Is there evidence for an association of antenatal corticosteroid treatment during the late-preterm period with an improvement in twins’ neonatal outcomes?In this cohort study, antenatal corticosteroid treatment of 1974 individuals with twin pregnancies and at risk for late preterm birth did not significantly reduce the risk of neonatal respiratory morbidity as well as other neonatal complications.The evidence is insufficient that antenatal corticosteroid treatment during the late-preterm period in twin pregnancies is associated with a lower risk of newborn morbidity. Antenatal corticosteroid treatment of individuals with singletons at risk for delivery during the late-preterm period has been academically recommended. However, the evidence on the use of antenatal corticosteroid treatment for twins at risk for delivery during the late-preterm period is still lacking.To evaluate whether antenatal corticosteroid treatment during the late-preterm period in twin pregnancies was associated with a lower risk of newborn morbidity.This retrospective cohort study of twin pregnancies delivered from February 1, 2013, to September 30, 2020, in a university-affiliated hospital in China included 1974 individuals with twin pregnancies who were at risk for late preterm birth (34 weeks and 0 days to 36 weeks and 6 days of gestation). Data were analyzed from June 30 to July 13, 2023.Antenatal corticosteroid treatment during the late-preterm period.The primary outcome measure was composite neonatal respiratory morbidity, defined as at least 1 of the following postnatal occurrences in at least 1 neonate of the twins: respiratory distress syndrome, mechanical ventilation, surfactant administration, transferred with respiratory complications, or neonatal death. Propensity score overlap weighting was used to analyze the association between antenatal corticosteroid treatment and the risk of neonatal outcomes.The study population consisted of 1974 individuals with twin pregnancies, including 303  who received antenatal corticosteroid treatment and 1671  who did not receive antenatal corticosteroid treatment. The propensity score overlap weighting showed no significant differences between the antenatal corticosteroid treatment group and the no–antenatal corticosteroid treatment group in the risk of neonatal primary outcome . None of the subgroup interaction tests were significant for the neonatal primary outcome in terms of gestational age at delivery, year of delivery, chorionicity, at least 1 infant small for gestational age, intertwin growth discordance, and infant sex, and neither was the sensitivity analysis of using propensity score matching and a different administration-to-birth interval and treating twin infants as individuals.This cohort study found insufficient evidence that antenatal corticosteroid treatment during the late-preterm period in twin pregnancies could be associated with a lower risk of newborn morbidity. This new finding can provide a reference for clinical practice. This cohort study evaluates whether antenatal corticosteroid treatment during the late-preterm period in twin pregnancies is associated with a lower risk of newborn morbidity. Only a few cohort studies, which were characterized by small sample sizes, unmatched comparisons, and great heterogeneity, have been reported. The results showed that the use of antenatal corticosteroid treatment was not consistently associated with an improving neonatal respiratory outcome.13 Therefore, the correlation between antenatal corticosteroid treatment for twins at risk of late-preterm birth and neonatal outcomes remains an unanswered question.Antenatal corticosteroids are generally administered to pregnant women at risk for premature delivery between 24 weeks and 0 days and 33 weeks and 6 days of gestation during both singleton and twin pregnancies to accelerate fetal lung maturation and decrease the incidence of neonatal mortality and morbidity.To better assess the association between antenatal corticosteroid treatment during the late-preterm period and neonatal outcomes of twin pregnancies, we conducted a retrospective cohort study of 2705 twin pregnancies. We used propensity score overlap weighting to make the maternal and neonatal baseline information comparable. Furthermore, to ensure the robustness of the findings, we performed subgroup analysis on potential interactions with the specific variables, including chorionicity, at least 1 infant small for gestational age (SGA), intertwin growth discordance, and infant sex, and we performed sensitivity analysis using propensity score matching and a different administration-to-birth interval and treating twin infants as individuals.STROBE) reporting guideline for cohort studies. The study was approved by the Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University, and the requirement for informed consent was waived because the data were deidentified.The retrospective cohort involved all individuals whose twin pregnancies were delivered from February 1, 2013, to September 30, 2020, at the Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China, a tertiary hospital. The individuals had been identified from the institution’s electronic medical record system, and their electronic records were extracted for the relevant data on maternal characteristics, pregnancy outcomes, neonatal outcomes, and antenatal corticosteroid treatment. Gestational age (GA) was calculated based on the last menstrual period, adjusted by the crown-rump length in the first trimester or the head circumference of the larger fetus with fetal ultrasonographic examination after 14 weeks of gestation in the case of spontaneous conception, as well as based on the timing of in vitro pregnancy fertilization conceived via assisted reproductive technology. Our study conformed to the Strengthening the Reporting of Observational Studies in Epidemiology (The inclusion criteria were as follows: (1) establishment of a prenatal medical record at our hospital and (2) receipt of the routine prenatal care offered by our hospital until delivery. The exclusion criteria were as follows: (1) GA of less than 34 weeks at delivery; (2) intrauterine death of 1 fetus; (3) major congenital abnormality or fetal chromosomal abnormality; (4) monochorionic monoamniotic twins; (5) chorioamnionitis or maternal fever; (6) complications unique to monochorionic twins, including twin-to-twin transfusion syndrome, twin reversed arterial perfusion sequence, or twin anemia-polycythemia sequence; (7) missing outcomes because of being transferred at birth without respiratory complications; or (8) antenatal corticosteroid treatment received before 34 weeks and 0 days of gestation .15 The high probability of delivery was defined as the spontaneous rupture of the membranes, intact membranes and at least 3 cm of dilation or 75% cervical effacement, or a preterm delivery expected for any other indication for which the attending physician decides to induce labor or perform cesarean delivery within 24 hours and 7 days. After birth, maternal characteristics and neonatal outcomes were compared between twins in 2 study groups: those who received at least 1 dose of dexamethasone from 34 weeks and 0 days to 36 weeks and 6 days of gestation and those who did not.The exposure was set as follows: at least 1 dose of antenatal corticosteroids was administered between 34 weeks and 0 days and 36 weeks and 6 days of gestation, in view of a high probability of delivery in the late-preterm period. The protocol for antenatal corticosteroid treatment comprises 4 intramuscular doses of 5 mg of dexamethasone 12 hours apart.The primary outcome measure was composite neonatal respiratory morbidity, defined as at least 1 of the following postnatal occurrences to either neonate: respiratory distress syndrome (at delivery), mechanical ventilation (within 72 hours of life), surfactant administration (at delivery), transferred with respiratory complications (at delivery), or neonatal death (within 72 hours of life). Transferred with respiratory complications was defined as having a poor respiratory condition, such as respiratory distress syndrome, for which the neonate was transferred to the local children’s hospital for further evaluation and treatment by neonatologists.The secondary outcomes were referred to as the use of continuous positive airway pressure (CPAP), the use of CPAP for more than 2 consecutive days, asphyxia, severe asphyxia, transient tachypnea, admission to the neonatal intensive care unit for any duration or for no less than 3 consecutive days, necrotizing enterocolitis, sepsis, hyperbilirubinemia, hyperglycemia, hypoglycemia, and acidosis. Hypoglycemia was defined as a glucose level of less than 40 mg/dL  tested at any time. The definitions of variables and outcomes are provided in eTable 1 in F test was performed for the comparison of quantitative data, and the χ2 test or the Fisher exact test was performed for the comparison of categorical data.Data were analyzed from June 30 to July 13, 2023. The baseline characteristics of the 2 study groups were compared. Continuous variables with normal distributions were presented as mean (SD) values, and categorical data were presented