Network-driven cancer cell avatars for combination discovery and biomarker identification for DNA damage response inhibitors.

Combination therapy is well established as a key intervention strategy for cancer treatment, with the potential to overcome monotherapy resistance and deliver a more durable efficacy. However, given the scale of unexplored potential target space and the resulting combinatorial explosion, identifying efficacious drug combinations is a critical unmet need that is still evolving. In this paper, we demonstrate a network biology-driven, simulation-based solution, the Simulated Cell™. Integration of omics data with a curated signaling network enables the accurate and interpretable prediction of 66,348 combination-cell line pairs obtained from a large-scale combinatorial drug sensitivity screen of 684 combinations across 97 cancer cell lines (BAC = 0.62, AUC = 0.7). We highlight drug combination pairs that interact with DNA Damage Response pathways and are predicted to be synergistic, and deep network insight to identify biomarkers driving combination synergy. We demonstrate that the cancer cell 'avatars' capture the biological complexity of their in vitro counterparts, enabling the identification of pathway-level mechanisms of combination benefit to guide clinical translatability.

Integrative Epigenetic and Molecular Analysis Reveals a Novel Promoter for a New Isoform of the Transcription Factor TEAD4.

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

Pathomechanisms of Prenatally Programmed Adult Diseases.

Based on epidemiological observations Barker et al. put forward the hypothesis/concept that an adverse intrauterine environment (involving an insufficient nutrient supply, chronic hypoxia, stress, and toxic substances) is an important risk factor for the development of chronic diseases later in life. The fetus responds to the unfavorable environment with adaptive reactions, which ensure survival in the short run, but at the expense of initiating pathological processes leading to adult diseases. In this review, the major mechanisms (including telomere dysfunction, epigenetic modifications, and cardiovascular-renal-endocrine-metabolic reactions) will be outlined, with a particular emphasis on the role of oxidative stress in the fetal origin of adult diseases.

Telomere Length and Telomerase Activity of Granulosa Cells and Follicular Fluid in Women Undergoing In Vitro Fertilization.

This study aimed to evaluate the interrelationship between telomere length, telomerase activity and oxidative DNA damage in patients undergoing in vitro fertilization (IVF). This single-center, observational clinical study comprised 102 unselected, consecutive patients with various infertility diagnoses. Granulosa cells (GCs) and follicular fluid (FF) were analyzed simultaneously for telomere functions and for the marker of oxidative DNA damage, 8-hydroxy-2-deoxyguanosine (8-OHdG). An Absolute Human Telomere Lengths Quantification qPCR Assay kit and Telomerase Activity Quantification qPCR Assay kit (Nucleotestbio, Budapest, Hungary), as well as an 8-OHdG ELISA kit (Abbexa Ltd., Cambridge, United Kingdom) were used for analyses. Similar telomere lengths were found in GCs and FF, however telomerase activity was markedly depressed, while 8-OHdG levels were markedly elevated in FF compared with those in GCs (p < 0.01). Telomere lengths were independent of telomerase activity both in GCs and FF. However, GC 8-OHdG was inversely related to telomerase activity in GCs and FF (p < 0.05). Importantly, 8-OHdG levels both in GCs and FF had significant negative impact on the number of the retrieved and MII oocytes (p < 0.01), whereas FF 8-OHdG was negatively related further to the number of fertilized oocytes and blastocysts (p < 0.01). In conclusion, we could not confirm the direct association of telomere function and reproductive potential. However, oxidative DNA damage, as mainly reflected by 8-OHdG, adversely affected early markers of IVF outcome and clinical pregnancies.

Self-management of vaginal cube pessaries may be a game changer for pelvic organ prolapse treatment: a long-term follow-up study.

INTRODUCTION AND HYPOTHESIS: Loss of anatomical support for the pelvic organs results in pelvic organ prolapse (POP). We hypothesized that daily self-management of a cube pessary might be a safe, feasible long-term treatment in women with symptomatic POP. METHODS: A cohort of 214 symptomatic POP patients (stage 2+) were enrolled prospectively (January to December 2015). Each patient was size-fitted with a space-filling cube pessary and completed a questionnaire online or by phone ≥5 years after her initial fitting. Change in quality of life (QoL) was measured with the Patient Global Impression of Improvement (PGI-I). RESULTS: Of 185 women included in our analyses, 174 (94%) were continuing to use their pessary 4 weeks post-insertion. Among those, 143 (82.2%) used the pessary successfully for ≥5 years. A large majority of these patients (88.8% [127 out of 143]) described their condition as much or very much improved compared with their pretreatment status (PGI-I). Adverse secondary effects (ASEs) were infrequent [15.4% (22 out of 143)]; when they did occur, they were mild, including smelly vaginal discharge (15 out of 22) and slight vaginal bleeding caused by the fitting procedure (6 out of 22). CONCLUSIONS: Daily self-management of cube pessaries was found to be a safe and effective treatment for improving POP-related symptoms and QoL in the long term.

