking nutrient deficiencies to activation of the maternal or fetal HPA axisOur study has several limitations. First, the sample size is not large enough. Enlarging the samples is necessary for the following study. Second, this is a single-center study. Although our research center is the largest in Quzhou, it is also very important to combine with other research centers to make our findings more applicable to all populations. Third, our research did not deeply explore each subtype of preterm birth, the special reason of which may suggest its underlying pathogenesis. Fourth, while many potential confounders have been adjusted, there may still be some factors such as nutrition and supplement intake of the mother influencing the results. However, the basic nutritional status of pregnant women might be reflected in pre-pregnancy BMI, and the nutritional intake during pregnancy or the supplement intake of the mother might be partially reflected in the GWG.In conclusion, the association between GWG and risk of preterm birth differs according to maternal age and pre-pregnancy BMI. Although inappropriate age and pre-pregnancy BMI are recognized as risk factors for pregnancy outcomes in precious studies, targeted recommendations in different clinical applications are actually needed. Obstetricians should take maternal age and pre-pregnancy BMI into account when they manage weight gain in pregnant women, so as to reduce the occurrence of preterm birth. The same is true of risk prediction models for preterm births. Otherwise, investigations to explore the underlying mechanisms for changing associations between maternal GWG and risk of preterm birth cannot be ignored in the future.The cohort study of mother–child pairs was conducted in Quzhou, a middle-underdeveloped city in south China. We enrolled women with single pregnancy who delivered live infants at Quzhou Maternal and Child Medical Association, Zhejiang University, which is the largest obstetric center in Quzhou city, between Jan 1, 2018 and June 30, 2019. Clinical variables were abstracted after enrolment of the cases from the medical records. The information was collected by study staffs and reviewed by a medical doctor using a standard data-abstraction form. This study was approved by the Human Ethics committee at Quzhou Maternal and Child Health Hospital , and written informed consent was obtained from each participant. The methods were performed in accordance with the relevant guidelines and regulations.In this study, we included all mothers who delivered a live singleton after more than 28 gestational weeks and available for GWG and pre-pregnancy BMI. The main reason for including only singleton live births in our study is that women with twin pregnancies have greater GWG than women with singleton pregnancies and are more likely to experience risks such as preterm birth. The guidelines recommended by Institute of Medicine (IOM) for GWG in twin pregnancies are totally different from singleton pregnancies. Gestational age staging was based on last menstrual period date and available obstetric assessment using ultrasound. Women who delivered stillbirth or underwent termination of pregnancy (n = 16) were excluded. Those with missing data on weight and height in the beginning of pregnancy or invalid delivery weight (n = 53) were excluded. Mothers with pre-existing hypertension or diabetes, which are strong risk factors for preterm birth, were also excluded (n = 58). In total, 4274 women and 4274 infants were included in this study.The clinical variables included: (1) General information: maternal age, education level, pre-pregnancy weight and height, delivery weight of pregnant women; (2) Gravidity, parity, maternal basic diseases, previous history of preterm birth; (3) Antenatal care visit, complications of current pregnancy, mode of conception; (4) Neonatal information: neonatal sex, gestational age.2), normal weight (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), and obese (≥ 30 kg/m2)8.Pre-pregnancy BMI was classified into four groups according to the 2009 IOM standards: underweight , very preterm birth (32–33 + 6 weeks’ gestation), and moderately preterm birth (34–36 + 6 weeks’ gestation).In our study, preterm birth was the main outcome, which was defined delivering less than 37 weeks. Slightly different from traditional classification systemMaternal and neonatal demographic and clinical features are reported as frequency (%) or means (± SD). The rates of preterm birth according to population characteristics were calculated. Categorical variables were analyzed by chi-squared tests. Logistic regression models were performed to estimate the odds ratios (ORs) and 95% confidence interval (CI) for preterm birth and its subgroups. Pre-pregnancy BMI, maternal age, education levels, parity, gravidity, previous history of preterm birth, mode of conception, prenatal care, timing of initiation of prenatal care, pregnancy-induced hypertension, gestational diabetes mellitus and sex of infant were adjusted in our analyses. Otherwise, to further investigate the relationship between GWG and preterm birth, we did stratified analyses and explore the joint effect of pre-pregnancy BMI and maternal age. Two-sided p values less than 0.05 were considered significant. All statistical analyses were done with SPSS 20.0 software.Supplementary Information