as numbers and percentages. The 10), advanced maternal age (≥35 years at delivery), primipara, assisted reproductive technology, gestational diabetes, hypertensive disorders during pregnancy, GA at delivery, preterm birth, mode of delivery , chorionicity (monochorionicity or dichorionicity), mean birth weight of both twin infants from the same mother, at least 1 infant SGA, intertwin growth discordance, and infant sex (the same or opposite).For adjusting between-group differences, propensity scores were developed to reflect the probability of each participant receiving antenatal corticosteroid treatment, with the use of multivariable logistic regression with exposure  as the dependent variable and all observed maternal-level and neonatal-level characteristics as the independent variables. The covariates covered baseline maternal sociodemographic and clinical factors, as well as neonatal characteristics, which were expected to be associated with neonatal outcomes. These covariates included year of delivery . Inverse probability of treatment weighting, standardized mortality ratio weighting, and overlap weighting, as well as the approach of PSM of 1 to 1, 2, 3, and 4 matching patients with the nearest neighbor matching algorithm , were performed to examine the association between antenatal corticosteroid treatment and neonatal outcomes. Standardized mean differences were used to select the optimal adjustment method. As the absolute standardized mean difference of all covariates was smaller than 10% to support the assumption of balance between study groups,We conducted 6 subgroup analyses of the primary outcome to examine the heterogeneity in clinically relevant subgroups: GA at delivery , year of delivery, chorionicity, at least 1 infant with SGA, intertwin growth discordance, and infant sex. The robustness of the results was examined via multiple sensitivity analyses. First, we performed 1:1 PSM as an alternative to overlap weighting because the absolute standardized mean difference of almost all covariates was smaller than 10% .A 2-tailed A total of 2705 individuals with twin pregnancies were eligible for the current retrospective analysis, of whom 731 met the exclusion criteria. Thus, the study population comprised 1974 individuals with twin pregnancies: 303  who received antenatal corticosteroid treatment between 34 weeks and 0 days to 36 weeks and 6 days of gestation and 1671  who did not receive antenatal corticosteroid treatment .P < .001). Also, several baseline characteristics were significantly different between groups, with the antenatal corticosteroid treatment group having a lower mean (SD) birth weight than the no–antenatal corticosteroid treatment group   in the antenatal corticosteroid treatment group delivered before 37 weeks and 0 days of gestation, while only 711 individuals (42.5%) in the no–antenatal corticosteroid treatment group did ( < .001) .After overlap weighting, the univariate logistic regression analysis showed no significant differences between the antenatal corticosteroid treatment group and the no–antenatal corticosteroid treatment group in the risk of neonatal primary outcome . Similar results were shown for the risk of being transferred with respiratory complications, respiratory distress syndrome, and surfactant administration. The risks of other neonatal complications, such as CPAP use, CPAP use for more than 2 days, asphyxia, severe asphyxia, transient tachypnea, hyperbilirubinemia, hyperglycemia, hypoglycemia, and acidosis, also did not differ significantly . SubgrouThe sensitivity analyses showed a similar result in terms of the association of antenatal corticosteroid treatment with neonatal outcomes after 1:1 PSM , or the confounding factors associated with antenatal corticosteroid choice.There has been considerable evidence supporting antenatal corticosteroid treatment during the late-preterm period for singleton pregnancies13 Instead of the conventional multivariable logistic regression previous studies used, we used the propensity score overlap weighting method to effectively eliminate the potential confounding factors, achieving the effect of randomized clinical trials to the greatest extent possible in retrospective studies.20 For outcome measures, based on earlier studies, such as the ALPS trial and the clinical realities of our center, we developed outcome indicators that could comprehensively reflect the association between antenatal corticosteroid treatment and neonatal outcomes among twins.11 As there are no randomized clinical trials on the effect of antenatal corticosteroid treatment in twin pregnancy to our knowledge, we believe that carefully designed observational studies can be of great help in treatment decision-making. Next, the results did not change significantly in the multiple subgroup and sensitivity analyses, showing that the results were robust enough to be convincing and reliable.To minimize the confounding factors in antenatal corticosteroid treatment of individuals with twin pregnancy, we implemented the following strategies. We took advantage of a cohort of 2705 individuals with twin pregnancies, which, to our knowledge, is larger than that of previously reported studies.24 Unlike the 2 previously reported investigations,13 our study aimed to explore the potential association of the administration-to-birth interval with outcomes; we found that antenatal corticosteroid treatment, regardless of the length of the interval, was not associated with improved neonatal respiratory outcomes, which suggested that our findings were not associated with the off-target phenomenon.As antenatal corticosteroids have shown the greatest benefit if the infant is delivered 24 to 48 hours after the initial dose and within 7 days of the administration, the off-target phenomenon of antenatal corticosteroid treatment refers to the absence of delivery within the 7 days after antenatal corticosteroid treatment.26 Although the beneficial association of corticosteroids with respiratory outcomes for early-preterm singleton infants is widely recognized, controversy still exists over their association with outcomes for early-preterm twin infants, with large-sample studies demonstrating no improvement in respiratory outcomes.28 Hence, the corticosteroid dosage currently in use is probably not optimal enough to promote fetal lung maturation in twin pregnancies.The lack of benefit associated with antenatal corticosteroid treatment in twin pregnancies may be attributed to the different pharmacokinetics of corticosteroids between twins and singletons. Corticosteroids are thought to have a shorter half-life and a higher clearance rate among twins, and the physiological changes in the maternal blood volume during twin pregnancy may affect drug distribution, all of which may lead, for twins, to lower effective therapeutic doses of corticosteroids that are intended to promote fetal lung maturation.Our study suggested that antenatal corticosteroid treatment for twin pregnancies at risk of late-preterm birth could not decrease neonatal respiratory morbidity but could increase the risk of neonatal hypoglycemia when administered 1 to 7 days prior to birth. Thus, antenatal corticosteroids should not be administered to those who do not have a high likelihood of preterm birth with twin pregnancies until more robust evidence is available. The current shortcomings in the use of antenatal corticosteroid treatment for twins should be properly addressed, such as the lack of data on optimal dosage, estimating preterm birth, and the long-term effect on multiple systems. Further randomized clinical trials are required to help determine optimal antenatal corticosteroid treatment recommendations for this special risk group of women.This study has several limitations. First, this cohort study performed a retrospective observational analysis. Although we used propensity score overlap weighting to balance the differences between groups, unmeasured and residual confounding could still exist. Second, although one of the primary outcome indicators was patient transferred with respiratory complications, the information on the prognosis of these newborns was inevitably lost, which increased the potential for bias. Third, due to the challenges in estimating the timing of delivery, only approximately 30% of the antenatal corticosteroid treatment group completed the full course of corticosteroids, which may have led to bias in the outcomes. Fourth, the findings need to be interpreted with caution because some neonatal outcomes, such as death in the late-preterm period, had a low incidence. Fifth, the cohort study spanned approximately 8 years, during which the changes in clinical practice, medical standards, and policy guidelines could have exerted a potential effect on the generalizability of the conclusion.In this cohort study, evidence was insufficient that antenatal corticosteroid treatment for a twin pregnancy during the late-preterm period could be associated with a lower risk of newborn morbidity. However, antenatal corticosteroid treatment 1 to 7 days before birth was associated with a higher risk of neonatal hypoglycemia. More well-designed prospective studies are required.


