According to a longstanding paradigm, aquatic amniotes, including the Mesozoic marine reptile group Ichthyopterygia, give birth tail-first because head-first birth leads to increased asphyxiation risk of the fetus in the aquatic environment. Here, we draw upon published and original evidence to test two hypotheses: (1) Ichthyosaurs inherited viviparity from a terrestrial ancestor. (2) Asphyxiation risk is the main reason aquatic amniotes give birth tail-first. From the fossil evidence, we conclude that head-first birth is more prevalent in Ichthyopterygia than previously recognized and that a preference for tail-first birth likely arose in derived forms. This weakens the support for the terrestrial ancestry of viviparity in Ichthyopterygia. Our survey of extant viviparous amniotes indicates that fetal orientation at birth reflects a broad diversity of factors unrelated to aquatic vs. terrestrial habitat, further undermining the asphyxiation hypothesis. We propose that birth preference is based on parturitional mechanics or carrying efficiency rather than habitat.The online version contains supplementary material available at 10.1186/s12862-023-02110-4. Chaohusaurus, Mixosaurus, Cymbospondylus, Besanosaurus, Shonisaurus, Qianichthyosaurus, Ichthyosaurus, Leptonectes, Stenopterygius, Maiaspondylus and Platypterygius [Stenopterygius and Ichthyosaurus are so far the only genera with multiple described gravid specimens, and the sample of Stenopterygius is vastly larger than that of any other taxon [Ichthyopterygia is a clade of Mesozoic reptiles adapted to marine life. One of the numerous adaptations that helped the group radiate in, or even invade the marine realm is viviparity . There iterygius –14. It ier taxon .Stenopterygius in 1749 (SMNS 2). The earliest discussions usually pertained to whether the preserved smaller specimens were fetuses or possible gastric contents resulting from predation or cannibalism [Stenopterygius  [Chaohusaurus was subsequently described with two fetuses in situ, one of which presumably in the birth canal, showing clear head-first orientation  [Viviparity in ichthyosaurs was first proposed in 1842 , followinibalism , 19. Bötnibalism  publisheiscissus ). He stureptiles , 21. Howreptiles , and the mother) . This le mother) . HoweverChaohusaurus, as one of the basal-most ichthyosaurs, still retained the ancestral head-first birth position [Mixosaurus already showed a tail-first parturition preference, and left out the nuances concerning the tail/head birth positions of Stenopterygius. This led to the hypothesis that birth orientation switched early in the evolutionary history of ichthyosaurs [Cymbospondylus : Besanosaurus: 1 specimen, the holotype, in utero, orientation suggesting tail-first birth [Shonisaurus: 1 specimen, unknown orientation [Qianichthyosaurus: 1 specimen in utero orientation suggesting tail-first birth [Ichthyosaurus: 3 specimens, all inconclusive [Leptonectes: 1 specimen, inconclusive [Maiaspondylus: 1 specimen, inconclusive [Platypterygius: 1 specimen in utero orientation suggesting tail-first birth [Motani and colleagues proposed that viviparous reproduction likely evolved in the terrestrial ancestors of Ichthyopterygia and that position . Their pposition  account hyosaurs . Subsequst birth ; Shonisaentation , 7; Qianst birth ; PMS, NKnclusive , 23 and nclusive ; Maiasponclusive ; and Plast birth .Delphinapterus leucas [Maiacetus showed an in utero orientation suggestive of head-first birth [Maiacetus showed the ancestral artiodactyl condition and proposed that Maiacetus possibly gave birth on land and that subsequent obligatorily marine cetaceans would have given birth tail-first [The assumption that derived ichthyosaurs should show a preference for tail-first birth was based on the idea of the prevalent “increased asphyxiation risk” hypothesis, which infers that head-first orientation of the fetus reduces the risk of drowning/asphyxiation during the birth process , 25. Thes leucas , 27. Aspst birth . The autil-first .Mixosaurus cf. cornalianus, one of which was previously studied [Mixosaurus is a fully marine adapted Middle Triassic ichthyosaur already displaying dorsal and caudal fins, a streamlined body and flippers [Mixosaurus prompted us to review the literature on ichthyosaur reproduction and add nuances and taphonomic concerns regarding the study of parturition in gravid female ichthyosaurs. We provide a short review of the reproductive strategies of other marine reptiles and briefly compare them to that of ichthyosaurs. Likewise, we survey birthing positions in certain extant viviparous mammals and reptiles to test the postulate that tail-first births are necessarily an adaptation to giving birth in water. Lastly, we propose a novel hypothesis on when and why parturition orientation may have switched from head-first to tail-first in Ichthyopterygia.Here we add to this discussion by documenting the orientation of fetuses in three specimens of  studied . Mixosauflippers . It is uflippers , 31. TheMixosaurus containing two fetal vertebral columns in the maternal body cavity and disarticulated fetal cranial and postcranial material outside the maternal cavity. In the more ventrally situated fetal vertebral column, two centra show clear rib facets. This vertebral column is preserved in ventral view, which means these facets should be parapophyses. The rib facets lie on the part of the lateral centrum surface closest to the maternal tail. The positioning therefore shows that the fetus is oriented facing the maternal tail, suggesting head-first parturition articulated vertebral columns and a proximal limb fragment including a humerus, radius and ulna. The more dorsally situated of the two vertebral columns shows clear rib facets. It is unclear in which view the column is visible, but likely in ventral view, because of the overlapping associated gastralia  and a disarticulated posterior limb. Fetal cranial material is located just dorsally of the maternal scapula in the maternal ribcage. The fetus therefore likely lies in utero  as well as an advanced degree of ossification in the chondrocranium suggests a developmental stage comparable to stage 4 of Stenopterygius [in utero suggesting tail-first birth and those in PIMUZ T 4830 are in head-first presentation close to the cloaca.Litter size is small in all three studied specimens of terygius , meaningStenopterygius ranged from 1 to 11; the largest litter was observed in one female , but many were observed to have only one fetus; the average was found to be 3.3 [Litter size in o be 3.3 .Stenopterygius (SMNS 81961 and SMNS 80234), we calculate that in roughly 54% (22/41) of the preserved gravid females tail-first fetal birth canal orientation is observed, whereas in 15% (6/41) of cases head-first birth canal orientation is present . The other cases (13/41) were deemed inconclusive, as the fetuses were not preserved in situ in the reproductive tract or were too disarticulated. Remarkably, in two cases (SMNS 16811 and MHH 1981/33), fetuses were preserved together in the maternal body cavity in both orientations . We are therefore cautious to conclude a climatic effect based on the limited data.Based on the data Böttcher provides plus our own observations (suppl data 1) on Mixosaurus here described do indeed display in utero tail-first orientation. Given the small sample size , it is difficult to conclude anything on preferred birth orientation in Mixosaurus, but it is at least not strictly tail-first. It is in our opinion unlikely that the fetuses of PIMUZ T 1902 and PIMUZ T 2262 would have been able to turn in utero as they are in an advanced stage of development when compared to Stenopterygius.Head-first birth position in the fetuses of PIMUZ T 4830 has been hypothesized previously based on the distribution of jaw elements towards the maternal pelvic girdle, in combination with the recovery of caudal vertebrae dorsal to the adult pectoral girdle . The twoStenopterygius.Our reanalysis of Böttcher’s data supports the tail-first birth orientation preference reported in the original study; in fact tail-first birth predominated by a factor of 3.6:1 in Stenopterygius at birth depends on the following questions: does either birth position lead to greater pregnancy complications, and if so, is our sample biased towards the disadvantageous orientation? To neither question do we have a fully satisfactory answer at this time. However, in modern odontocetes, both birth orientations are recorded and even though they have a large preference for tail-first birth, neither position has been observed to result in major complications during birth [in utero orientations, we are unlikely to see a strong bias towards complicated pregnancies in the fossil record of Stenopterygius.Whether or not our observations reflect preferred orientation in the sample of ng birth –36. Thisin utero. Fetal turning is unfortunately not well studied in any viviparous animal. Extant lepidosaurs would be the closest living viviparous relatives of ichthyosaurs. In viviparous squamates (as in their oviparous counterparts), fetuses are tightly curled through lateral flexion, forming a compact rounded mass tightly enclosed within the fetal membranes [Stenopterygius [in utero orientation therefore equals birth orientation is plausible, and we will use this assumption throughout the rest of the discussion.The other factor that we have to take into account is potential fetal turning embranes , 38. In terygius ); subseqterygius , 9, 14. Stenopterygius, the sample size is far too small to assess preferred birth orientation. However, in general head-first birth is more common in the small sample size of more basal Triassic taxa , whereas all specimens of the derived pelagic Merriamosauria  in which orientation can be assessed show tail-first presentation in their single specimen or average sample, as does Mixosaurus. We therefore hypothesize that a (slight) preference of tail-first parturition is more prevalent from the base of the Merriamosauria and that likely all more basal members either had a slight head-first birth preference or no preference either way .As stated, the major difference between ichthyosaur fetuses and those of extant lepidosaurs lies in their conformation during development, whereby lepidosaurs are generally curled  are not , 38. The are not . Either  are not , 56, pre are not , 14, 22, are not . In vivia snakes , 59, theCarsosaurus unsurprisingly shows an in utero condition closely matching extant viviparous squamates [The aigialosaur quamates . In the quamates , 60. TheHyphalosaurus baitaigouensis. Two gravid females have been found: one containing fetuses in the body cavity and one containing, and associated with, embryos in eggs [in ovo and in utero, although in ovo there seems to be more curving [Hyphalosaurus (1 published gravid specimen), and the data presented here.The exact phylogenetic position of Choristodera is uncertain, but it is regularly recovered as sister taxon to either Lepidosauromorpha or Archosauromorpha . Within  in eggs , 63. Fur in eggs  as well  in eggs , 43. The curving . The fetDinocephalosaurus [in utero position, suggestive of a possible tail-first birth. Moreover, there is an isolated expelled fetus attributed as cf. Dinocephalosaurus [Dinocephalosaurus.Only one viviparous archosauromorph taxon has so far been discovered: the Middle Triassic losaurus , 47. Thelosaurus . The spelosaurus . Given tOutside of Cetacea, aquatic and semi-aquatic mammals do not show a clear preference in fetal orientations at birth. In Sirenia, both head-first