Panthera leo) are notoriously secretive during the final stages of pregnancy and postpartum. Behavioral indicators depicted by movement patterns obtained by remote detection of collared female lions in the Kruger National Park were necessary for the monitoring of birth timing. Over the study period, eight plus a potential three parturition incidences of collared females were recorded. Of the variables measured , range size during the month of parturition was the most indicative movement pattern of a successful birth. By backdating the potential birth month of the litters, date of conception was calculated and our results revealed a correlation between the birthing peaks of preferred prey during the month of conception. Birth timing in conjunction with remote sensing and ecological factors were thus identified behaviors associated with denning.The role of social cues in the reproduction of social mammals, particularly carnivores, has been thoroughly studied and documented in literature. However, environmental cues such as resources of water, food, and shelter have been identified to a lesser extent. Pregnant lions ( Behavioural indicators depicted by movement patterns obtained by remote detection of collared female lions in the Kruger National Park can reduce the search area for reproductive lions. The information informs management on activities such as landscapes to include in protected areas, avoiding fire or other disturbing activities. A similar pattern was recorded for lions (Panthera leo) in Nairobi National Park, Kenya  of the Kruger National Park (KNP) in the 1960s and in lions in the 1990s  do lions show seasonality of reproduction, (b) is birth timing a function of ecological factors, and (c) is fecundity a function of ecological factors?To test these hypotheses, we first determined the potential month when a birth occurred through several indicator variables collected through GPS radio‐telemetry and GPS satellite. The indicator variables measured were the number of fixes at a localized site, the elapsed time of consecutive fixes at a localized site, and the distance between the fixes. We then verified whether a successful birth occurred according to the following: (a) the duration of stay at a particular denning site using GPS fix clusters; (b) core and total home range size during the potential birth month, and then (c) variation in daily net displacement (km) during the wet and dry seasons; and (d) variation in daily net displacement (km) during the potential birth month. These variables were chosen because they are the best indicators of changes in prenatal and postnatal behavioral patterns. To test for the season of reproduction, we measured movement patterns by calculating net displacement as a function of energy expenditure and range size as a function of resource availability. To test for the association of ecological factors on birth timing, we calculated prey biomass and rainfall as a function of resource availability. To test the association of fecundity with ecological factors, we compared the KNP with other game reserves as benchmarks.2Sclerocarya birrea and Acacia nigrescens), shrubveld (Colophospermum mopane), thickets (Acacia spp. and Combretum spp.), plains (Themeda triandra and Bothriochloa radicans), and bushveld  and comprises woodlands  took place between February and March 2010 while in the northern region, where prey biomass and rainfall is low, captures (N = 13) were conducted from August to September 2010. We also captured lions throughout the study period when collars needed to be changed. Of the collared females in the northern region, only 10 were used for this analysis because of inconsistent data when collars failed or females were not seen for more than three months.In each pride captured, one adult female was fitted with a collar. In the southern region, where prey biomass and rainfall is high, captures (www.awt.co.za) . All collars had Global Positioning System (GPS) and Very High Frequency (VHF) radio capability. Prides in the southern region had collars with cellular Global System for Mobile (GSM) GPS download while the prides in the northern region had satellite GPS download. Body condition was scored 1–5 according to the Packer and Whitman   when the animal was within cellular network range. Satellite GPS collars uploaded fixes every 4 hr onto the http://mstrackweb8.skygistics.com) and two www.awt.co.za) and (http://www.awt.awetelemetry.com). Variations in vegetation structure, terrain, climatic factors, and power lines may have affected collar upload frequency, accuracy, and precision . Satellite collars were the most reliable with low failure‐to‐download incidences.Behavioral indicators such as range use, diet and movement patterns have been used by Packer, Pusey, and Eberly  to identDuration at a site was calculated by measuring the amount of time spent at a denning site or at a locality. The duration of stay was calculated by identifying clusters defined as the number of consecutive fixes within a 200 m radius. The information included longitude, latitude, altitude, temperature, date, and time. We used the data from the GPS fixes to measure daily movement patterns of the collared lionesses.2.1c was calculated. This adjusts the