HLA class II molecules are key factors determining susceptibility to autoimmune disorders, and their role in immune-mediated skin conditions such as psoriasis has been extensively investigated. However, there is currently little understanding of their role in antibody-mediated skin diseases such as autoimmune blistering disorders. We researched the available literature using PubMed to narratively review the current knowledge on HLA associations in antibody-mediated blistering skin pathologies. Our results summarized the risk alleles that are identified in the literature, together with certain known protective alleles: in the pemphigus group, alleles HLA-DQB1*0503 and HLA-DRB1*0402 are most commonly associated with disease; in the pemphigoid group, the most studied allele is HLA-DQB1*0301; in epidermolysis bullosa acquisita, few genetic studies are available; in dermatitis herpetiformis, the association with haplotypes HLA-DQ2 and HLA-DQ8 is strongly established; finally, in linear IgA bullous disease, specific HLA alleles may be responsible for pediatric presentations. Our current pathogenic understanding of this group of disorders assigns a key role to predisposing HLA class II alleles that are able to bind disease autoantigens and therefore stimulate antigen-specific autoreactive T cells. The latter engage B lymphocytes that will produce pathogenic autoantibodies. The distribution of HLA alleles and their disease associations are variable across demographics, and an in-depth pathogenetic understanding is needed to support associations between HLA alleles and disease phenotypes. Additionally, in a personalized medicine approach, the identification of HLA alleles associated with the risk of disease may become clinically relevant in identifying susceptible subjects that should avoid exposure to known triggers, such as medication, when possible. Human leukocyte antigen (HLA) molecules are transmembrane glycoproteins expressed on nearly all nucleated cells in the body. The HLA region is located on the short arm of chromosome 6 6p21), commonly referred to as the major histocompatibility complex (MHC). There are two major classes of HLA antigens: HLA class I  and HLA class II ; these HLA molecules exhibit a high degree of polymorphism, with numerous alleles at each specific locus. The primary function of these glycoproteins is to regulate the immune response; in particular, class I and class II MHC molecules present peptides to CD8 cytotoxic T cells and to CD4 T cells, respectively. The HLA system has also gained prominent importance in the field of human tissue transplantation, where molecular HLA typing is routinely performed to allow for adequate matching between donor and recipient and to prevent rejection [, commonlThe aim of our work is to review the current knowledge around HLA class II in antibody-mediated blistering cutaneous diseases, notably pemphigus group, pemphigoid group, dermatitis herpetiformis (DH), epidermolysis bullosa acquisita (EBA), and linear IgA bullous disease (LABD). To achieve this objective, PubMed was searched from inception to August 2023 using the terms “HLA” and “pemphigus” or “pemphigoid” or “dermatitis herpetiformis” or “epidermolysis” or “linear IgA disease”. Papers written in the English language were screened by the authors to retrieve results relevant to the topic of this review; moreover, the reference list of included papers was analyzed to identify additional results.HLA types play a crucial role in the development of pemphigus by facilitating the presentation of self-antigens to Th cells, activating humoral immunity, and generating self-antibodies. These antibodies disrupt intercellular adhesion, leading to the formation of erosions and blisters . The twoPV involves autoantibodies targeting desmoglein 3 (Dsg3) and, occasionally, desmoglein 1 (Dsg1) . The disPopulation studies uncovered further links between specific class II alleles and PV within distinct ethnic groups. HLA-DRB1*0402 is associated with PV in over 90% of Ashkenazi Jews, while HLA-DQB1*0503 is associated with non-Jewish populations. HLA-DRB1*1404 emerges as an important risk factor within Indo-Asian populations. Furthermore, investigations highlight an association between PV and non-classic HLA class Ib alleles . HLA g polymorphisms significantly associate with Jewish PV patients, while those of HLA-E—already implicated in multiple autoimmune conditions—are shared between Caucasian and Ashkenazi Jewish, potentially disrupting immune tolerance in these patients ,16,17. AIn PF, autoantibodies target Dsg1, while those targeting Dsg3 remain undetected. Consequently, PF primarily affects the skin, with mucosal surfaces having no involvement . EndemicPNP is a type of pemphigus disease often associated with both malignant and benign neoplasms, frequently of hematologic, and lymphoid origin . In PNP,PH is categorized as a clinical variant of pemphigus, merging the clinical attributes of DH with the immunopathological characteristics of pemphigus ,35. A caAP is marked by the presence of IgA autoantibodies targeting both desmosomal and non-desmosomal keratinocyte cell surface components. The predominant types within IgA pemphigus are subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN). In SPD, the autoantigen Dsc1 has been identified, whereas the antigen in the case of IEN is variable . AssociaAutoimmune blistering disorders belonging to the pemphigoid group are defined by the presence of pathogenic autoantibodies targeting agents of the epithelial basement membrane zone (BMZ)—dermal epidermal junction or epidermal stromal junction—of the skin and mucosae. When there is a clinical suspicion, a specific diagnosis is confirmed by histology and direct immunofluorescence studies and supported by the serum detection of pathogenic autoantibodies. Histology is significant for subepidermal blister formation associated with a dermal infiltrate of neutrophils and eosinophils. In current clinical practice, serologic studies based on indirect immunofluorescence and ELISA may be sufficient for diagnosis, especially when the clinical presentation is clearly suggestive. The HLA-DQβ1*0301 allele (HLA-DQ7) has been reported as a shared susceptibility gene for all subsets in the pemphigoid group . To suppBullous pemphigoid (BP) usually affects elderly subjects and is the most commonly observed autoimmune blistering disorder (AIBD), partly owing to the constant aging of the general population. Its management may be challenging owing to the fragility and frequent comorbidities of patients, which may limit therapeutic options and increase overall morbidity. The onset of disease is the result of an interaction between genetic predisposition and potential triggering factors, including certain drugs. The role of HLA in mediating predisposition to autoantibody-mediated skin blistering conditions is established in the literature, with HLA class II genes such as DR and DQ reporting high prevalence in subjects with BP . HLA-DQBThe definition of mucous membrane pemphigoid (MMP) encompasses a heterogeneous disease phenotype with two key features: a predilection for mucosal involvement and a scarring tendency, which explains its progressive course, leading occasionally to serious sequelae. This is particularly relevant for ocular cicatricial pemphigoid, where scarring affects the conjunctival surfaces, leading to severe vision impairment. Autoantibodies targeting different antigens of the BMZ are detected in MMP: BP180, laminin 332, and integrin beta4 subunit in ocular MMP. The HLA class II allele most studied in MMP is DQB1*0301, the association of which has been highlighted in Caucasian populations and in subsets of patients with ocular cicatricial pemphigoid ,52,53. APemphigoid gestationis (PG) is a rare though well-characterized dermatosis of pregnancy that usually develops in the later periods of gestation or postpartum. A cutaneous vescicobullous eruption is the result of IgG1 autoantibodies produced against BP180. The development of disease is related to HLA class II mismatch in the placenta, and specifically to maternal exposure to paternal tissue, being the placental genome of paternal origin . PG has EBA is a rare diagnosis, associated with a spectrum of clinical variants: a “classical” presentation consisting of an acquired, chronic, trauma-induced, subepidermal blistering disease; a BP-like presentation; and other variants with features reminiscent of linear IgA dermatosis or MMP ,58. The DH is a chronic, autoimmune, blistering skin disorder intrinsically connected to gluten sensitivity . CurrentLinear IgA bullous disease (LABD) is a rare autoimmune blistering dermatosis with linear IgA deposits along the basement membrane zone. It occurs after puberty, with most patients presenting after the age of 60. Clinically, it manifests with pruritic papules, vesicles, and bullae, usually distributed on extensor surfaces; mucosal involvement is common, with erosions or ulcers and, sometimes, mucosal scarring . In the The prominent involvement of HLA genes in the initiation of AIBDs is supported by the key role of autoreactive Th2 cells in the pathogenesis of this group of disorders, that is ultimately characterized by specific autoantibody production. Autoreactive T cells are considered crucial for disease initiation but are also detected in healthy individuals; therefore, blistering diseases may occur following an imbalance between Th2 responses against cutaneous antigens, which are associated with disease, and Th1 regulatory responses, which maintain immune tolerance and are dysfunctional in disease . AccordiGenetic associations of HLA genes with AIBDs have been reviewed to highlight alleles that are related to increased susceptibility or to protection from the development of these disorders . These aSubjects that carry susceptibility genes to autoimmunity are particularly exposed to developing AIBDs when exposed to triggering factors, such as certain medications. Therefore, the identification of HLA alleles associated with the risk of disease may become clinically relevant in a personalized medicine approach: specific genetic testing may allow for a better patient selection and avoid the exposure of predisposed subjects to trigger drugs whenever possible.Additionally, variability in HLA class II alleles is able to influence the quantity and specificity of autoantibodies that are produced in these disorders. An in-depth understanding of their pathogenesis is needed to support associations between HLA alleles and severe disease phenotypes. Finally, the onset of autoimmune comorbidities in subjects with AIBD and the possibility of simultaneous disorders mediated by two different antibodies may be dependent on the co-occurrence of multiple HLA risk alleles. This may explain an epitope spreading phenomenon, through which an antigen can be presented to the immune system by two different HLA class II molecules and trigger the secretion of two different autoantibodies .