and tail-first births have been recorded –67. SuchOrcinus orca and 14% in captive belugas, with head-first deliveries being labeled a “natural variation” [in utero orientation is at least close to 50/50 in the genus Balaenoptera  and observations of birth in the genus Megaptera  are generally tail first [As for cetaceans, while tail-first deliveries predominate, they are far from universal. For example, one literature summary noted that the rate of head-first births was 7% in the killer whale riation”  . If fetuses born head-first were indeed prone to increased asphyxiation risk in secondarily aquatic tetrapods, we would expect to see a higher preference for tail-first births early in the evolutionary history of all aquatic, viviparous clades, due to strong stabilizing selection for this trait. This is not really the case (Appendix 1); we therefore deem the “increased asphyxiation risk” hypothesis to be unsupported by the data.Keichousaurus [Keichousaurus it would therefore be advantageous to give birth head- (or rather neck) first.Placental mammals that are born head-first generally share the characteristics that they have a flexible neck, long appendicular elements and a wide pelvic girdle. The peristaltic movement of the uterus and vagina push on the pelvic girdle of the fetus. This is mechanically convenient as the pelvic girdle is one of the widest parts of the fetal body. Moreover, early expulsion of the head and neck ensures that these will not be in an awkward position, or even at risk of damage, if the head gets stuck in the birth canal. Ichthyosaurs and cetaceans morphologically share the evolution of a streamlined body, including the loss of a discrete neck and fusion, or shortening of the cervical vertebral column. This makes the cervical region in these clades less vulnerable during parturition. Moreover, the pelvic area in cetaceans and ichthyosaurs is extremely reduced over evolutionary history, making the fetal cranium larger than the pelvis. It is therefore mechanically and energetically advantageous to peristaltically push on the cranium. We therefore hypothesize that peristaltic expulsion mechanics during parturition favor a tail-first birth in cetaceans and ichthyosaurs, which would explain the parturition preference switch over evolutionary history in the two clades. This hypothesis would also explain the preferred birth orientation in the eosauropterygian ousaurus . The necin utero (tail-first parturition) could have been slightly more advantageous than cranial posterior in utero orientation because in thunniform ichthyosaurs (and maybe cetaceans), the maternal center of mass would not be displaced as far from the anteriorly positioned center of buoyancy (the lungs) if the heavily ossified fetal crania were oriented anteriorly rather than posteriorly, thus reducing maternal energy expenditure on trim control [Alternatively, it is possible that with the loss of the discrete neck and reduction of the pelvic girdle and hind limb, head-first or tail-first orientation are somewhat equal in terms of maternal energy use and danger to the fetus. In this case, maternal stress induced from the orientation during pregnancy is the main selection pressure for fetal orientation. For example, having the cranium directed anteriorly  control .Mixosaurus were (re)examined. They show tail-first parturition preference in two cases (PIMUZ T 1902 and PIMUZ T 2262) and head-first parturition preference in the third case (PIMUZ T 4830). So far, the proposed hypothesis was that ichthyosaurs gave birth tail-first because of asphyxiation risk of the neonate during birth in the aquatic medium. Moreover, the head-first birth recorded in the early diverging ichthyosaur Chaohusaurus was proposed to be a remnant of the terrestrial origin of viviparity for the ichthyosaur clade. With the new data on Mixosaurus, the recently discovered head-first in utero orientation of Cymbospondylus and a review of the known literature on ichthyosaur in utero and parturition orientation, we propose that a slight preference for tail-first parturition possibly arose at the base of the Merriamosauria and that more basal ichthyopterygians either had a head-first preference or little preference. We want to stress that we should not be surprised to find either parturition orientation in future finds across phylogeny.Three specimens of pregnant in utero head-first orientation during pregnancy, which leads to tail-first birth, could have been advantageous energetically or in terms of trim control.Moreover, the “increased asphyxiation risk” hypothesis is an unlikely explanation for birth preference based on our review of parturition preferences of modern amniotes. We propose two new hypotheses on why ichthyosaurs might have switched birth preference, namely: (1) it may have been mechanically advantageous to push on the cranium rather than the pelvis during ichthyosaur birth, due to gradual decrease of pelvic size, reduction of a discrete neck and increased body streamlining in parvipelvian ichthyosaurs; (2) in utero, and the potentially large percentage of complicated births (including the non-preferred parturition orientation).We advise caution when assessing parturition preference in fossil viviparous taxa as it is uncertain what percentage of the sample shows birth complications, e.g., still births Mixosaurus were collected from the Besano Formation at the Monte San Giorgio locality during scientific excavations in the 20th century led by Emil Kuhn-Schnyder and Bernhard Peyer respectively. The locality is at the Swiss-Italian border and has recently become a UNESCO World Heritage site. The Besano Formation is Anisian-Ladinian (Middle Triassic) in age and has yielded many well-preserved vertebrate fossils [The three specimens of  fossils –84. See  fossils and Röhl  fossils  for an oMixosaurus; however, gravid females are relatively rare. In addition to the material previously described . We suggest to adopt the term fetus in future. This is more in congruence with the terminology of other viviparous vertebrates in which a conceptus is referred to as embryo up to the onset of organogenesis, and referred to as a fetus from that point until birth. We have therefore used “fetus” throughout the manuscript, since all referred specimens have started ossification and therefore all organs are inferred to have begun development.Up to this point, Chaohusaurus, but excluding Hupehsuchia and Omphalosauridae (Nasorostra) [We have also adopted the term Ichthyopterygia sensu . This enorostra)  of whichBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


The study objective was to describe trends in partner-accompanied birth between January 2019 and August 2021 and examine the associations of partner-accompanied birth with women’s psychological distress and partners’ housework and parenting. A total of 5605 women who had a live singleton birth between January 2019 and August 2021 and had a partner participated in this nationwide internet-based survey between July and August 2021 in Japan. The percentages of women’s intentions and actual experience of partner-accompanied births were calculated per month. Associations of partner-accompanied birth with scores on the Kessler Psychological Distress Scale (K6) ≥10, partners’ participation in housework and parenting, and factors associated with having a partner-accompanied birth were examined using a multivariable Poisson regression model. The proportion of women who had partner-accompanied births was 65.7% between January 2019 and March 2020, dropping to 32.1% between April 2020 and August 2021. Partner-accompanied birth was not associated with a K6 score ≥10, but was significantly associated with the partner’s daily housework and parenting . Partner-accompanied births have been substantially restricted since the beginning of the COVID-19 pandemic. The right to a birth partner should be protected, while addressing infection control. Since the declaration of the COVID-19 pandemic in early 2020, medical institutions worldwide have grappled with ways to provide the necessary medical care while protecting their patients and staff from the infectious disease. One difficult decision that hospitals have faced is whether to allow the accompaniment of birth partners  during labor and delivery.The World Health Organization (WHO) has issued a strong recommendation supporting women’s right to have a chosen companion during labor and delivery, even during the COVID-19 pandemic ,3,4,5,6.Despite the WHO’s strong recommendations, many countries have reported inconsistencies regarding the restrictions on birth partners. In a survey, around 33% of US women reported that they were not permitted to have a birth partner due to the pandemic . In a stOur study aimed to examine the level of access to birth partners for mothers who experienced labor and delivery during the pandemic in Japan. Additionally, the study aimed to identify obstetric and family function-related factors associated with the presence or absence of a birth partner. Given the importance of emotional and practical support for mothers during delivery, understanding the impact of COVID-19 on mothers’ access to birth partners has clinical and public health implications.This was a nationwide cross-sectional internet-based survey conducted as part of the Japan COVID-19 and Society Internet Survey (JACSIS). The study participants were sampled from the pooled panels of an internet research agency , which had approximately 2.2 million panelists registered as of 2019 . This stWe asked the women about their opinion during pregnancy on a partner-accompanied birth (“Did you wish to have a partner-accompanied birth during pregnancy?”) and their experience of whether they had a partner to accompany them during the birth (“Did your partner accompany you during the birth?”). In this study, partner-accompanied birth was defined as having a “partner in life,” regardless of marital status, accompany the labor and delivery process.Psychological distress was measured using the Japanese version of the Kessler Psychological Distress Scale (K6) ,18,19. TThe question regarding partner participation in housework and parenting was “Does your partner contribute to housework and parenting?” The response options were “always,” “sometimes,” “not very much,” and “not at all.” The responses were categorized into “always” or “not always.”satogaeri shussan in Japanese ; and mode of delivery .The covariates were selected based on previous studies on this topic ,10,22. TP < 0.05 was considered statistically significant. All analysis was performed using Stata version 15.1 .Descriptive analysis was performed to summarize the distribution of study participants’ basic characteristics. The percentage of women who had the intention to give birth accompanied by their partner and who had actually done so was calculated per month (between January 2019 and August 2021) to describe the trend over time. The number of participants who delivered between January and June 2019 and August 2021 was small. Thus, we grouped them as those who delivered between January and July 2019 and between July and August 2021. Further, the associations between partner-accompanied birth and postpartum outcomes  and factors associated with partner-accompanied birth were examined using multivariable Poisson regression models with adjustment for the aforementioned covariates. We used Poisson regression models because the prevalence of the outcomes exceeded 10% . For theThis study was approved by the Bioethics Review Committee of Osaka International Cancer