AIC for small sample sizes. The AIC∆ was then calculated, which is the difference between the AIC value of all candidate models and the candidate model with the lowest AIC score. The Akaike weight (wj) was then derived from the candidate model averages to determine the relative likelihood of the model  to identify which of the explanatory variables  were most indicative of the likelihood of a birth. These models do not force data into predefined artificial scales, thus allowing for nonlinearity and nonconstant variance structures in the data  using LoCoh  . Net displacement was calculated as the distance (km) between consecutive GPS fixes. This was then transformed into a daily net displacement by taking the average distance between fixes in a 24‐hr period. The schedule and frequency of GPS fixes for all collars were uploaded every four hours and commenced in February of 2010 in the southern region and in August 2010 in the northern region until December 2012. When collars failed, only GPS fixes from observations were recorded for movement purposes. The data, however, could not be used for calculating net displacement. Of the total fixes collected, the cellular GSM‐GPS collars had a 15% failure rate and the satellite GPS collars had a 10% failure rate over the three year study period.Range size  and daily net displacement were measured in ArcGIS 9Duration of stay at denning sites was calculated as the number of consecutive days spent within a 200 m radius. The radius of 200 m was chosen because this indicates localized movement and displacement. An excess of 200 m would suggest relocation away from an area  were then calculated.The peak conception time of the southern and northern regions was compared using one‐way ANOVA and linear regression. The GLM's were used to determine whether there was a relationship between births and conceptions of lion in the southern and northern regions and (a) rainfall, (b) prey biomass, and (c) prey lambing and calving. Births and conceptions were calculated for each region separately and then compared. A Poisson regression on a binomial distribution was used for response variable: birth and conception, and explanatory variables: rainfall, prey calving, and lambing. In this distribution: 1 = successful birth or likely conception and 0 = no births or conception; 1 = peak prey births and 0 = low prey births; and 1 = wet season and 0 = dry season. The AICTo answer the first hypothesis, we used the movement pattern outcomes from the potential birth month calculations to identify reproductive activity. We calculated potential month when a birth occurred by the following metrics collected through GPS radio‐telemetry: (a) the duration of stay at a particular denning site by identifying GPS fix clusters; (b) core and total home range size during the potential birth month; (c) variation in daily net displacement (km) during the wet and dry season; and (d) and variation in daily net displacement (km) during potential birth month.To address the second hypothesis, we calculated the association between the birth timing and ecological factors by measuring known birth and conception with (a) prey biomass, (b) lambing and calving of preferred prey, and (c) rainfall. We used verified cub births to identify whether there was an association between the occurrence of births and conception incidences and ecological factors.To address the third hypothesis, we measured the fecundity of female lions in the KNP; (a) age at first birth, (b) litter size, and (c) birthing intervals. We compared the fecundity of lionesses in the KNP with other lion populations.2.1.1p < .05). Data of the collared pregnant and nonpregnant females were taken for the duration of the study between 2010 and 2012. Range size of core and total home ranges in the southern and northern region were compared using the one‐way ANOVA (p < .05). Data came from both collared females (N = 20) and adult female pride members that were verified as having birthed cubs (N = 12). The data used from the noncollared females were only for identifying birth and conception and not for range size or net displacement We used GLM's to identify which of the movement patterns, range size, daily net displacement, as explanatory variables were most indicative of the likelihood of a birth.To address our first hypothesis, do lions show seasonality of reproduction, we analyzed movement patterns through net displacement, range size, and duration of stay at a site. Direct observations were conducted by ground proofing or camera traps to visually verify the presence of cubs and the collared female's reproductive state. To determine spatial use, the range size in a month of a birth was compared against the range size in the same month of a collared nonpregnant female in the same region. Once a litter was recorded, the variables of clusters (the number of consecutive fixes within a 200 m radius), daily net displacement, and range size for lions were grouped by region (southern and northern) and compared. The net displacements of collared pregnant and nonpregnant lionesses were compared using one‐way ANOVA , kudu (Tragelaphus strepsiceros), zebra (Equus quagga burchellii), and wildebeest  to identify whether there was variance in the fecundity of age at first birth, litter size, and birth