Local formation and action of estrogens have crucial roles in hormone dependent cancers and benign diseases like endometriosis. Drugs that are currently used for the treatment of these diseases act at the receptor and at the pre-receptor levels, targeting the local formation of estrogens. Since 1980s the local formation of estrogens has been targeted by inhibitors of aromatase that catalyses their formation from androgens. Steroidal and non-steroidal inhibitors have successfully been used to treat postmenopausal breast cancer and have also been evaluated in clinical studies in patients with endometrial, ovarian cancers and endometriosis. Over the past decade also inhibitors of sulfatase that catalyses the hydrolysis of inactive estrogen-sulfates entered clinical trials for treatment of breast, endometrial cancers and endometriosis, with clinical effects observed primarily in breast cancer. More recently, inhibitors of 17beta-hydroxysteroid dehydrogenase 1, an enzyme responsible for formation of the most potent estrogen, estradiol, have shown promising results in preclinical studies and have already entered clinical evaluation for endometriosis. This review aims to provide an overview of the current status of the use of hormonal drugs for the major hormone-dependent diseases. Further, it aims to explain the mechanisms behind the -sometimes- observed weak effects and low therapeutic efficacy of these drugs and the possibilities and the advantages of combined treatments targeting several enzymes in the local estrogen formation, or drugs acting with different therapeutic mechanisms. In case of prostate cancer, although some US projections expect a decrease in number of cases by 2040 . Breastry 2023; . Endometry 2023; . Prostatry 2023; . The numry 2023; . A risinry 2023;  as is th by 2040 , other c by 2040 .Hormone-dependent diseases include also a number of non-cancerous conditions  like endIn women, large part of hormone dependent cancers, like endometrial, ovarian and a proportion of breast cancers, occur after menopause when ovaries have ceased to produce steroids. Therefore, these tumours depend on the local (intracrine) estrogen formation from inactive precursors of ovarian or adrenal origin, like dehydroepiandrosterone sulfate (DHEA-S), DHEA, androstenedione and estrone sulfate  acts as a 17-hydroxysteroid reductase and has a high catalytic efficiency for the conversion of estrone to the most potent estrogen, estradiol. The action of HSD17B1 is opposed by the oxidative enzyme HSD17B2 responsible for inactivation of estradiol back to estrone .Local/intracrine estrogen biosynthesis thus comprises two complementary pathways, the aromatase pathway from DHEA or androstenedione and the sulfatase pathway from estrone-S . HSD17B1These pathways are not only involved in the intracrine estrogen synthesis in women’s cancer, but they have roles also in prostate cancer, where estradiol can be formed from testosterone produced in Leydig cells or from circulating estrone-S and can, similarly to the situation in female cancers, stimulate proliferation of cancerous cells . Besidesin-vivo phase); however, such strategy in unlikely to be applicable in clinic since studies showed blocking one single OATP is not sufficient to inhibit steroid hormone uptake, but blocking simultaneously multiple OATPs will disturb the metabolic homeostasis and the protection against toxins  at the uptake/efflux level, for instance by interfering with the activity of OAT/OATPs  estrDrugs that are currently used for treatment of hormone-dependent diseases act at the pre-receptor and receptor levels. Among the molecules that act at the receptor level, the selective ER receptor modulator tamoxifen has a central role in the treatment of breast cancer. Tamoxifen was discovered by serendipity by  and was https://www.elenaendometriosisstudy.com/#!/) and it is in the preclinical phase for endometrial and breast cancer  and integrated with PubMed searches. We specifically focussed on breast, endometrial, ovarian, prostate cancers and endometriosis, whereas we will not further discuss uterine fibroids and PCOS since the role of the local steroids in these conditions is underexplored.In the next sections, we will elaborate on the importance of aromatase, the sulfatase pathways and the HSD17B1-2 redox balance in hormone dependent diseases. We will then continue with an overview of the clinical studies that target the local formation of estrogens. To this end, we searched the Clinical Trial Database from the U.S. National Library of Medicine  promoters and transcription factors  and exemestane .In total, 452 completed clinical trials based on the use of aromatase inhibitors for breast cancer are currently registered in the database Clinical Trials, and for 163 trials results are available, including those that have led to the approval of the third generation aromatase inhibitors in current clinical practice and are briefly discussed above. Since the clinical trials investigating the efficacy of aromatase inhibitors in breast cancer have been the focus of several recent reviews , and sinIn the last two decades, a number of trials were initiated that evaluated the effects of aromatase inhibitors in combination with other targeted drugs, with 153 trials registered in the database Clinical Trials, and 59 trials with results . Among tThere are 28 trials where an aromatase inhibitor has been evaluated in patients with endometrial cancer, nine have been completed and for four trials results are available .Clinical trial NCT00997373 evaluated Letrozole treatment (2.5 mg/day) as parallel assignment in 24 patients with grade 1 or grade 2 endometrial cancer 2–3 weeks before hysterectomy or repeat biopsy. This study explored changes at Ki-67 expression in tissue samples and found decreased expression of this proliferation marker after treatment with the aromatase inhibitor.Two trials chaired by Dr. Slomovitz explored the efficacy of combining an aromatase inhibitor with an inhibitor of the mTOR signalling. Phase 2, single group assignment trial NCT01068249  evaluateAnother phase 2 trial, NCT02657928 , investiAromatase inhibitors have been studied in ovarian cancer patients in 41 trials, seven completed, five terminated and for five trials, results are available . Phase 2Phase 2/3 trial NCT02101788  comparedThe phase 2 trial NCT02657928  investigPhase 2, single group assignment trial NCT02283658 aimed at evaluating the efficacy of Letrozole combined with the mTOR inhibitor Everolimus. In total, 19 out of the 20 recruited patients completed the trial and 47% (95%CI: 24-71) were free of disease at 12 weeks , 21% (95%CI 6-46) had a therapeutic response based on CA-125 level .Aromatase inhibitors were investigated also in patients with endometriosis, nine trials have been registered, seven completed and for one trial, NCT02203331, results are known . This raSix clinical studies investigated effects of aromatase inhibitors in prostate cancer patients and two were completed, but the results of these studies have not yet been published.STS is expressed in female and male reproductive organs  in postmenopausal women  and middh plasma .In tissues and cells that express estrone-S transporters from the OATP superfamily  and STS,In the last decades, a series of STS inhibitors has been synthetized and tested in cell based and animal models. The field of STS inhibitors has been covered by several reviews  and willBased on information available at the database Clinical Trials, inhibitors of the sulfatase have been tested in patients with breast cancer, endometrial cancer, and patients with endometriosis.In breast cancer the first phase 1 clinical trial with an STS inhibitor has been performed in 2003 and 2004 . FourteeAfter this first phase 1 clinical trial, seven trials were registered in Database Clinical Trials and two have been completed  and acceptable safety profile .Two phase 2 trials in breast cancer patients were prematurely terminated . The first trial started in 2011 and aimed to assess the short term intra-tumoral and peripheral effects of Irosustat administered for 14 days prior to surgery in postmenopausal women with newly diagnosed primary invasive ER positive breast cancer. This study terminated after recruiting only two patients due to the futility analysis. The second trial IPET investigated the effects of Irosustat on growth of breast cancer as a primary endpoint. Postmenopausal women with early, hormone sensitive, treatment naïve breast cancer received 40 mg of Irosustat once daily for 2 weeks. The effects of Irosustat were evaluated by PET scans (Positron Emission Tomography). Secondary endpoints were the assessment of pharmacodynamics profile and safety and tolerability of Irosustat. Thirteen patients were recruited but the study was unfortunately terminated prematurely due to challenging recruitment.Two clinical trials in endometrial cancer patients are registered in the database Clinical Trials  were executed with xenografts or transgenic animals expressing the human enzyme  and non-steroidal, where molecules mimic the steroid scaffold . In caseer cells . Furtherer cells . The thet cancer . Tumour t cancer . More rerpowered .in-vivo models of various conditions. Husen et al. induced breast cancer by subcutaneous injection of MCF7 cells in mice. Animals were ovariectomised and estrone with or without the inhibitor was delivered using osmotic minipumps. The authors observed a reduction in breast cancer growth of over 80% .Only the C15 estrone derivative developed by Organon Finland, former Forendo pharma (compound FOR-6219/OR-6219) reached the clinical phase for endometriosis with three clinical trials registered in the database Clinical Trails . Phase 1Targeting the local generation of steroid hormones represents a novel and promising approach for hormone dependent conditions as it is now clear that a large part of the hormones that sustain disease conditions are generated locally from inactive precursors and via the use of alternative routes that are not targeted by current medications. For instance, DHEA-S is a depot for local androgen synthesis in prostate cancer patients also after hormone therapy using 17a-hydroxylase-17,20-lyase (CYP17A1) inhibitors , and STSThe development of resistance to hormone-deprivation therapy is a major concern, and combination of drugs instead of monotherapy can extend the sensitivity period. Studies have also shown that inhibition of aromatase can increase the expression and activity of STS in breast cancer . TherefoHormonal drugs require effective hormone signalling, and other intracellular signalling pathways (such as Akt-PI3K-mTOR) can compromise the response to hormonal drugs by taking over the proliferative actions . In thisAn important aspect of the use of hormonal drugs is that not all patients may be responsive, especially (but not only) in the field of oncology. In this context, it is becoming increasingly clear how to predict a response to hormonal drugs in patients, which will help to target the treatments to responsive patients only .For the future, full characterisation of disease features by immunohistochemistry, and molecular analyses or biomarkers is needed to stratify patients based on expression of the drug target, likelihood of response to treatment and risk of recurrence and medical treatment.Drugs targeting hormone action have been introduced decades ago, but identification of new drug targets, dual action drugs, combination with other targeted treatments can provide benefit to the cancer patients and patients with other non-oncological but still distressing conditions.