Institute, Japan (20084). All procedures followed the ethical guidelines for medical and health research involving human subjects enforced by the Ministry of Health, Labor, and Welfare, Japan. Informed consent was obtained electronically before proceeding to the survey, through which all participants were informed that they could withdraw at any time during the study. Their data were collected anonymously and their confidentiality strictly protected. As an honorarium for study participation, the participants received credit points (“Epoints”) after completing the questionnaire.Of the 5605 women included in the analysis, 14.9%, 48.4%, and 36.7% delivered between January 2019 and March 2020, April and December 2020, and January and August 2021, respectively . The proThe proportion of women who wished for partner-accompanied birth did not change over the observational period, with an average of 71.2% between January 2019 and March 2020 and 78.1% between April 2020 and August 2021 . HoweverA K6 score ≥ 10 was observed in 14.2% and 13.2% of women who had their partners accompanying them at birth and those who did not, respectively . The adjDaily housework and parenting by the partner was reported by 52.5% and 50.5% of women who had their accompanying partner at birth and those who did not, respectively . The adjFactors associated with partner-accompanied birth are presented in Our study examined mothers’ level of access to a partner-accompanied birth during the pandemic in Japan and identified obstetric and family function-related factors associated with the presence or absence of a birth partner. Our results identified that partner-accompanied birth rates declined to 32.1% after April 2020 and remained similarly low through our data collection period ending August 2021. Partner-accompanied births were significantly associated with (1) partners’ daily participation in housework and parenting, (2) the birth being their first , and (3) births at smaller facilities rather than larger hospitals. Further, the rate of partner-accompanied births was negatively associated with mothers giving birth in their region of origin, where their own parents may reside. There was no significant change in the rate of partner-accompanied births in relation to (1) timing during the pandemic or (2) the infection risk within the region . Additionally, partners’ daily participation in housework and parenting was associated with fewer reports of maternal psychological distress.Only 32.1% of laboring mothers were allowed partner-accompanied childbirth during the pandemic in Japan. Previous research has documented that pregnant women greatly benefit from the presence of a support person of their choice during labor and childbirth to provide physical, emotional, and psychological support. Furthermore, there is strong evidence of better maternal and fetal outcomes when birth partners are present. While infection control is extremely important, it is also vital to balance other potential consequences , in thisRegarding the balance of infection control and the consequences of infection prevention measures, COVID-19 positivity rates within the community should be considered. Our results found that partner-accompanied births remained low throughout the assessed time frame (April 2020 to August 2021) and did not change with location or timing. Despite the declining rates of COVID-19 positivity after the pandemic’s start  and befoAnother factor associated with higher rates of accompanied births was partner involvement in housework and parenting. Our study documented that partner involvement was associated with fewer reports of maternal psychological distress. Japan has ranked 89 out of 189 countries in the Women’s Workplace Equality Index , and theOur results show that pregnant women who returned to their region of origin for childbirth had lower rates of partner-accompanied birth. It is unclear whether these women had other support persons present, such as their families. We hypothesized that when pregnant mothers returned to their region of origin, their partners remained in their regular residence, which could have posed a geographical challenge.Partner-accompanied birth was not associated with suspected maternal psychological distress, although previous studies have reported that women without partner-accompanied births showed a high prevalence of psychological distress ,33. A poTo our knowledge, this is the first study to describe the trend of partner-accompanied birth across Japan before and during the COVID-19 pandemic using data from a large sample and to demonstrate that partner-accompanied birth was strongly restricted by the pandemic. However, our study has some limitations. First, we limited our assessment of accompaniment at birth to only partners and did not examine the accompaniment of other support persons, such as parents or friends. Second, our study did not include policy changes regarding partner-accompanied birth at the facility level over the observational period, which could have directly affected the change in partner-accompanied birth rate. However, we adjusted for facility type and mode of delivery in the analysis, and our findings partially explained variations of the partner-accompanied birth policy by facility type or mode of delivery. Third, the data collection survey time frame for some responders was inconsistent, which may have affected memory and experience recall. Fourth, the study findings may have been affected by access to the internet, and participation in the study may have been affected by mental health or domestic problems. Finally, our findings do not explain causal relationships due to the cross-sectional design and should be carefully interpreted.Our study documented the significantly and consistently decreased rates of partner-accompanied birth during the COVID-19 pandemic in Japan, regardless of the level of community infection risks. While infection control is extremely important, it is also vital to balance it with potential consequences of the infection control measures themselves. A woman’s access to trusted emotional, psychological, and practical support is associated with reductions in various negative childbirth outcomes, and depriving them of such support yields consequences. The WHO continues to strongly recommend respecting the woman’s right to a chosen companion during labor and delivery, even in the pandemic. Considering the consistently low levels of partner-accompanied births in Japan during the pandemic, it could be advised that the regulation surrounding accompaniment in hospitals be more flexibly managed in response to the infection risks of the community and individual.


We aimed to investigate child mortality, perinatal morbidities and congenital anomalies born by women with substance misuse during or before pregnancy (DP or BP).Taiwan Birth Registration from 2004 to 2014 linking Integrated Illicit Drug Databases used to include substance misuse participates. Children born by mothers convicted of substance misuse DP or BP were the substance-exposed cohort. Two substance-unexposed comparison cohorts were established: one comparison cohort selected newborns from the rest of the population on a ratio of 1:1 and exact matched by the child’s gender, child’s birth year, mother’s birth year and child’s first use of the health insurance card; another comparison cohort matched newborns from exposed and unexposed mothers by their propensity scores calculated from logistic regression.The exposure group included 1776 DP, 1776 BP and 3552 unexposed individuals in exact-matched cohorts. A fourfold increased risk of deaths in children born by mothers exposed to substance during pregnancy was found compared to unexposed group : 2.07–9.97]. Further multivariate Cox regression models with adjustments and propensity matching substantially attenuated HRs on mortality in the substance-exposed cohort . Raised risks of perinatal morbidities and congenital anomalies were also found.Increased risks of child mortality, perinatal morbidities or congenital anomalies were found in women with substance use during pregnancy. From estimates before and after adjustments, our results showed that having outpatient visits or medical utilizations during pregnancy were associated with substantially attenuated HRs on mortality in the substance-exposed cohort. Therefore, the excess mortality risk might be partially explained by the lack of relevant antenatal clinical care. Our finding may suggest that the importance of early identification, specific abstinence program and access to appropriate antenatal care might be helpful in reducing newborn mortality. Adequate prevention policies may be formulated. According to the United Nations Office on Drugs and Crime report, over 280 million people aged between 15 and 64 used at least one illicit drug in the past year, accounting for 5% of the population  database was consolidated in 2015 to the Health and Welfare Data Science Center (HWDSC) of the Statistics Department of the Ministry of Health and Welfare in Taiwan government. The IIDD including police records for substance misuse contained information of crime records, Birth Registration, Household Registration, National Health Insurance Databases, National Mortality Database and the other dozen databases. After integration, the HWDSC provided the research team with a de-identified secondary data for on-site analysis. All data or ID numbers were encrypted, making sure that no access to any personal identifiable information could be gained. Besides research team performed de-identified secondary data analysis under the strict third-party organization’s supervision (HWDSC), all individual data could not bring out the HWDSC. As for the reliability of using criminal records as a proxy measure of substance exposure, research has shown that approximately 50% of incarcerated individuals, including those sentenced for non-drug-related offenses, are believed to meet the diagnostic criteria for drug abuse or dependence . Written consent from the study participants was waived because the data were collected from a population-based database of de-identified secondary data. Taiwan Food and Drug Administration, Department of the Ministry of Health and Welfare approved this study to publish by an official document.n = 3552) for subsequent comparisons.This study used Birth Registration and Birth Notification files from 2004 to 2014 and linked to the IIDD that contained police records to identify mothers transferred by police for substance misuse DP or BP. Children born to these mothers were the substance-exposed cohort. A non-substance-exposed comparison cohort was established selecting children from the rest of the population on a ratio of 1:1 and matched by the child’s gender, child’s birth year, mother’s birth year and child’s first use of the health insurance card. To make the characteristics from DP, BP and the unexposed three groups similar for comparisons, and more importantly, we wanted to compare DP vs. the unexposed and BP vs. the unexposed group in the same regression model, a second-stage exact match selecting subjects with similar characteristics from the substance-exposed DP and BP groups to later compare with their exact-matched counterpart was established. For instance, in n = 4156) for subsequent comparisons of the risk of mortality and other outcomes between PS-matched exposed and unexposed cohorts. Children without any use of the health insurance card were excluded.However, since there were many other parameters of individual characteristics