intervals in the southern and northern regions. A Kruskal–Wallis test was used to determine whether there was a difference between the fecundity in the KNP when compared to other parks in east Africa , South Africa , and historical KNP data. We compared parks that were relatively of similar size and functioning under natural ecological and environmental conditions rather than smaller reserves that highly manage the lion populations. All statistical analyses were run in R version i386 2.15.1 Ink R Core Team (http://www.R-project.org/)  using the Rcmdr package  daily net displacement than the prides in the northern region . When compared, the pregnant females in the southern region had a shorter daily net displacement  than the pregnant females in the northern region ; . When the southern and northern pregnant females were compared with nonpregnant females during the birth month, the pregnant females had a shorter step length (distance between consecutive GPS fixes), than the nonpregnant females . There was a 51%, reduction in net displacement in the northern (from 9.48 to 5.81 km) and southern (from 7.40 to 3.08 km) regions during the birth month  and total home range  compared with the core range  of nonpregnant females . During the birth month, total home range of collared pregnant females  was smaller compared with the total monthly home range  of the nonpregnant females . The best candidate GLM models to predict a successful birth incident and denning site comprised core range and the combination of core and total range . The average amount of time spent at a locality during a month when a birth event occurred was shorter for nonpregnant females  than that of pregnant females .During a birth month, the amount of time spent within a 200 m radius differed, but not significantly, between nonpregnant and pregnant females  and in the north  regions  and in the north  region  and northern  regions . Nor was there a significant difference for litter size ; . There was insufficient data from the pregnant females to compare birthing intervals , which averaged 2.9 years (SE = 1.1) in the south.From the data collected in this study, there was no significant difference in the average age at first birth for lionesses in the southern . Nor was there a significant difference found in the fecundity of KNP between this study and 1978 , especially the fawns, during pup‐rearing months (Dellinger et al., In Kenya, spotted hyena births coincide with response to season variation and resource availability (Holekamp et al., Our third hypothesis, fecundity alters as a result of ecological factors, was not supported by our results. Firstly, litter size, age at first reproduction, and birthing interval were not significantly different between the resource‐rich southern region and poorer resourced northern regions. Secondly, when compared with historical data Smuts, , and datUsing movement patterns as behavioral indicators, we were able to detect the denning sites remotely and identify reproductive activities of lions. In existing protected areas, conservation practitioners can be more selective in their management of denning sites to avoid burning and minimize disturbance during denning. In the event of poaching activity on a reserve, conservation mangers will be better suited to direct law enforcement to the denning sites. Although this study did not focus or take into account poaching of wild lions, the lessons learned could be applicable to addressing such incidences. Kruger National Park has not been identified as a source of lion poaching. Lions that have been killed outside the park are ad hoc animals that have left the park and entered neighboring communal property. Throughout Africa, retaliation killing for livestock lose is one of the primary human–wildlife interactions leading to death and population declines of lions (Tumenta et al., This study provided insight into areas of study not previously questioned such as the importance of conception rather than just the birthing. Population dynamics could consequently be a function of poor conditions during conception and gestation. Similar to the importance of understanding the role of landscape features and habitat quality in lion denning site selection, so is understanding the triggers and drivers of conception. This study suggests that space use in the form of landscape features and habitat quality have an impact on reproduction. Although this study was conducted in an area with known bTB disease prevalence, disease did not appear to be a significant driver.It is important to note that this study did not explore social cues of synchronized births of pride members or the effect of male takeovers. To strengthen our analysis, social cues in the form of male takeovers, estrus synchrony, and crèche formations could be incorporated. Nevertheless, ecological and environmental cues that influenced the conception period were identified, particularly regarding the influence of births of preferred prey. Identifying the resources and landscape features associated with denning sites is an important next step to explore the effect of seasonality and elucidate denning site characteristics.None declared.All authors contributed equally to this work including the conception, data analysis, and manuscript synthesis.