Polycystic kidney diseases (PKDs) are genetic disorders characterized by the formation and expansion of numerous fluid-filled renal cysts, damaging normal parenchyma and often leading to kidney failure. Although PKDs comprise a broad range of different diseases, with substantial genetic and phenotypic heterogeneity, an association with primary cilia represents a common theme. Great strides have been made in the identification of causative genes, furthering our understanding of the genetic complexity and disease mechanisms, but only one therapy so far has shown success in clinical trials and advanced to US Food and Drug Administration approval. A key step in understanding disease pathogenesis and testing potential therapeutics is developing orthologous experimental models that accurately recapitulate the human phenotype. This has been particularly important for PKDs because cellular models have been of limited value; however, the advent of organoid usage has expanded capabilities in this area but does not negate the need for whole-organism models where renal function can be assessed. Animal model generation is further complicated in the most common disease type, autosomal dominant PKD, by homozygous lethality and a very limited cystic phenotype in heterozygotes while for autosomal recessive PKD, mouse models have a delayed and modest kidney disease, in contrast to humans. However, for autosomal dominant PKD, the use of conditional/inducible and dosage models have resulted in some of the best disease models in nephrology. These have been used to help understand pathogenesis, to facilitate genetic interaction studies, and to perform preclinical testing. Whereas for autosomal recessive PKD, using alternative species and digenic models has partially overcome these deficiencies. Here, we review the experimental models that are currently available and most valuable for therapeutic testing in PKD, their applications, success in preclinical trials, advantages and limitations, and where further improvements are needed. Caenorhabditis elegans—with PKD proteins often localized to the cilium or basal body.2 The nonsyndromic PKD forms include autosomal dominant PKD (ADPKD) and autosomal recessive PKD (ARPKD). ADPKD is the most common (approximately 1:1000) and is typically adult-onset; PKD1 (approximately 79%) and PKD2 (approximately 15%) are the major genes, but mutations to at least six other loci are minor causes .311PKD1 and PKD2 encode polycystin-1 and -2 (PC1 and PC2), which form a functional complex at the primary cilium. Within ADPKD, there is considerable phenotypic heterogeneity ranging from limited cyst development and normal kidney function in old age to very early-onset disease, including neonatal lethality, with genic and allelic factors important.13 ADPKD is typically monoallelic, but biallelic inheritance has been rarely described, including in very early-onset presentations.1417 Most patients with ADPKD become hypertensive; liver cysts are common and sometimes result in clinically significant polycystic liver disease (PLD); and there is an increased predisposition to intracranial aneurysms.18 ARPKD has an incidence of approximately 1:20,000, leads to neonatal lethality in approximately 25% of patients, and is mainly caused by biallelic mutations in PKHD1.1926 ARPKD is typically characterized by massively enlarged cystic kidneys in utero or perinatally that can result in childhood ESKD and congenital hepatic fibrosis (CHF).202729 There is also phenotypic heterogeneity in ARPKD, partly due to allelic effects, with some patients presenting as adults with more prominent CHF.30Polycystic kidney diseases (PKDs) can be broadly categorized into two main groups: nonsyndromic (affecting mainly the kidney and liver) and syndromic (affecting numerous organ systems). PKDs are considered ciliopathies because of defects in the functioning of primary cilia—data first provided by a study of 3135 These diseases exhibit significant phenotypic and genetic heterogeneity and overlap; >10 different genes are mutated in each syndrome.36 The broad range of phenotypes likely reflects the role of primary cilia in orchestrating multiple signaling pathways, especially during development. However, each syndrome has distinct features. Meckel syndrome is the most severe with encephalocele or other central nervous system defects, CHF, and polydactyly and causes perinatal lethality.37 Whereas NPHP, JBTS, and BBS are typically childhood disorders associated with tubulointerstitial nephritis and cysts (NPHP), with the addition of cerebellar vermis hypoplasia, polydactyly, and CHF (JBTS), or cognitive impairment and obesity (BBS).32Syndromic PKDs are rarer and usually recessively inherited, including Meckel syndrome, Joubert syndrome (JBTS), nephronophthisis (NPHP), and Bardet-Biedl syndrome (BBS).in vitro) have arisen by spontaneous mutations (generated by nonspecific mutagenesis and screening) and generated by homologous recombination or more recently gene editing tools, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (in vivo). These models have been invaluable for understanding PKD pathogenesis, disease etiology, genetic interactions, and testing therapeutic candidates. Developing models that properly recapitulate organ properties (in vitro) and the human disease phenotypes (in vivo) has been a challenge, but numerous positive preclinical studies have been performed in vitro and in orthologous and nonorthologous animal models, even if the number progressing to clinical trials and US Food and Drug Administration approval has been more limited. Here, we review the orthologous in vitro and in vivo models suitable for therapeutic testing that are currently available for nonsyndromic and syndromic PKDs. We also discuss model optimization, success of clinical and preclinical trials, recommendations for the appropriate setup of preclinical trials, and advantages and limitations of these models.Experimental models of these disorders in animals and from human patient material , and ex vivo kidney culture .3848 In this review, we mainly focus on the in vitro PKD model that is the most informative for therapeutic testing, organoids generated from human pluripotent stem cells. Renal epithelial 3D spheroid cultures and ex vivo embryonic mouse kidney cultures have also been extensively used for testing the efficacy of compounds aimed to ameliorate or slow PKD cyst formation and growth. However, the mechanism(s) mediating cyst expansion and amelioration in these models seem less clear because large spheres and cysts form and regress in relatively shorter periods, suggesting targeting of secretory mechanisms rather than a combination of mechanisms as observed in PKD. The reader is directed to ref. in vitro PKD models.A number of in vitro structures that recapitulate many but not all the features of the parent organ, allowing for the assessment of kidney features of patients with PKD in vitro, and can be generated from embryonic stem cells or induced pluripotent stem cells (iPSCs) by creating the appropriate differentiation conditions.48 Indeed, kidney organoids contain both epithelial nephron and supporting cell ultrastructures, providing the most advanced in vitro models to date. They are great tools for investigating various cellular features in live cells from the patient tissue context and have been adapted for high-throughput analyses. This approach has advanced greatly over the past ten years, with several studies performed in the PKD field demonstrating that organoids can be generated from iPSCs of patients with ADPKD49 or knockout of PKD1 or PKD2 in human embryonic stem cells using CRISPR/Cas9.50 These systems are amenable to automated high-throughput screens that could be used for disease modeling and drug screening.41 Although organoids provide a unique and specialized PKD model, there are a few shortcomings: (1) primarily formation of proximal tubules, without collecting ducts, limiting recapitulation of all aspects of ADPKD phenotypes; (2) significant variability observed in PKD patient-derived iPSCs for organoid formation and tubule structure, making CRISPR/Cas9-generated models more useful at present; (3) lack of critical kidney features, such as vascularization, kidney-specific microenvironment, and fluid flow; and (4) the developmental stage of the parent cells dictating organoid maturation.5153 To circumvent some of these issues, next-generation organoid models are currently being established, including generation of adult kidney tubular organoids  and development of organoid-on-a-chip technologies , which may provide a better model for an adult-onset disease (ADPKD) and one that is more representative of the kidney, respectively.5355Organoids are complex tissue-like 3D multicellular Caenorhabditis elegans and Drosophila melanogaster), lower vertebrate models (Xenopus laevis and Danio rerio), and numerous mammalian models .5659 We have narrowed our focus to animal models that are most frequently used or best suited for drug screening and preclinical testing. For a broader review, see ref. 48A variety of PKD animal model systems have been used in research, including invertebrate models (Danio rerio) do not have a mammalian-like metanephros, they have a pronephros in embryonic stages (most frequently used) and a mesonephros in adulthood that contains many segments similar to the mammalian kidney . For a broader review, see refs. Although zebrafish .63 However, disruption of PKD genes in zebrafish can also lead to obstruction of the cloaca, which results in reduced fluid flow, and increased fluid retention, complicating the interpretation of cystic phenotypes in some cases.77 Another common feature among zebrafish models of PKD is body or tail curvature, which may be associated with changes in collagen expression in the ADPKD morphants (pkd1a/b and pkd2).78 Some models also develop laterality (pkd2) or liver defects (tsc2), both often associated with mutation of PKD/primary cilia genes .63 Most studies in zebrafish have been performed at embryonic stages; however, some recent studies have evaluated the phenotypes of adult fish . There are also differences between morphant and stable knockout/knock-in models for some PKD genes, particularly ADPKD models, where morphants typically exhibit more severe phenotypes (body curvature in pkd1a/b morphants and pronephric cysts in pkd2 morphants), potentially because of maternally contributed transcripts.61Most zebrafish models develop pronephric cysts, although some do not .Most of the PKD animal models generated to date have been mouse models because of the shared structural similarity of the mammalian kidney Figure ; the avaPkd1 and Pkd2, but because heterozygotes of fully inactivating alleles (modeling the human disease) only develop a few cysts and homozygotes are not viable79 , other approaches are required to model the progressive human disease. These approaches can mainly be divided into conditional models (see below) and gene dosage models, which are based on the principle that the level of the functional PKD gene product or protein dictates disease initiation and severity, developed by introducing incompletely penetrant or hypomorphic alleles . Dosage models are often assayed biallelically, resulting in a global reduction but not loss of functional protein, mimicking the haploinsufficiency seen in humans, with the level of the functional PC complex correlated with disease severity . A rat model with a spontaneous splicing mutation in Pkhd1 inducing a frameshift (Pkhd1PCK/PCK) develops a slowly progressive PKD and PLD phenotype, more like ADPKD than ARPKD . These studies highlight that there are likely compensatory mechanisms present in rodents, for Pkhd1/fibrocystin that do not exist, or are modulated in humans. Because the phenotypes of these models vary so widely (rapid progression versus slowly progressive or mild) and do not always mimic the human disease state, many of these models are most useful for only one particular disease stage . Because the nonrenal phenotypes can be severe, sometimes resulting in death within the first month of life, or only a limited number of animals survive postnatally, these models can be difficult to use for kidney-related intervention studies . However, by the deliberate or spontaneous generation of incompletely penetrant alleles, mouse and rat models suitable for therapeutic testing have been developed . These and other spontaneous models (Tg737orpk/orpk and Bicc1bpk/bpk mice and Pkhd1PCK/PCK rats) have helped identify disease genes, have been invaluable for studying disease mechanisms, and have been used extensively for preclinical trials.8187 Although the main focus of this review is on ADPKD orthologous disease models, many insights and applications in ADPKD have been obtained through study of these nonorthologous spontaneous models , highlighting overlap in PKD mechanisms and the value of these models.Many mouse models mimicking syndromic PKDs have also been developed, but global loss of these loci, by definition, often results in a broad range of disease phenotypes  conditional, where recombination is controlled by the spatial and temporal expression of a specific gene promoter, including targeting a particular nephron segment at a particular developmental stage/time point  conditional inducible, where recombination is spatially controlled by expression of the controlling gene, but temporal expression is induced; and (3) inducible, where recombination is spatially broad (because of a more widely expressed promoter), but temporally controlled by an induction agent  results in severe, rapidly progressive disease, whereas disruption in >P14 yields a much milder, slowly progressive disease . Two separate recombinases (cre and Flp) were used for gene inactivation and reactivation, respectively, and inducible expression of the transgenic Pkd1 or Pkd2 allowed phenotypic assessment of wild-type re-expression at particular time points.102 This strategy is attractive as an alternative because this allows for spatial and temporal gene inactivation and variant reactivation, and historically, the generation of transgenic models has been more time and cost-effective than the generation of knock-in models.A more controllable approach to obtain viable models is the use of conditional or inducible mouse models. Because there have been many studies across numerous PKD types, we focus on conditional and inducible ADPKD models as an example Table . Howevert Figure ; 2) con con106 T74 Unfortunately, the one currently available ADPKD model, a PKD1+/− miniature pig model, has slowly progressive PKD with cysts forming at 5 months of age and the kidney structure only beginning to change at 24 months, with some liver cysts , STAT6/p100, YAP/yes-associated protein/transcriptional co-activator with PDZ-binding motif), TGFβ, nuclear factor kappa B, rotein kinase B, hedgehog, cyclic AMP, and calcium.98 Many of these defects have also been observed in human in vitro models; however, differences in cell origin, mechanism of immortalization, and cell line history  complicate these interpretations.38 Expansion of these analyses in kidney organoids, with similar procedures used for model generation, will be greatly beneficial. Furthermore, recapitulating these findings in multiple orthologous models across species will be very helpful in narrowing down relevant cellular defects and disrupted signaling pathways.All the PKD models discussed earlier have helped understand PKD mechanisms. Although the pig model is challenging, it has been used to examine glucose metabolism, cell proliferation, increased glycolysis, mammalian target of rapamycin and extracellular signal regulated-kinase signaling, and decreased oxidative phosphorylation and phosphorylation of 5′ adenosine monophosphate-activated protein kinase as potential disease mechanisms.Pkd1; Pkd2 mouse models have shown an additive effect, in terms of the cystic phenotype, consistent with the dosage of the polycystin-complex being a determinant of disease severity.112 Similarly, digenic Pkd1 and Pkhd1 studies in mice and rats have shown synergistic enhancement of the renal cystic phenotype, indicating a related function of PC1 and fibrocystin and ultimately providing models that more accurately recapitulate human ARPKD.80 The relationship between ADPKD and autosomal dominant PLD was illustrated by enhanced PKD on conditional removal of Prkcsh or Sec63 in the kidney when Pkd1+/− or Pkd2+/− alleles were present.96 These digenic disease enhancements contrast with the reduction in PKD severity associated with removal of primary cilia by conditional loss of Kif3a or Ift20 in kidney tubules, compared with loss of Pkd1 or Pkd2 alone.113 This suggests a PC1-dependent inhibition and cilia-dependent activation pathway that promote cystic growth. Consistent with a polycystin-dependent inhibition pathway, reactivation of Pkd1 or Pkd2 transgenic expression after induced loss of the endogenous genes in kidney tubules showed that the cystic phenotype can be halted or even reversed by re-expression of the ADPKD genes.102 These results have important implications for future therapies re-expressing the ADPKD gene or repairing the endogenous defect.Interbreeding of different animal models or genetic interaction studies has also provided valuable information about shared pathways and disease mechanisms. Digenic in vivo models, developing systems for moderate to high-level drug screening is a priority for the field. So far, zebrafish models are best suited for this screening, with only low-throughput screening possible in mammals. The body curvature and pronephric cyst phenotypes in zebrafish PKD models have been used for relatively high-throughput drug screening and identifying appropriate doses and toxicity.114 To date, two large