that might be possible confounders, such as mother’s different comorbidities or different medical utilizations  in the exposed and unexposed cohorts. Adverse health outcomes included any of the following congenital anomalies: congenital heart diseases , congenital spinal cord or other nervous  system defects and perinatal morbidity .et al., et al., et al., et al., et al., et al., The PS was calculated by including covariates of the child’s gender, child’s birth year, the year of child’s first use of health insurance card, mother’s age at giving birth, birth orders  and the method of delivery . Model 2 adjusted for covariates in Model 1 and fifth minute APGAR score, premature birth and low birth weight. Analyses were performed by exact-match cohorts and PS-matched cohorts, respectively.From 2004 to 2014, there were 1,969,040 newborns in the Birth Registration file, which were then linked to the IIDD that contained police transfer records. There were 18,285 mothers of newborn babies caught by police because of substance misuse between years 2004 and 2014 .Among the 18,285 newborns from the substance-exposed cohort, 2,078 and 8,772 newborns were born by mothers exposed to substance DP or BP, respectively. Numbers of 10,850 newborns from unexposed mothers were selected and 1:1 matched by child’s gender, birth year, first use of the health insurance card and mother’s birth year. After the second exact matching, 1,776 subjects from each two substance-exposed subgroups (DP or BP) and 3,552 subjects from the unexposed group were selected. Results from the exact match are shown in p < 0.001, p = 0.023), perinatal morbidities (p < 0.001), congenital anomalies (p = 0.009), including congenital heart (p = 0.027) and nervous system defects (p = 0.027), prematurity (p < 0.001) and low birth weight . Causes of death were mainly accidents and injuries , followed by unknown causes (ICD-10: R00-R99), and morbidities of the perinatal period or congenital malformations .The mortality rate in the DP group , 8.83–22.33] per 10,000 person-years) was similar with that in the BP group  but higher than the unexposed group  during the follow-up period among the exact-matched cohorts. Significant differences were found comparing child mortality, perinatal morbidities, congenital anomalies, prematurity and low birth weight among the exact-matched cohorts. The highest rates were all shown in the subgroup of children born by mothers with substance-exposure DP  and PS-matched unexposed groups . NewbornThe novelty of this study is the use of a large nationwide population-based cohort data to compare risks of child mortality, congenital anomalies and perinatal morbidities in children born by mothers with or without heroin, amphetamine and ketamine exposures during vs. before pregnancy. The exact and propensity-matched comparisons provided further adjustments for more potential confounders than past studies. Hence, main additions of this study to existing evidence were the long-term approach to follow-up on outcomes of mortality or other developmental diseases using a large population-based representative cohort data, the inclusion of more substances, comparisons between DP vs. BP, DP vs. unexposed and BP vs. unexposed, and more covariates with strict matching methods to adjust for potential confounders that have not been able to be controlled before. Results from exact and PS-matched comparisons revealed elevated risks of mortality and perinatal morbidities among mothers who used substances DP and/or BP. A higher risk of congenital nervous system defects was found among substance-exposure DP mothers than the exact-matched unexposed group. Adjustments or PS-matching by mother’s social economic status, birth years, age at birth, comorbidity index, antenatal outpatient visits or medical expenses substantially attenuated the HRs on mortality in the substance-exposed cohort.et al., et al., et al., et al., et al., et al., et al., et al., We found excess mortality and perinatal morbidities in children born by mothers with substance exposures DP after PS-matching (and not in the exact-matching comparisons). This finding was consistent with previous research reporting higher mortality in fetus or children born by mothers exposed to opioid, heroin, amphetamine, methamphetamine or methadone compared to that from the general population (Fang et al., et al., Compared to results from exact-match controls, the elevated risk of mortality decreased from 4-folds to 1.6-folds in the PS-matched cohorts. Such risks remained within similar ranges after further adjustments. Since the PS-matched cohorts controlled for more potential confounders than the exact-matched cohort, the statistical power of PS-matched cohort analysis is larger than the exact-matched analysis. Our finding therefore suggested that when comparing mothers with more similar comorbidities, psychosocial conditions or antenatal medical utilizations after PS-matching, the risk of death would not be overestimated. It may also indicate that improving mother’s access to clinical attention or prenatal care may help decrease child mortality. Literature has reported that pregnant amphetamine users were significantly less likely to receive antenatal care than the general population (Smith et al., et al., et al., et al., et al., et al., et al., We reported raised perinatal morbidities in children born by mothers with substance use DP or BP compared to exact-matched counterparts. However, after PS-matching and added low birth weight, preterm births and fifth minute APGAR score into the adjustment model, substance use DP no longer lead to significantly increased risks of perinatal morbidities. This finding may suggest the importance of recognizing and managing risk factors for prematurity and low birthweight. Literature has consistently reported that amphetamine or heroin exposures DP were associated with premature delivery or lower birth weight compared to general newborns (Hulse After adjusting for fifth minute APGAR score, premature birth and low birth weight, significantly increased risk of perinatal morbidity was found only in the subgroup of mothers with substance exposure BP and not those with DP. It is possible that the raised risk of perinatal morbidities in these children was also more likely to be related to mother’s domestic stress, social or economic conditions or even the maternal physical or psychiatric care in prison during gestation, which we were not able to control for. Adequate interventions or programs for such psychosocial conditions after the identification of pregnant substance users may be suggested.et al., et al., In our results, although higher risk of congenital spinal cord or other nervous system defects was found among children born by mothers with substance exposure DP compared to their exact-matched counterparts, no significantly higher risks of any congenital heart or brain anomalies were found when comparing PS-matched groups. Such finding was in line with previous research concluding that intrauterine substance exposure–induced teratogenic risks were not significant (Oei This large population-based cohort study found that exposures to substances DP or BP were associated with increased risk of child mortality, congenital anomalies and perinatal morbidities. Major strengths of this study included careful linkages to several nationwide databases large enough to provide sufficient statistical power. The identification of mothers having substance use was from police records and courts’ sentences with clear confirmation of substance abuse being convicted. Two stages of exact-match and PS-matched multivariate regression models not only supplemented further adjustments of potential confounding variables but also provided more similar distributions on characteristics among the three cohorts. Comparisons of both the exact match and PS-matching in our study demonstrated that PS-matching did not over-pair potential confounders or mediators.et al., Key limitations would be the lack of other risk factors that were not available in the dataset, such as smoking status, alcohol use or other residual confounders associated with substance use and mortality. Therefore, the possibility and impacts of prenatal polysubstance misuse should still be considered. Second, since the study sample was drawn from police criminal records, it is possible that these substance users only accounted for a small percentage of real users. Being transferred by police for substance misuse can be an indicator of substance exposure to some degree, but it is important to keep in mind that it is not a comprehensive or definitive measure of an individual’s substance use patterns, amount or frequency, the aspects of exposure, level of dependence or overall health and wellbeing. Also, law enforcement data may not capture the full extent of substance use in the population, particularly among those who do not come into contact with the criminal justice system (Hser In our study, we found elevated risks of child mortality, perinatal morbidities and congenital anomalies born by mothers exposed to substances DP. Such findings may be partially explained by intrauterine substance exposures, as well as mothers’ demographic, psychosocial conditions or degree of prenatal medical care. The worrying phenomenon indicates the need of early identification, specific abstinence program and access to appropriate antenatal care. Provisions of health promotion programs on educating women with substance use ways of protecting maternal and securing fetal physical conditions are also required.


Neonatal intraventricular hemorrhage prevention bundles for preterm infants commonly defer daily weighing for the first 72 h, with reweighing occurring on day 4. Clinicians rely on maintaining stable sodium values as a proxy of fluid status to inform fluid management decisions over the first 96 h after birth. Yet, there exists a paucity of research evaluating whether serum sodium or osmolality are appropriate proxies for weight loss and whether increasing variability in sodium or osmolality during this early transitional period is associated with adverse in-hospital outcomes.To evaluate whether serum sodium or osmolality change in the first 96 h after birth was associated with percent weight change from birth weight, and to assess potential associations between serum sodium and osmolality variability with in-hospital outcomes.This retrospective, cross-sectional study included neonates born at ≤30 gestational weeks or ≤1250 g. We evaluated associations between serum sodium coefficient of variation (CoV), osmolality CoV, and maximal weight loss percentage in the first 96 h after birth with in-hospital neonatal outcomes.R2 = 0.01–0.14). For every 1% increase in sodium CoV, there was an associated 2-fold increased odds of surgical necrotizing enterocolitis and 2-fold increased odds of in-hospital mortality . Sodium CoV was more strongly associated with outcomes than absolute sodium maximal change.Among 205 infants, serum sodium and osmolality were poorly correlated with percent weight change in individual 24-h increments (In the first 96 h, serum sodium and osmolality are poor proxies for assessing percent weight change. Increasing variability of serum sodium is associated with later development of surgical necrotizing enterocolitis and all-cause in-hospital mortality. Prospective research is needed to evaluate whether reducing sodium variability in the first 96 h after birth, as assessed by CoV, improves newborn health outcomes. Intraventricular hemorrhage (IVH) remains an