Ixodes ricinus and endophilic I. trianguliceps on two mountain slopes in Norway by assessing larval infestation rates on bank voles (Myodes glareolus).During the last decades a northward and upward range shift has been observed among many organisms across different taxa. In the northern hemisphere, ticks have been observed to have increased their latitudinal and altitudinal range limit. However, the elevational expansion at its northern distribution range remains largely unstudied. In this study we investigated the altitudinal distribution of the exophilic During 2017 and 2018, 1325 bank voles were captured during the spring, summer and autumn at ten trapping stations ranging from 100 m to 1000 m.a.s.l. in two study areas in southern Norway. We used generalized logistic regression models to estimate the prevalence of infestation of both tick species along gradients of altitude, considering study area, collection year and season, temperature, humidity and altitude interactions as extrinsic variables, and host body mass and sex as intrinsic predictor variables.I. ricinus and I. trianguliceps infested bank voles at altitudes up to 1000 m.a.s.l., which is a substantial increase in altitude compared to previous findings for I. ricinus in this region. The infestation rates declined more rapidly with increasing altitude for I. ricinus compared to I. trianguliceps, indicating that the endophilic ecology of I. trianguliceps may provide shelter from limiting factors tied to altitude. Seasonal effects limited the occurrence of I. ricinus during autumn, but I. trianguliceps was found to infest rodents at all altitudes during all seasons of both years.We found that both This study provides new insights into the altitudinal distribution of two tick species at their northern distribution range, one with the potential to transmit zoonotic pathogens to both humans and livestock. With warming temperatures predicted to increase, and especially so in the northern regions, the risk of tick-borne infections is likely to become a concern at increasingly higher altitudes in the future. During the last decades increasing temperatures have been shown to have an impact on the distribution of species across a wide range of taxonomic groups , 2. DepeIxodes ricinus has been recorded at altitudes up to 1070 m.a.s.l. [I. trianguliceps, has been recorded at altitudes as high as 2300 m.a.s.l. in Switzerland [I. ricinus, it is likely to be less exposed to limiting environmental conditions, especially temperature, and hence able to survive in more extreme environments [Along with other organisms, certain ectoparasites, such as ticks, have undergone similar range expansions –16. Tickm.a.s.l. , 26, andm.a.s.l. , and in m.a.s.l. . Accordim.a.s.l. , it may ronments . With anronments , 28, andronments –33.I. ricinus have occurred at its geographical center in Europe [I. ricinus has undergone a northward range shift similar to that observed in other Scandinavian countries [Most studies investigating the altitudinal dynamics of n Europe , 29, 34,n Europe , the incountries –38, withountries  and consountries . It has ountries . Even soI. ricinus and the specialist tick I. trianguliceps in Norway, by studying the infestation rates of both species on a commonly found rodent, the bank vole (Myodes glareolus). Because of the nest-dwelling behaviour of I. trianguliceps, we expect it to be less limited by altitude than I. ricinus.To the best of our knowledge, no recent field studies have investigated the altitudinal distribution of ticks at their northern distribution range in Europe. The aims of this study are to determine the altitudinal distribution patterns of the generalist tick Betula pubescens) and spruce (Picea abies) as the dominant tree species, and blueberries (Vaccinium myrtillus) as the dominant species at ground layer. Below the tree line the vegetation is mostly homogeneous. Above the tree line the vegetation is primarily dominated by common heather  and blueberries. Boulder fields occur frequently throughout the gradient, and the highest peak found on the plateau is 1288 m.a.s.l. Most of the data collection points were located on topographically open hillside. Temperature and precipitation normals for the study areas can be found in Appendix Alnus glutinosa) as the dominant tree species. At ground layer the vegetation is dominated by blueberries, and different species of ferns and tall perennial herbs. Above the tree line common heather, dwarf birch (Betula nana), common juniper (Juniperus communis) and crowberry (Empetrum nigrum) are the dominant species. Surrounding the study area are several mountain peaks exceeding 1500 m.a.s.l.This study was carried out along two mountain slopes in Norway during 2017 and 2018 , tundra vole (M. oeconomus), grey red-backed vole (Myodes rufocanus), wood mouse (Apodemus sylvaticus), yellow-necked mouse (A. flavicollis) and house mouse (Mus musculus). The common