drug screening studies have been performed in zebrafish models of PKD, evaluating the efficacy of 115  and 2367  compounds, respectively (Table 115 Although morphants can and have been used for these large screens (histone deacetylase inhibition), use of genetic models is favored because morpholino injections at this scale are typically less practical. Use of zebrafish models for drug screening seems to be an underutilized tool in the field currently. Although kidney organoids do not recapitulate all features of PKD, they do form cysts and potentially provide an opportunity to gather drug screening information from human samples with an individualized medicine approach. High-throughput assays have been developed more recently in this regard but have not yet been used.42Because the PKD phenotype is best recapitulated using Supplemental Table 2). Many of these are supported by data from preclinical trials performed using many of the animal models described in this review , for the compounds with results (n=9), there is some consistency between the findings in humans and animal models n=5 but also some differences . Studies testing the efficacy of pravastatin (HMG-CoA reductase inhibitor), octreotide and pasireotide , bosutinib (Src/Bcr-Abl tyrosine kinase inhibitor), and tolvaptan  have all shown some consistency between clinical and preclinical trial results, demonstrating improvement in ADPKD phenotypes , vasculature, a kidney-specific microenvironment, and fluid flow  kidney changes between mice and humans, and a significant proportion of therapies have not translated to humans ; fewer models/tools are available, given the difficulty with genetic manipulations in the past; and they require a larger housing footprint , as discussed earlier, have shown great advances in recent years,145 and a combination of animal and human models for preclinical and other studies would be of great value and will hopefully aid in translation of novel therapies into clinical trials and human patients—a major hurdle in the past.Overall, all the experimental models discussed in this review have value in several different areas, and each have advantages, but also have limitations. When selecting a model for your studies, the application and resources available are likely the most important things to consider. In addition, using multiple models is always a good strategy, whether it is multiple animal models or a combination of animal and Although the development of the animal models described earlier has led to countless important findings in the PKD field, there are still some gaps that we should consider filling. These include identifying and generating improved animal models for ARPKD, expanding our repertoire of rat models, and considering ways to improve and expand the currently available pig models. Carefully considering the design and execution of preclinical trials, using multiple models, if possible, should also be considered as an important goal to improve the translation of these trials to humans.


Maintenance of optimal leaf tissue humidity is important for plant productivity and food security. Leaf humidity is influenced by soil and atmospheric water availability, by transpiration and by the coordination of water flux across cell membranes throughout the plant. Flux of water and solutes across plant cell membranes is influenced by the function of aquaporin proteins. Plants have numerous aquaporin proteins required for a multitude of physiological roles in various plant tissues and the membrane flux contribution of each aquaporin can be regulated by changes in protein abundance, gating, localisation, post-translational modifications, protein:protein interactions and aquaporin stoichiometry. Resolving which aquaporins are candidates for influencing leaf humidity and determining how their regulation impacts changes in leaf cell solute flux and leaf cavity humidity is challenging. This challenge involves resolving the dynamics of the cell membrane aquaporin abundance, aquaporin sub-cellular localisation and location-specific post-translational regulation of aquaporins in membranes of leaf cells during plant responses to changes in water availability and determining the influence of cell signalling on aquaporin permeability to a range of relevant solutes, as well as determining aquaporin influence on cell signalling. Here we review recent developments, current challenges and suggest open opportunities for assessing the role of aquaporins in leaf substomatal cavity humidity regulation. Crop productivity is directly impacted by water availability which is influenced by the climate . Our cliHordeum vulgare) and Sorghum bicolor  family. Many but not all types of aquaporins can function as water channels . AquaporBrassica species   . AquaporZea maize PIP2;5 expression and interacted with ZmPIP2;5 through binding potentially influencing water uptake during stress  cell-specific comparison of aquaporin encoding gene expression levels in mesophyll cells, bundle sheath cells and vascular tissue cells revealed that the sets of aquaporin PIP, TIP, NIP and SIP isoforms that were most highly expressed in these different cell types varied  entry into guard cells which then impacts the influence of abscisic acid signaling triggering stomatal closure through Open Stomata 1 (OST1) protein kinase mediated phosphorylation   . There amovement . It may unctions .2O2 to accumulate within and outside of plant cells  by which H2O2 is transported within and between cells for signalling and detoxification are of significant interest for improving crop abiotic stress tolerance  and Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1)  . Perturblimation . These r2O2, relatively less is known about intracellular H2O2 movement. It is possible that some aquaporins could contribute to influencing sequestration of excess H2O2 into the vacuole  Testing membrane protein function using systems such as proteoliposomes, living cell-based heterologous systems, electrophysiological approaches and use of fluorescent biosensors; and (4) functional assessment in planta using expression pattern analysis, testing protein subcellular localisation; phenotyping plants with loss of protein function like T-DNA mutants or RNAi mutants or plants with either loss of function or enhanced expression; for example approaches like CRISPR-Cas9 (The dynamics of aquaporin regulation are tissue, cell and organelle specific . TranscrSPR-Cas9  or TALENSPR-Cas9  have beeAquaporin protein abundance does not always scale with mRNA levels . The regThere are likely to be many aquaporins involved in regulating the flux of water in and out of leaf cells, and different aquaporins would be expected to have different roles in different leaf cell types. This means that leaf transcriptome analysis derived from data where whole leaves were sampled is unlikely to reveal which specific aquaporin transcript changes are relevant to regulating leaf water potential. To narrow down which aquaporins may be most of interest it would be applicable to use cell-specific RNA-seq analysis involving laser dissected leaf epidermal, mesophyll, vasculature and guard cells, similar to the approach taken by The proteomics approach by Tagging candidate aquaporins of interest using fluorescent markers and expressing them in plants can enable assessment of the localisation of the target aquaporins in different tissues and cells, including assessment of whether the localisation is around the entire cell membrane, internalised or in a polar pattern . The tagExpressing aquaporins of interest in forms that control for post-translational modification effects in a range of genetic backgrounds can help to tease out what signals regulate aquaporin influence on water transport Figure 4In summary, key opportunities for future research include Figure 5Narrowing down on the number of aquaporin candidates that might be involved in influencing changes in leaf humidity. This could involve measuring cell-specific aquaporin transcript abundance over a time-course following changes in VPD , checkinin planta, and investigating the associated impact on cell signaling pathways and aquaporin permeability (Exploring whether PTMs influence the function of the aquaporin candidates in heterologous systems and eability .Resolving whether leaf humidity differs in plant material where candidate aquaporin function has been lost, increased or modified. This will involve factoring in the potential for the aquaporin candidates to be subject to PTMs influencing their function .Agricultural productivity loss due to insufficient soil and atmospheric water availability is a major threat to food security . ManipulStudying and engineering aquaporin function in crops is challenging due to the large numbers of aquaporin isoforms present and because aquaporin function and regulation is complex. Aquaporins can transport many different molecules, and influence signaling pathways, and their localisation, gating and permeability are dynamic. However, optimising the regulation of aquaporins can contribute to adapting crop plants to environmental conditions where water availability is sub-optimal by enabling crops to have enhanced control of the flux of water, osmolytes and signaling molecules relevant to rapidly adjusting tissue hydration.Resolving how different aquaporins contribute to different hydraulic features in plants such as leaf humidity regulation, stomatal function, root-to-shoot solute transport, root solute uptake, cell and tissue osmoregulation, and stress signaling is expected to position the field with key information needed to devise strategies for improving crop productivity in challenging environmental conditions.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