important and potentially preventable cause of long-term neurocognitive morbidity in preterm infants. Strategies to reduce the risk of IVH during the most vulnerable 72 h after birth frequently include midline positioning and minimizing handling, including deferring daily weights . With deAlthough correlation between serum sodium and weight loss has been established for older children and adults, complex fluid and electrolyte shifts occur as preterm infants transition from fetal-to-neonatal physiology, and these shifts may impact the reliability of electrolytes as proxies for hydration status . Moreoveabsolute change in serum sodium (the difference between sodium at 2 specific time points) and IVH [1) is serum sodium a reasonable proxy for weight loss in the first 96 h after birth, and 2) which correlates best with adverse health outcomes, absolute change or CoV of serum sodium values? The University of Washington Neonatal Intensive Care Unit (NICU) implemented an IVH Prevention Bundle in 2018 that first included daily weights, followed by a period in which daily weights were not obtained for the first 3 d after birth. Using a retrospective cross-sectional analysis of this population, we now address these 2 questions.Studies have demonstrated an association between  and IVH . The coeA retrospective cross-sectional study was performed including 205 neonates born ≤1250 g or ≤30 wk gestation and admitted to the University of Washington Level IV NICU in Seattle, Washington between February 8, 2018 and March 19, 2021. Infants with congenital anomalies, diagnosed genetic conditions, and those who did not survive the first 96 h after birth were excluded from all analyses. Institutional Review Board approval was received from the University of Washington (Study 00006091). The study was performed in accordance with the Declaration of Helsinki (revised 2013).n = 109). The percent weight change and change in serum sodium and osmolality were measured daily.An IVH Prevention Bundle was implemented in 2 phases, the first with and the second without daily weighing. From February 8, 2018 to October 31, 2019, the bundle included daily weights in the first 72 h after birth; between November 1, 2019 and March 19, 2021, the IVH Prevention Bundle did not permit weighing until 96 h after birth. To determine the association between percent weight change and serum osmolality or serum sodium, data from all neonates with daily weight measurements and serum biomarkers recorded on days 1 through 4 after birth were utilized  , any neclateral) ,21, NEC Infants diagnosed with IVH within the first 96 h after birth were excluded from analyses assessing IVH as an outcome as our current unit protocol is to obtain the first cranial ultrasound at 7–10 d after birth; any cranial ultrasound performed earlier was per individual clinician’s decision. Common reasons for doing off-protocol early head ultrasounds at our institution include, but are not limited to: severe hypotension, unexplained acute anemia, increasing vasopressor requirements, or to guide discussions about redirection to comfort care; thus, we reasoned this was a sicker, atypical group and they were excluded. Infants who did not live long enough to be diagnosed with clinical outcomes of interest were excluded from that specific analysis. Thus, infants who did not survive 1 week after birth were excluded from IVH  and NEC analyses; infants who did not survive to 36 wk postmenstrual age were excluded from PDA and BPD analyses. Only infants who were discharged home were included in analyses examining length of stay .At our institution, it is protocolized to provide minimal amounts of sodium-containing fluids within the first 48–72 h after birth. Starting on day 2, 1–2 mEq/kg of sodium is provided via parenteral nutrition. Umbilical arterial lines commonly use sodium acetate and are routinely used in infants <28 wk gestation, starting after birth. No other parenteral or enteral supplementation is provided during this period.U tests for categorical and continuous variables, respectively. Linear regression models were used to predict percent weight change for each 24-h period during the first 96 h after birth, using either change in serum sodium or serum osmolality as a predictor after adjusting for gestational age (GA), small-for-gestational-age (SGA) status  [Demographic variables for those exposed to IVH bundles with and without daily weighing were compared by Fisher’s exact tests and Mann-Whitney nal age) , vasopreTo control for potential known confounders, we utilized logistic regression models to predict categorical in-hospital outcomes using either serum sodium CoV, osmolality CoV or absolute maximal sodium change over the first 96 h after birth. Models were adjusted for the following potential confounders: GA, whether the infant was weighed in the first 0–72 h, SGA status, vasopressor use in the first week after birth, average daily urine output within 96 h after birth, average daily fluid intake within 96 h after birth, weight loss <5% within 96 h after birth, weight loss >15% within 96 h after birth, lowest sodium <135 mEq/L or lowest osmolality <280 mEq/L within 96 h after birth, highest sodium >145 mEq/L or highest osmolality >300 mEq/L within 96 h after birth, and highest glucose within 96 h after birth . GlucoseThese variables were selected because of their known association with alterations in serum sodium values, GA , SGA 2727, urine1) serum sodium CoV, 2) osmolality CoV, and 3) absolute maximal sodium change with the length of hospital stay after adjusting for GA, daily weighing, SGA status, vasopressor use in first week after birth, average urine output within 96 h after birth, average fluid intake within 96 h after birth, weight loss <5% 0–96 h after birth, weight loss >15% 0–96 h after birth, lowest sodium <135 mEq/L or lowest osmolality <280 mEq/L within 96 h after birth, highest sodium >145 mEq/L or highest osmolality >300 mEq/L within 96 h after birth, and highest glucose in the first 96 h after birth.Linear regression models evaluated the association of the To investigate the optimal thresholds for absolute maximal sodium change and sodium CoV, the cutpointr library in R was used to identify thresholds that provided the highest sum of sensitivity and specificity to predict mortality . These tP) level of less than 0.05 was considered significant.Statistical analyses were performed in Prism version 9.2.0 (GraphPad Software) and R version 4.1.1  . A proban = 109) and without (n = 96) daily weights are presented in Infants included in the IVH Prevention Bundle with (R2 = 0.12). Urine output was significantly associated with percent weight change from day 2 to day 3 . Fluid intake, urine output, and maximum glucose were significantly associated with percent weight change from day 3 to day 4 after birth . GA, vasopressor use, and SGA were not significantly associated with percent weight change in any 24-h period.The percent weight change associated with change in serum sodium and osmolality for sequential 24-h intervals after birth is shown in R2 = 0.14). Associations between the other variables and percent weight change in the osmolality models were similar to those seen in the sodium models.Similar to changes in serum sodium, changes in serum osmolality were only significantly associated with percent weight change on day 2 to 3. For every increase of 1 mEq/L of serum osmolality, there was an associated increase in percent weight loss by 0.18% in the adjusted model , and other life-saving procedures altering and confounding serum electrolyte values. The adjustments within our regression models may also limit clinical applicability at this time, and we strongly recommend future, prospective studies seeking to reproduce our findings and seek understanding of clinical generalizability. Secondly, the study was retrospective and observational; we did not have an a priori sample size calculation, but will use these results to inform future, prospective studies. This study design prevents any inference of causality, and all conclusions are only associations. However, these associations provide insight into the need for more rigorous research into this transitional period and if any fluid management, nutritional, or feeding strategies can improve overall newborn health outcomes.In conclusion, serum sodium and osmolality values were only weakly associated with weight change over the first 96 h after birth in our cohort of newborns born at ≤30 wk gestation or with ≤1250 g birth weight and, therefore, were poor proxies for weight loss. However, increasing serum sodium variability, as measured as CoV, in the first 96 h after birth was associated with greater odds of adverse in-hospital health outcomes including development of surgical NEC and all-cause in-hospital mortality in this same preterm population. Our study’s findings further highlight that in a head-to-head evaluation with absolute maximal sodium change, sodium CoV is a more robust measure of sodium variability with stronger associations with all adverse health outcomes herein studied. Ultimately, our study’s findings do not support the practice of using serum sodium or osmolality as proxies for weight loss or to determine fluid management decisions. Although preventing severe weight loss is a priority, our findings highlight the importance of tight regulation of sodium values to prevent significant variation. Further research is needed to evaluate the impact of mitigating significant fluctuations in serum sodium, as measured by CoV, in the first 96 h after birth on preterm in-hospital morbidities.The authors reported no funding received for this study.The authors report no conflicts of interest.


Occludin (OCLN) is an important tight junction protein and has been reported to be abnormally expressed in the development of malignant tumors. However, its biomarker and carcinogenic roles in kidney renal clear cell carcinoma (KIRC) are less investigated.The Cancer Genome Atlas database and Human Protein Atlas database were used to analyze the expression of OCLN in KIRC. UALCAN database and methylation-specific PCR assay were used to evaluate the methylation level of OCLN in KIRC. Univariate and multivariate Cox regression analyses were performed to model the prognostic significance of OCLN in KIRC patient cohorts. The correlation between OCLN expression and the immune cell infiltration, immune-related function and immune checkpoints were explored. Finally, EdU, scratch assay and transwell experiments were conducted to validate the role of OCLN in KIRC development.in vitro.The expression of OCLN was significantly downregulated in KIRC, compared with normal renal tissues (p<0.001). Patients with low OCLN expression showed a worse prognosis and poorer clinicopathological characteristics. Functional enrichment analysis revealed that OCLN was mainly involved in biological processes such as immune response, immunoglobulin complex circulating and cytokine and chemokine receptor to mediate KIRC development. Immune-related analysis indicated that OCLN could potentially serve as a candidate target for KIRC immunotherapy. OCLN overexpression inhibited proliferation, migration and invasion of KIRC cells in vivo experiments will be conducted to decode its molecular mechanism in KIRC carcinogenesis in the future work.OCLN was validated as a candidate prognostic biomarker and therapeutic target of KIRC based both on computational and experimental approaches. More  The incidence of renal cell carcinoma (RCC) is rising gradually and it accounts for approximately 