shrew (Sorex araneus), pigmy shrew (S. minutus) and water shrew (Neomys fodiens) are also found in these areas. The bank vole is a reservoir host for several tick-borne pathogens, such as B. burgdorferi [Babesia microti [Candidatus Neoehrlichia mikurensis [A. phagocytophylum [I. trianguliceps [I. ricinus [The bank vole is a commonly found rodent throughout Europe and occurs virtually everywhere in Fennoscandia . It is tgdorferi , Babesia microti , Candidakurensis  and A. ptophylum . It is pguliceps  and is h ricinus , 57. BecI. trianguliceps in this study, we focused solely on the capturing of hosts. However, examining tick burdens accurately from live small mammals can be difficult [http://www.grahnab.se), and the other plot with lethal traps . The traps in both plots were arranged in a 4 × 5 grid, with a spacing of 10 m between each trap. Live traps were baited with a slice of apple for hydration and whole oats for caloric value, and a lining of sawdust was provided on the trap floor as insulation. Lethal traps were baited with peanut butter for practical reasons as it is easily applied to the inside of the trap body. Trap type does not influence tick burden size on the captured animals [Ticks are commonly collected by two methods: cloth-dragging for questing (unfed) ticks and host examination for feeding ticks. The cloth-dragging method is only applicable for the collection of exophilic species, and only when the vegetation is dry. During most days of data collection the vegetation was either partly wet or weather conditions did not allow for cloth-dragging. Additionally, as we aimed to include the infestations of ifficult ; therefoifficult , and alt animals . At each animals . For thi animals , all aniAt the end of every trapping season, captured bank voles were examined for ticks in the laboratory, as a full-body post-mortem examination provides the highest degree of sensitivity . The dayI. ricinus and I. trianguliceps collected, respectively, were larvae, only the larval stage was included in the analysis.Ticks were determined for life stage and species under a Zeiss Discovery V20 stereomicroscope , using an established publication key as reference . BecauseI. ricinus larvae and I. trianguliceps larvae was performed separately. As is usually the case with tick presence on small mammals, neither tick species was evenly distributed on the hosts [I. ricinus and I. trianguliceps larvae present, respectively. We therefore chose to use the presence or absence of larvae as the response variable and applied generalized linear modelling with a binomial distribution, i.e. logistic regression. The probability of encountering a tick was defined as prevalence. As predictor variables we considered altitude , study area , collection year (2017 and 2018), season  and humidity (%). Because altitude and temperature were negatively correlated , temperature per se was not used as a predictor variable in order to avoid introducing collinearity into the model. Two-way interactions between season and altitude, study area and altitude, year and altitude, study area and year, as well as year and season were included in the starting model. We also considered individual bank vole body mass (in grams) and sex  as intrinsic co-variates. Starting with a full starting model containing all variables and the two-way interactions listed above, we used a backward step model selection process to progressively remove non-significant predictors, by comparing the residual deviance and degrees of freedom of nested models using a Chi-square test (χ2), until an optimal model that contained only significant predictor variables (α = 0.05) was found. To visually represent the infestation probabilities of both species across study areas, collection years and seasons, we created multiple regression line plots using the ggplot2 package in R [The statistical analyses were performed using the software package R version 3.5.3 . The anahe hosts , and 13.age in R .This study was carried out in strict accordance with regulations issued by the Norwegian Environment Agency, and a permit was provided prior to the start of the sampling  for the duration of the trapping period. The trapping protocol for animal capture was approved by the Animal Ethics Committee of the Department of Nature, Health and Environment (University of South-Eastern Norway). All efforts were made to minimize animal suffering.I. ricinus were 7.4-fold more numerous than those of I. trianguliceps . Larval burdens ranged from one to 100 ticks for I. ricinus  and from one to 29 for I. trianguliceps (mean ± 95% CI: 0.5 ± 0.1), and more than half of the bank voles (57.4 %) were infested with at least one larva of either tick species. For I. ricinus and I. trianguliceps, respectively, 46.8 and 18.0% of voles carried at least one larva. Of all the voles, 7.3 % were infested with at least one larva of both tick species. The total number of ticks collected