International Journal of Molecular Sciences encompasses six contributions: four original articles and two reviews highlighting novel advancements to understanding FGF signaling pathways and how they modulate cellular processes in health and disease.Fibroblast growth factors (FGFs) and their cognate receptors (FGFRs) are important biological molecules with a wide array of pleiotropic functions. Consisting of 22 distinct mammalian FGF ligands and four highly conserved transmembrane receptor tyrosine kinases FGFR1–4), this vast molecular network plays a role in nearly every organ of the body and many other biological processes throughout the course of life, including development, metabolism, tissue repair, and angiogenesis [–4, this The articles in this Special Issue largely focus on two hormones, FGF21 and FGF23, which play important roles in regulating metabolic and skeletal functions in the body. FGF21 is produced primarily by the liver in response to various metabolic stresses and has been shown to improve insulin sensitivity and lipid profiles in animal models . In indiCzaya et al.  thoroughTo discover new FGF23-mediated cardiac pathologies, Böckmann et al.  evaluateSilva et al.  also tooFGF23 not only plays a role as a potential biomarker in cardiovascular disease but also in chronic pulmonary disease . Gulati Switching focus to another FGF ligand, Martínez-Garza et al.  reviewedFgf21 mRNA expression levels, respectively [Lastly, Scholle et al.  investigectively ,21. ThisGiven the broad range of topics covered in just a few articles for this Special Issue, the widespread involvement of FGFs in development and disease is abundantly clear. The use of FGFs as biomarkers in disease identification, the modulation of associated signaling pathways, and even the inhibition of the ligands or receptors themselves is a quickly evolving field that may lead to many therapeutic discoveries. These studies and those that build on them will contribute greatly to our understanding of FGF signaling in health and disease over the coming years.


It has been 11 years since Brent Stockwell identified and named ferroptosis . FerroptTo examine progress in the ferroptosis field and advances in basic research and clinical applications focusing on ferroptosis, we launched a Research Topic named Ferroptosis in Cancer and Beyond in early 2022, which was a great success. Given the rapid progression in this field, we opened a second call for this same Research Topic in late 2022, which has now been successfully closed. This second volume brings together 13 papers, including 6 research articles and 7 reviews. These articles outline recent information about ferroptosis from both basic research and clinical translation angles. The papers are briefly introduced below.McKale Montgomery and Cameron Cardona reviewed the regulatory processes regarding iron homeostasis, from absorption, metabolism to its participation in ferroptosis, and discussed the essential roles of various IRPs in ferroptosis and their potentials to be therapeutically maneuvered in cancer treatment. To explore how ferroptosis is regulated at the post-translational level, Zhang et al. introduce emerging evidence for the O-GlcNAc modification (O-GlcNAcylation) in ferroptosis in a review article and discuss the crosstalk between O-GlcNAcylation and ROS and related antioxidant defense systems. The authors elucidate the role of O-GlcNAcylation in proteins involved in iron metabolism and the regulation of lipid metabolism and peroxidation during ferroptosis. Furthermore, the underlying mechanisms including mitochondria dysfunction and endoplasmic reticulum alteration brought by O-GlcNAcylation are discussed. In their original research study, Nikulin et al. identified ELOVL5 and IGFBP6 may modulate the sensitivity of breast cancer cells to ferroptosis, possibly via enhancing the activity of GPX4, an antioxidant enzyme that plays a critical role in ferroptosis. Through analysis of the transcriptomic database and validation with HPLC-MS, the knockdown of either ELOVL5 or IGFBP6 was shown to cause remarkable changes in the production of long and very long fatty acids. In addition, the knockdown of ELOVL5 or IGFBP6 in MDA-MB-231 cells promotes cell death induced by PUFAs, and the potential benefit of PUFAs addition for improving chemotherapeutic effects was proposed in the condition of low IGFBP6 (and maybe ELOVL5) gene expression.To comprehensively understand the contribution of iron regulatory proteins (IRPs) to ferroptosis, Tan et al. GSTP1 is fundamental to maintaining cellular oxidative homeostasis and is involved in ferroptosis. Based on increasing evidence showing that iron metabolism, lipid peroxidation, and GSH level are modulated by radiotherapy, the authors elaborated on the potential to control GSTP1 levels to enhance the efficacy of radiotherapy in cancer treatment. More pathways in ferroptosis induced by radiotherapy and their implications for radiotherapy were reviewed by Giovanni Luca Beretta and Nadia Zaffaroni, and other strategies were proposed to improve the efficacy of radiotherapy, including enhancing ionizing radiation by other reagents or selectively inducing ferroptosis with metal-based nanoparticles. Lu et al. introduced all kinds of therapies for glioblastoma, including immunotherapy, radiotherapy, and chemotherapy, and discussed how ferroptosis participates and affects the efficacy of different therapeutic treatments. In an original research article, Shi et al. found that dihydroartemisinin (DHA), an adjuvant drug-enhancing chemotherapy, induced cervical cancer death via initiating ferroptosis and explored the involvement of ferritinophagy induced by DHA. Furthermore, DHA was also shown to have a synergistic role with doxorubicin (DOX) in promoting cervical cancer cell death.Glutathione S-transferase P1 (GSTP1) was proposed to be a potential target to tackle radioresistance in cancer therapy by Jiang et al. analyzed the differential gene expression in CD8+ T cells from CD8+ highly or low infiltrated samples in acute myeloid leukemia (AML) and conducted extensive bioinformatics analysis, and six ferroptosis-related genes (FRGs) were identified to generate a prognostic prediction model, which was validated to be helpful to risk stratification and prognostic prediction of AML patients. Han et al. identified several ferroptosis-related genes (FRGs) which correlate well with the immune microenvironment and establish a model to predict the prognosis of cervical cancer patients. Further mechanisms underlying iron homeostasis, ROS and lipid peroxidation, GPX4-GSH, and other regulator systems in cervical cancer were discussed in a review by Xiangyu Chang and Jinwei Miao. In another review, Lai et al. specifically elaborated on the influence of steroid hormone signaling on ferroptosis and discuss the involvement of ferroptosis in gynecologic cancers and potential therapies targeting ferroptosis for the treatment of gynecologic cancers.Growing evidence has revealed the impact of T cell infiltration in the development of various types of cancer. Li et al. established a prognostic prediction model for colorectal cancer (CRC) patients with 8 FRGs among which NOS2 is one of the most significantly affected examples and was validated with the CRC mouse model and the involvement of NF-κB pathway was elucidated. To investigate whether ferroptosis is associated with colon adenocarcinoma (COAD), Baldi et al. identified a 4-gene signature that distinguishes high-risk and low-risk patients, and those FRGs were further shown to be implicated in many pathological related pathways and a variety of miRNAs and transcription factors were found to be involved. These researches consolidated the idea that disease-associated cell death has a specific gene expression profile relevant to the prognosis of the patient (With data from FerrDb and TCGA database,  patient .Taken together, this second volume of the Research Topic Ferroptosis in Cancer and Beyond adds new knowledge to this field, furthering research and the clinical translation of ferroptosis.