3% of all cancer types . The cauin vitro and in vivo  cohort and pathological sample through histological staining, OCLN was found to be one of the potential biomarkers for diagnosis and prognosis of KIRC. Immune-related analysis indicated that OCLN could serve as a potential target for immunotherapy in KIRC. The As shown in http://timer.cistrome.org/). The mRNA sequencing data including 542 KIRC specimens and 72 normal renal tissue specimens and the corresponding clinical information were downloaded from TCGA database on Mar, 23rd, 2023 . As illustrated in https://www.proteinatlas.org/).The expression of OCLN between tumor and adjacent normal tissues was analyzed by TIMER2  . Moreover, a stratified analysis based on patients’ age, gender, tumor grade, clinical stage, and N stage was performed.The OCLN promoter methylation level between KIRC and normal renal tissues was analyzed in the UALCAN database  (http://https://cn.string-db.org/).The functional enrichment analysis of differentially expressed genes (DEGs) was performed at the gene ontology  (GO) andUnivariate and multivariate Cox regression analysis were conducted using “survival” R package to evaluate the association between OCLN expression and KIRC prognosis. In addition, a nomogram was constructed by the “rms” R package  and “reghttp://kmplot.com/) databases were used to analyze the relationship between the expression of OCLN and OS in KIRC patients in different immune cell subsets. “ESTIMATE” was used to calculate the stromal score and immune score for KIRC. The relationship between stromal/immune score and OCLN expression was analyzed, the median value was set as the dividing line of high and low score. ssGSEA analysis was performed using the “GSEABase” and “GSVA” R packages to obtain scores for immune-related functions, and the scores were calibrated to compare differences in immune-related functions between high and low OCLN expression groups. The correlation between the OCLN expression and the expression level of immune checkpoint‐related genes was further analyzed.“CIBERSORT” package  was usedProteins were extracted from KIRC and adjacent normal renal tissues by RIPA lysate  supplemented with proteinase inhibitor. Proteins were loaded into an SDS-PAGE gel, and then transferred into a PVDF membrane. The membrane was blocked with 5% skim milk for 2h at room temperature and then incubated overnight with the corresponding primary antibody  at 4°C. Finally, the membrane was incubated using horseradish peroxidase-conjugated secondary antibodies and exposed by an enhanced chemiluminescence (ECL) kit (Beyotime).2O2 at room temperature for 15 minutes and then washed in PBS. Next, 5% BSA was incubated for 1 h at 37°C to block nonspecific binding sites. The sections were incubated with primary antibodies  overnight at 4°C. IHC was performed by SP Rabbit & Mouse HRP Kit (DAB) .The paraffin-embedded specimens were cut into 4 µm thick section for immunohistochemical staining. All sections were dewaxed with xylene and then rehydrated in gradient alcohol. The sections were incubated with 3% H2 in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin solution . According to the manufacturer’s protocol, the plasmids were transfected into 769P cells using lipofectamine_2000 transfection reagent . After 48 h of transfection, RIPA buffer  was used to extract total proteins.The human KIRC cell line (769P cells) were maintained at 37°C in a humidified atmosphere containing 5% COThe EdU assay kit was purchased from Beyotime Company . According to the manufacturer’s protocol, transfected cells were treated with EdU reagent for 3h. Then, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with 0.3% Triton X-100 for 15 min at room temperature. Finally, cells were stained with fluorescent dye and Hoechst. The Nikon TI2-D-PD inverted microscope (Japan) was used to capture the images.μL of pipette tip was used to scratch the cell plate and the cells were washed by PBS. Then, cells were continued to be cultured with 1% fetal bovine serum  at 37°C for 24h. The photographs were taken at 0h and 24h. For the transwell assay, after 48 hours of transfection, 5×104 cells were seeded in a 24-well Transwell chamber  with a layer of Matrigel matrix glue  (matrix glue: Serum-free medium=1:6). After 36h, the cells in the upper chamber were fixed with 4% paraformaldehyde for 20 min at room temperature and stain with crystal violet  for 20 min. The Nikon TI2-D-PD inverted microscope (Japan) was used to capture the images.For the wound healing assay, after 48 hours of transfection, 20 5-Azacytidine was purchased from MedChemExpress Company . 769P cell were seeded into six-well plates at appropriate density the day before dosing. After 48h of addition of different concentrations of 5-Azacytidine, the cells were used for subsequent processing. TRIzol  was used to extract the total RNA from 769P cells. Then, the HiScript III RT SuperMix for qPCR (+gDNA wiper)  was used to perform the reverse transcription reactions. 2X Rapid Taq Master Mix  was used to perform gene amplification followed by PCR products added to a 1% agarose gel for electrophoresis. The procedure for protein extraction and western blot were the same as above. The detailed primer sequences are as follows:GAPDH forward:5′- TCGACAGTCAGCCGCATCT′ and Reverse: 5′-CTAGCCTCCCGGGTTTCTCT-3′;OCLN Forward: 5′- ACAAGCGGTTTTATCCAGAGTC′ and Reverse: 5′- GTCATCCACAGGCGAAGTTAAT-3′After 48h treatment with 5-Azacytidine, genomic DNA was extracted from 769P cells with the FastPure Cell/Tissue DNA Isolation Mini kit , according to the manufacturer’s instructions. Next, the DNA(1μg) was treated with EpiArt Magnetic DNA Methylation Bisulfite kit following manufacturer’s instructions . The methylated and unmethylated Twist were amplified using 2xEpiArt HS Taq Master Mix . The detailed primer sequences were as follows: Methylated: 5′-TTTAAGGTTTTATTCGAAGTAGGC-3′(forward), 5′-GTTACGACCCGAAAAACGAA-3′(reverse); the unmethylated: 5′-TTAAGGTTTTATTTGAAGTAGGTGG-3′(forward), 5′-ACATTACAACCCAAAAAACAAA-3′(reverse). The PCR products were separated in a 1.5% agarose gel.All data were analyzed using R software (v4.2.2). Univariate and multivariate Cox regression analysis were used to identify independent prognostic factors. All experiments were independently performed at least three times. All statistical analyses were conducted using GraphPad Prism 9.0 . The Student’s t-test was used to analyze the differences between two groups, and p<0.05 was considered to be statistically significant.By TIMER database mining, the expression level of OCLN was obtained from a pan-cancer perspective. As shown in The correlation between OCLN expression and corresponding clinicopathological features was analyzed to explore the clinical role of OCLN in KIRC. As shown in To further explore the underlying mechanism of OCLN low-expression in KIRC, DNA methylation analysis was performed. As known, DNA methylation is an important event in the epigenetic modification of the genome and is closely related to disease process. The DNA methylation levels of OCLN for KIRC were tested using the UALCAN database. As shown in To further evaluate the prognostic value of OCLN in KIRC, univariate and multivariate Cox regression were performed. The univariate Cox analysis indicated that OCLN expression (p<0.001), age (p<0.001), grade (p<0.001), and stage (p<0.001) were significant factors for overall survival of KIRC patients  experiments were performed respectively to validate the expression of OCLN in clinical samples. As shown in Moreover, 769P cell line was selected for follow-up functional experiments to confirm the results of bioinformatics analysis. As shown in Kidney cancer is the third most common urinary malignancies, and the clear cell carcinoma is often observed as a typical pathological subtype. Surgical resection is the ideal method for treatment of KIRC patients, unfortunately a great number of patients are suffered from tumor recurrence after surgery , 31. Curin vitro experiments showed that the expression of OCLN increased significantly when the methylation level in KIRC cells was inhibited. The low expression of OCLN was associated with tumorigenesis and progression of KIRC. In the TCGA-KIRC clinical cohort, the expression level of OCLN was negatively related to tumor grade, clinical stage, T stage and M stage. In addition, KIRC patients with lower OCLN expression had a poorer prognosis. Univariate and multivariate analysis showed that OCLN was an independent prognostic factor. In vitro experiments showed that OCLN overexpression inhibited the proliferation, migration and invasion of KIRC cells. Therefore, these findings indicated that OCLN may act as a tumor suppressor for KIRC.OCLN is known to be presented in all the tight junctions. The expression of OCLN in KIRC was significantly lower than that in normal renal tissues in this study. Generally, the downregulation of tumor suppressor genes expression may be due to transcriptional repression, promoter methylation, or increased protein degradation. Mo et al. demonstrated that calprotectin subunit S100A9 can semimethylate the promoter of OCLN in melanoma cells, resulting in reduced expression of OCLN . In thisin vitro and in vivo (To investigate the underlying molecular mechanism of OCLN in KIRC carcinogenesis, an OCLN-related protein-protein interaction network was constructed and the correction analysis was performed. Based on network modeling, a total of 14 genes were highly relevant to OCLN, and some of them have been reported to be strongly associated with the development of KIRC. For example. Li et al. demonstrated that CLDN7 expression was reduced in KIRC, and overexpression of CLDN7 inhibited the proliferation, migration and invasion ability of KIRC cells in vivo . It coulThe tumor microenvironment is composed of tumor cells, a variety of stromal cells, cytokines, chemokines, etc. –38. It iin vivo experiments. Second, the mechanism between OCLN expression and the infiltration of immune cells was not well explained. More clinical and pathogenic experiments will be conducted in the future work to investigate the carcinogenesis of OCLN during KIRC evolution.In summary, this study deciphered the role of OCLN in KIRC clinical characteristics, prognosis, immunotherapy. OCLN could be considered as a potential biomarker for KIRC personalized medicine. It should be concerned that several limitations still need to be considered. First, the conclusions were not confirmed by This study showed that OCLN was downregulated in KIRC, and the low OCLN expression was associated with poor prognosis in KIRC. Moreover, the expression of OCLN was associated with immune cell infiltration and could act as a novel potential biomarker and therapeutic target for KIRC prognosis and immunotherapy. Further experiments need to be conducted to explore the molecular mechanisms of OCLN in KIRC.The original contributions presented in the study are included in the article/The studies involving humans were approved by the ethics committee of The First Affiliated Hospital of Soochow University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.ZJ, YK, and CW contributed equally to this study. YL and YH designed and supervised the study jointly. ZJ, ZF, ZT, YS and YL performed and validated the bioinformatics analysis. ZJ, YK, CW and YH designed the experimental scheme and performed the experiments. ZJ, YK, CW, YL and YH drafted and revised this manuscript. YL and YH provided support for publishing funds. All authors contributed to the article and approved the submitted version.