from voles at every altitude is shown in Fig. During 2017 and 2018, a total of 43,920 trap nights was performed, capturing 1325 bank voles . Altogether 5372 tick larvae were collected from the trapped voles at all altitudes of the gradient. Larvae of I. ricinus presence , and a significant interaction between altitude and season  illustrates that this effect was stronger in the autumn than in the spring and summer  shows that the negative altitude effect was stronger in Lærdal than in Lifjell  and significantly lower among voles captured in the autumn  , but the seasonal effect, with lower prevalence in the autumn, was much stronger in 2017 than in 2018, as indicated by a significant interaction between year and season  , with especially the autumn prevalence being lower in Lærdal  , ticks were found significantly more often found on male hosts than on female hosts, resulting in a significant sex effect in the best model .While host weight did not affect the prevalence of I. trianguliceps on bank voles was ultimately found to be much simpler than that for I. ricinus   and significantly higher in 2018 than in 2017  , with the overall prevalence of I. trianguliceps being higher among voles captured in the autumn and with an equal prevalence in spring and summer  nor host sex  affected the prevalence of I. trianguliceps presence on bank voles.Neither host weight  was zero, while still a substantial proportion of the hosts were infested at lower altitudes. Bown et al. [Capreolus capreolus) as tick hosts reduces the larval burden of I. ricinus on small rodents, but not that of I. trianguliceps. The generally higher I. ricinus prevalence at lower altitudes in our study might therefore be caused by I. ricinus being able to make use of cervids as hosts, since we expect such hosts to be more common at the lower altitudes. At higher altitudes it is possible that both tick species are limited by the lower overall temperatures and/or the shorter snow-free season, but that I. trianguliceps is less affected due to its endophilic nature. It is reasonable to assume that I. ricinus cannot be active for as many days at 1000 m as at lower altitudes, which would reduce the probability of finding a host, ultimately leading to a lower population density. Ixodes trianguliceps, however, seeking a small mammal host within its burrows, is not as limited by low ambient temperatures [Although both tick species decreased in abundance with increasing altitude, both were found at all altitudes of the gradient. We expected altitude to have a stronger effect on n et al.  showed teratures  and shouI. ricinus and I. trianguliceps in these areas of Norway is still unknown. Nonetheless we have documented I. ricinus presence far above what has previously been reported [Persistence of tick populations at high altitudes is not necessarily restricted by survival, molting success or oviposition , 30; ratreported .I. ricinus was generally highest in the spring. Studies in Switzerland, Italy and the UK have shown I. ricinus larvae to have a bimodal activity pattern, peaking in the spring and autumn [I. ricinus larvae are not considerably inhibited by summer drought in this region and that the relatively cool and humid conditions of the two regions, compared to further south in Europe, enables it to continue questing as long as temperatures are high enough.For all altitudes the prevalence of d autumn , 44, 70 d autumn . In our I. trianguliceps were infesting voles during all active seasons of the year, but in contrast to I. ricinus, prevalence was highest in the autumn. Because I. trianguliceps is dependent on a very humid environment [I. trianguliceps at this part of the year.Larvae of ironment , it is pI. ricinus larvae, with males having higher infestations than females. As males roam around more than females [I. ricinus. However, no significant difference was found in infestation with I. trianguliceps larvae between male and female voles. As both male and female voles probably utilize burrows to a similar extent, both sexes should be equally exposed to infestations of I. trianguliceps larvae, which is in agreement with earlier findings [Higher levels of parasitism of male hosts compared to female hosts is a common observation –75, and  females , 77, thefindings , 46.I. ricinus and I. trianguliceps are present at much higher altitudes than what has previously been documented in Norway. With ticks expanding to higher altitudes there is an increasing risk of human and livestock infection with tick-borne diseases. The occurrence of tick-borne infections has both social and economical consequences, but to date these problems have been restricted to low-altitude areas in this region. Although human cases of tick-borne infections viaI. trianguliceps are rare [ I. ricinus ticks in Norway [In this study we show that both are rare , this spare rare , 68, 78.n Norway –81, but n Norway , the norn Norway . This wi