Under-five mortality remains a global public health concern, particularly in East African countries. Short birth interval is highly associated with under-five mortality, and birth spacing has a significant effect on a child’s likelihood of survival. The association between short birth intervals and under-five mortality was demonstrated by numerous observational studies. However, the effect of short birth intervals on under-five mortality has not been investigated yet. Therefore, this study aimed to investigate the impact of short birth intervals on under-five mortality in East Africa using Propensity Matched Analysis.A secondary data analysis was conducted based on the most recent Demographic and Health Survey (DHS) data of 12 East African countries. A total weighted sample of 105,662 live births was considered for this study. A PSM analysis was carried out to evaluate the effect of short birth intervals on under-five mortality. Under-five mortality was the outcome variable, while the short birth interval was considered a treatment variable. To determine the Average Treatment Effect on the population (ATE), Average Treatment Effect on the treated (ATT), and Average Treatment Effect on the untreated (ATU), we performed PSM analysis with a logit-based model using the psmatch2 ate STATA function. The quality of matching was assessed statistically and graphically. The common support assumption was checked and fulfilled. We have employed Mantel-Haenszel bounds to examine whether the result would be free from hidden bias or not.The prevalence of short birth intervals in East Africa was 44%. The under-five mortality rate among mothers who had optimal birth intervals was 39.9  per 1000 live births while it was 60.6  per 1000 live births among mothers who had a short birth intervals. Propensity score matching split births from mothers into treatment and control groups based on the preceding birth interval. In the PSM analysis, the ATT values in the treated and control groups were 6.09% and 3.97%, respectively, showed under-five mortality among births to mothers with short birth intervals was 2.17% higher than births to mothers who had an optimal birth interval. The ATU values in the intervention and control groups were 3.90% and 6.06%, respectively, indicating that for births from women who had an optimal birth interval, the chance of dying within five years would increase by 2.17% if they were born to mother with short birth interval. The final ATE estimate was 2.14% among the population. After matching, there was no significant difference in baseline characteristics between the treated and control groups , which indicates the quality of matching was good.We conclude that enhancing mothers to have optimal birth spacing is likely to be an effective approach to reducing the incidence of under-five mortality. Our findings suggest that births to mothers with short birth intervals have an increased risk of death in the first five years of life than births to mothers who had an optimal birth interval. Therefore, public health programs should enhance interventions targeting improving birth spacing to reduce the incidence of under-five mortality in low-and middle-income countries like East African countries. Moreover, to achieve a significant reduction in the under-five mortality rate, interventions that encourage birth spacing should be considered. This will improve child survival and help in attaining Sustainable Development Goal targets in East African countries. Under-five mortality is the probability of dying before five years of age, which is the key global indicator of child health . DespiteThe World Health Organization (WHO) currently recommends an interval between the last live birth and the next pregnancy of at least 24 months, a birth interval of 33 months . Birth iChild survival is significantly influenced by birth spacing, and under-five mortality is substantially correlated with birth spacing that is not optimal . NumerouThere is limited empirical evidence on the causal impact of preceding birth intervals on perinatal, infant, and child mortality. The adverse consequences of a short interval for infant and child survival and maternal mortality and morbidity have been attributed to the biological effects related to the “maternal depletion syndrome” or more generally the woman not fully recuperating from one pregnancy before supporting the next one , 17.As advocated by the WHO, optimal birth spacing is one of the most effective strategies to reduce child mortality in low-and middle-income countries . ExtensiPropensity Score Matching (PSM) is a methodological technique that aimed to remove bias by matching treated ) and un-treated ) pregnant mothers with similar conditional probability to receiving the treatment (SBI). In this study, we matched the mothers with OBI to mothers with SBI with similar propensity score values for birth intervals , 22. TheA secondary data analysis was done based on the most recent Demographic and Health Survey (DHS) data of the 12 East African countries conducted from 2012 to 2021. DHS is a community-based cross-sectional study conducted in a five-year interval to generate updated health and health-related indicators.The DHS survey of 12 East African countries  was used for the current study. The total sample size at the East African level was 105,721. Only mothers who had a previous history of birth were included in this study.The outcome variable was under-five mortality. It was coded as “0” if the child was alive and “1” if the child died within the first 59 months of age. Pregnant mothers who had a previous history of birth were considered for this study (primigravida mothers were excluded). The treatment group is those mothers who had a short birth interval and the control group is those who had an optimal birth interval. According to the WHO recommendation, the birth interval was divided into two categories: “short birth interval,“ defined as less than 33 months, and “optimal birth interval,“ defined as 33 months and above. Many maternal pre-intervention characteristics have been included in the model as it ensures a better chance that the propensity score matching assumption holds. Variables that affect short birth intervals and under-five mortality but which are not affected by the treatment (SBI) were included. Variables such as residence, country, and mother’s level of education, household wealth status, sex of household head, marital status, number of birth before the current birth, maternal age, and media exposure were considered for matching.In randomized control trials, study participants are assigned to either the treatment or control group through randomization that controls both known and unknown confounders between the groups (no systematic difference between groups). whereas, because researchers cannot randomly assign study participants to either of the groups in observational studies, the imbalance of the observed variables introduces bias and affects the exposure’s causal effect. In situations where confounding variables can be measured, we can adjust for and treat the imbalance between groups. A function of the observed covariates so-called the balancing score can be used to correct the imbalance between the groups. Based on the balancing score, the observed variables should be independent of the assignment of the treatment i.e. short birth interval or optimal birth spacing.The propensity score method is commonly used to balance the inequality of confounding variables in observational studies. After adjusting for the observed covariates using propensity scores, the difference in outcomes between the child born to mothers with short birth intervals and the child born to mothers with optimal birth intervals will be an unbiased estimate of the effect of short birth intervals on under-five mortality. A propensity score is the likelihood that a pregnant woman received a treatment  given all the observed covariates. It is a conditional probability of receiving treatment (SBI) and thus always has a value between 0 and 1. The larger the propensity score, the more likely a woman is to have SBI. The treatment variable of interest must be dichotomous in a propensity score analysis.Propensity score analysis usually starts with an assessment of the imbalance of the baseline covariates between the treatment and control groups. This can be assessed by significance tests like an independent t-test for continuous variables and chi-square for categorical variables.In the propensity score model, the exposure variable is considered a treatment variable, and the dependent variable is considered an outcome variable. Based on the relationship between treatment and outcome variables, observed covariates can be categorized into three groups: covariates only related to treatment assignment; covariates related to both treatment assignment and outcome ; and covariates only related to outcome. However, PSM only can be included. The likelihood of the woman having SBI based on the selected confounders is reduced to a propensity score for each woman. This propensity score is generated for each subject from the selected confounders. Since the treatment variable of interest is dichotomous, the common methods adopted to produce propensity scores are either logistic regression or discriminant analysis.Propensity score matching was used to assess the impact of short birth intervals on under-five mortality. The PSM is a statistical technique that aims to address the primary drawback of causal inference in observational research designs when randomization cannot be used to establish the treatment and control groups. This approach involves forming matching sets of control and treatments of individuals whose propensity scores are similar. After a matching sample has been established, the effect of SBI can be assessed by comparing under-five mortality directly between SBI and OBI women in the matched sample.teffects psmatch STATA command). Besides, we used a t-test to access the balance for all covariates before and after matching, with a 5% level of significance or more considered indicative of imbalance.The PSM approach was employed to compare under-five mortality among births with optimal birth intervals and births with short birth intervals. The PSM approach was chosen because the birth interval was not randomly assigned to both groups and can be affected strongly by observable and non-observable variables. Variables such as socio-demographic and obstetrical-related variables, which have a significant association with birth interval and under-five mortality were selected as PSM variables. Births with short birth intervals were matched to births with optimal birth intervals using logit regression  showed significant differences (p < 0.05) in under-five mortality between the SBI and OBI groups. After matching, the abovementioned variables showed a non-significant difference  in under-five mortality between mothers with SBI and mothers with OBI, suggesting that PSM significantly reduced the between-group difference in the observed characteristics.iT be under-five mortality for those ith birth with short birth interval (treatment group), and AiC denotes under-five mortality for mothers with optimal birth intervals. The observed outcome can be written as iT- AiC ) and our interest is to estimate the average effect of treatment (SBI) on the treated (ATT), We aim to estimate the average effect of short birth intervals on the treated. Assume Aocument} . This capstest command to assess the covariate balance, we tested the following matching methods: nearest neighbor matching with and without replacement, and radius matching with calipers from 0.01. The psmatch2 command was used to generate the ATT, ATU, and ATE for the matching method that produced the highest quality of matches. The common support option was also employed to produce higher-quality matches.Overall variables for matching were selected before the treatment . The commonest assumptions of PSM such as common support and selection on unobservable were assessed graphically and statistically. During analysis, the common support option was considered to limit the balancing propensity to only mothers with treatment (SBI) whose propensity score for under-five mortality lay within ranges of propensity scores for controls. Using the The quality of matching was evaluated based on the balancing of the covariates between the treated and control groups. Firstly, the quality of matching was assessed by computing the standardized bias before matching and after matching. This bias is computed as the percentage difference.2 and Likelihood ratio test of the joint insignificance of all the covariates from the logit estimation of the conditional treatment probability before and after matching.The bias is computed as the percentage difference of the sample means in the treated and matched control groups as a percentage of the square root of the average of the sample variances in both groups. Though no hard and fast rule exists on the level of standardized difference that would indicate an imbalance, a difference of less than 10% is taken to indicate a negligible difference. Secondly, the pseudo Rmhbounds STATA command [A sensitivity analysis was conducted to test the robustness of the PSM estimates . As the  command .http://www.dhsprogram.com. There are no names of individuals or household addresses in the data files.Since the study was a secondary data analysis of publically available survey data from the MEASURE DHS program, ethical approval, and participant consent were not necessary for this particular study. We requested DHS Program, and permission was granted to download and use the data for this study from A total of 105,721 pregnant mothers who had a previous history of birth in 12 East African countries were included in this study. Of them, 46,489 (44%) of the mothers had a short birth interval. Under-five mortality among mothers who had optimal birth interval was 39.9  per 1000 live births whereas it was 60.6  per 1000 live births among mothers who had a short birth interval. All the baseline characteristics showed significant differences (p < 0.05) across the birth interval category (OBI vs. SBI) before matching  showing the improvement of quality matching in the model. The median bias reduced from 14% in the unmatched to zero after matching (Table 2 and LR tests. The pseudo-R2 was < 0.001 and the LR-test had become insignificant (p = 0.25), showing that there was no systematic difference in covariate distribution between the treated and control groups  Table .In public health, healthcare decision-makers attempted to assess how the public health interventions among the treated populations have changed. This study investigated the impact of short birth intervals on under-five mortality using propensity score matching analysis. This approach is one of the best approaches to assess the impact of a certain intervention in observational studies by constructing an adequate comparison group since randomization can’t be applied.Previous studies found that short birth interval was a significant predictor of under-five mortality and they have reported a statistically significant association between birth interval and under-five mortality in East African countries. However, we tried to estimate the impact of short birth intervals on under-five mortality and overcome the concern of selection on the unobservable. Women with short interbirth intervals were socio-economically, demographically, and birth-related characteristics are different at baseline than women who had an optimal birth interval. Therefore, conducting a standard regression analysis of under-five mortality between women with SBI and women with OBI would yield biased estimates of the impact of short birth interval on under-five mortality and the result would not be adjusted for endogeneity bias.As to our review of literature, we found that existing literature showed that no study practically attempts to remove the selection bias/adjust at baseline to rectify the causal inference of short birth interval which leads to under-five mortality. Therefore, this study is the first study that attempts to quantify the effects of short birth intervals on under-five mortality using propensity scores after removing the possible selection bias.Using PSM we found that births to mothers with short birth intervals had a 2.12% higher risk of death in the first five years of life compared to those births to women who had an optimal birth interval. Furthermore, for those births born to mothers who had an optimal birth interval, if they would have been born to mothers with short interbirth intervals, their chances of mortality in the first five years would have been increased to 2.17%.The effects of short interbirth intervals on under-five mortality can be attributed to the fact that short birth intervals are associated with increased preterm birth, low birth weight, and small gestational age births, which in turn contribute to under-five mortality. The WHO recommends a healthy pregnancy gap of at least two years (24 months) , which eEven though this study offers important insight into the effect of birth interval on under-five mortality, the result should be interpreted in light of the following limitations. The matching was done based on the observed variables, there is a possibility of residual confounding. Moreover, DHS is a cross-sectional study and it’s prone to social desirability and recall bias. Despite the abovementioned limitations, the study has the following strengths. First, this study is based on nationally representative DHS data with a high response rate. Secondly, DHS uses a standardized questionnaire for the data collection which is consistent across all 12 countries. Furthermore, this study is the adjustment for potential confounders using the PSM approach in the estimation of the association between birth interval and under-five mortality.The short interbirth interval was found significant risk factor for under-five mortality in East African countries. Births to mothers with short birth intervals had a higher chance of dying in the first five years of life. These findings highlight that community and healthcare-based interventions that target enhancing optimal birth spacing should be prioritized and scaled up in East African countries to reduce the burden of under-five mortality in East African countries.


A hydatid cyst is a parasitic infection that can occur in multiple organs of the body, mostly in the liver. The ovary is one of the very rare locations for those cysts.The authors report a case of a 43-year-old woman with a primary hydatid cyst, the patient presented with left lower quadrant abdominal pain for 2 months. Ultrasound of the abdomen showed evidence of a multivesicular, fluid-containing cystic lesion in the left adnexa. The mass was excised and a hysterectomy with total left salpingo-oophorectomy was performed. Histopathology confirmed it to be a hydatid cyst.The clinical presentation of an ovarian hydatid cyst can differ, ranging from asymptomatic for years to dull pain if it compresses on the neighbouring organs or tissues, it may even cause a systemic immunological reaction if it ruptures.Cyst excision when possible is the best treatment, percutaneous sterilization techniques, and drug therapy may also be applied in certain cases. HighlightsOvaries are rare places for hydatid cysts, yet a hydatid cyst should be among the differential diagnosis for a fluid-filled cyst on the ovary, especially in endemic areas.Hydatid cysts can mimic an ovarian cyst or a tumour and thus can lead to bad complications during excision in case it ruptures or in the case of a wrong diagnosis.Radical excision when possible can lead to great results and can prevent the possibility of rupturing which can lead to a deadly allergic reaction and can also decrease the recurrence possibility.1.This article has been reported in line with the SCARE criteria2.Echinococcosis is a zoonosis, that mammals form its intermediate host, humans can be accidental hosts as they do not play a role in the natural cycle. Dogs are the main definitive host. Hydatid cyst disease occurs through infection with the larval stages of the taeniid cestodes of the genus Echinococcus or through ingestion of free eggs that are found in food (unwashed vegetables), water, and infected soi. Those eggs are passed in the faeces of infected dogs and then ingested by humans. The larva then penetrates the intestinal wall and migrates via the bloodstream to all parts of the body3.The liver is the most common location of hydatid cysts, and the second most common organ is the lungs3.About 10% of the cysts can appear in unusual body sites. The ovary is one of the very rare locations for the cysts that causes ovarian hydatid disease (HD). It is morphologically similar to other locations and can occur primarily or secondary. Usually, the secondary is more common and is associated with liver or lung echinococcosis, or in the context of multiorgan hydatid cysts3.Ovarian HD can stay asymptomatic for years but it can become symptomatic when a cyst compresses on the neighbouring organs or tissues, and the most common presentation is abdominal or pelvic pain4.Imaging has an important role in the diagnosis and staging of the disease, imaging techniques include ultrasound which is one of the most common imaging modalities and can be used for the following process after treatment also, besides ultrasound we have computed tomography and MRI that can be useful when ultrasound does not show the cysts clearly or surgical treatment is required4.Aside from imaging diagnosis, different serological tests have been introduced to diagnose hydatid cystsIn this paper, we report a rare case of a woman with an ovarian hydatid cyst, that was diagnosed and treated successfully in the obst&gyn hospital at the University of Aleppo.A 43-year-old woman presented to the hospital clinic with left lower quadrant abdominal pain increasing during menstruation for the last 2 months. There were no associated symptoms. She is a smoker but non-alcoholic, she does not take any medication, and her familial history has nothing important.Her past menstrual history was normal, she is gravida 8 and para7. No significant weight loss was reported lately. There has a history of a lipoma mass removal in the right flank. During the examination, the patient was not pale or jaundiced. No palpable organs or masses were detected. Laboratory investigations revealed normal liver and kidney functions with normal serum CA-125 and CEA. Ultrasound of the abdomen showed normal size and thickness of the uterus, and evidence of a multivesicular, fluid-containing cystic lesion in the left adnexa (9.5×8.5×4 cm approximately) while the right adnexa, liver, spleen, and kidneys were all normal , which all were normal.During the surgical operation, the mass did not show the properties of a normal cyst. So, a hysterectomy with total left salpingo-oophorectomy was performed Fig. . The his5.Echinococcosis is a zoonotic disease caused by tapeworms. It is divided into two forms: cystic echinococcosis, which is caused by Echinococcus granulosus sensu lato, and alveolar echinococcosis, which is caused by Echinococcus multilocularis5. The main risk factors for contracting the disease are contact with infected dogs and raising livestock5.The disease is prevalent in certain areas, including western China, Central Asia, South America, Mediterranean countries, and eastern Africa6. The clinical presentation for hydatid cysts can differ a lot ranging from asymptomatic for years to inducing systemic immunological reactions if it ruptures7. The onset of symptoms is related to the cyst’s size, position, organ, and correlation with adjacent tissues7.HD is caused by the larval stage of Echinococcus granulosus, and it can develop in almost any part of the body. The liver is the most common location for hydatid cysts, accounting for 75% of cases. The lungs are the second most common site, accounting for 15% of cases. The remaining 10% of cases occur in other areas of the body, such as the brain, cavernous sinus, submandibular gland, thyroid gland, heart, pleura, chest wall, retrocrural tissue, kidney, spleen, pancreas, peritoneal cavity, inguinal canal, breast, bone, and in some cases, the ovaries8.As mentioned in our case, an ovarian hydatid cyst may mimic an ovarian tumour, which can lead to unnecessary procedures. Therefore, a precise diagnostic method is crucial. The gold standard method is abdominal ultrasonography, which has a sensitivity of 93%. Computed tomography may also be used. Other available diagnostic methods include serological tests, such as IgG against the parasite antigen with ELISA (sensitivity 95%) and the indirect hemagglutination test9.Treatment options for hydatid cysts include surgery, which is the first choice according to the WHO, even though it does not prevent recurrence9.However, chemotherapy with benzimidazole compounds  and the recently developed PAIR procedure (puncture-aspiration-injection-re-aspiration), which destroys the cyst’s germinal layer, offer additional treatment options for CE cases. It is worth mentioning that chemotherapy is not a satisfactory cure, as only about 30% of patients can expect a cure, and 30–50% can expect improvement after 12 months of follow-up7.In some cases, it may be reasonable to monitor the cyst rather than immediately pursuing treatment. This is because a proportion of cysts can calcify over time and become completely inactiveHydatid cyst should be considered among the differential diagnosis of a well-circumscribed ovarian cyst on echography, especially in endemic areas. It can be primary or secondary and can be asymptomatic for years. It is usually misdiagnosed as an ovarian cyst or an ovarian tumour and it can be well treated through radical excision and drug therapy. It should be followed to diagnose any recurrence. Careful excision is a must especially when an expected ovarian cyst does not show the properties of a normal cyst during surgery, as the rapture of the cyst can cause a dangerous allergic reaction.Not applicable.Written informed consent was obtained from the patient for the 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.The authors declare no source of funding for this manuscript from any organization nor any institution.H.S., H.B., Z.M. and T.J.A.-H. wrote the manuscript. M.T., K.K. and R.Z.did the surgical operation and reviewed the manuscript. A.M.A.-H. reviewed and supervised the manuscript.The authors want to declare no conflict of interest.None.Kinda Kellawi.All data underlying the results are available as part of the article and no additional source data are required.Not commissioned, externally peer-reviewed.