Validation and translation of the Hungarian version of the Australian Pelvic Floor Questionnaire (APFQ-H).

INTRODUCTION AND HYPOTHESIS: The aims of the study were the translation, cultural adaptation, and validation of self-administered Australian Pelvic Floor Questionnaire (APFQ) on a Hungarian population. METHODS: The validation was performed in 294 women over 18 who agreed to answer the Hungarian version of the APFQ. The validation of the questionnaire included evaluation of content/face validity, internal consistency, construct validity, test-retest reproducibility, discriminant validity and convergent validity. RESULTS: Acceptable and good internal consistency was observed in all four dimensions [McDonald's ω (95% confidence interval) coefficients were > 0.7 for each dimension: bladder 0.888, bowel 0.790, prolapse 0.895 and sexual function 0.738]. Test-retest analyses revealed high reproducibility with intraclass correlation coefficients (bladder 0.83, bowel 0.92, prolapse 0.96 and sexual function 0.87). Prolapse symptom score correlated significantly with Pelvic Organ Prolapse Quantification (POP-Q), and bladder score correlated significantly with the results of the International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ UI SF) (convergent validity). Scores distinguished between patients with pelvic floor disorders and controls (high discriminant validity). CONCLUSIONS: Hungarian version of the self-administered APFQ is a reliable and valid instrument for evaluating symptom severity and impact of pelvic floor dysfunction on the quality of life of Hungarian women.

The Role of L-Arginine-NO System in Female Reproduction: A Narrative Review.

Accumulating evidence are available on the involvement of l-arginine-nitric oxide (NO) system in complex biological processes and numerous clinical conditions. Particular attention was made to reveal the association of l-arginine and methylarginines to outcome measures of women undergoing in vitro fertilization (IVF). This review attempts to summarize the expression and function of the essential elements of this system with particular reference to the different stages of female reproduction. A literature search was performed on the PubMed and Google Scholar systems. Publications were selected for evaluation according to the results presented in the Abstract. The regulatory role of NO during the period of folliculogenesis, oocyte maturation, fertilization, embryogenesis, implantation, placentation, pregnancy, and delivery was surveyed. The major aspects of cellular l-arginine uptake via cationic amino acid transporters (CATs), arginine catabolism by nitric oxide synthases (NOSs) to NO and l-citrulline and by arginase to ornithine, and polyamines are presented. The importance of NOS inhibition by methylated arginines and the redox-sensitive elements of the process of NO generation are also shown. The l-arginine-NO system plays a crucial role in all stages of female reproduction. Insufficiently low or excessively high rates of NO generation may have adverse influences on IVF outcome.

[Conservative and surgical treatments performed for symptomatic pelvic organ prolapse and stress urinary incontinence in Hungary.] / A kismedencei szervek süllyedésének és a noi terheléses vizeletvesztésnek a konzervatív és sebészi kezelése Magyarországon.

INTRODUCTION AND OBJECTIVE: As clinical guidelines are available for the treatment of stress urinary incontinence, but not pelvic organ prolapse, in Hungary, the treatment of pelvic organ prolapse varies widely throughout the country and is not standardized. Due to the establishment of the Hungarian Continence and Urogynecological Association, we aimed to determine current conservative and surgical treatment trends and strategies for these conditions in Hungary and compare them with international practice. METHOD: An online questionnaire consisting of 20 multiple-choice, checkbox, multiple-choice grid, and short-answer items was sent to 40 urology and 65 gynecology departments in Hungary in September and October 2021. RESULTS: The overall response rate was 24.76%. Almost all (96.15%) respondents reported that conservative treatment options were offered as first-line therapy to patients with symptomatic pelvic organ prolapse. For symptomatic anterior-wall prolapse, anterior repair, and laparoscopic sacrohysteropexy/sacrocolpopexy were the preferred surgical options (by 28.96% and 27.42% of respondents, respectively). For apical prolapse, laparoscopic sacrofixation was the first-choice treatment (35.88%). For stress urinary incontinence, mid-urethral sling insertion with a transobturator (61.53%) or retropubic (15%) approach was the preferred intervention. CONCLUSION: The low overall response rate in this study alone reflects the current condition of Hungarian urogynecology. Our findings might provide a good basis for the improvement and refinement of diagnosis and therapy for female urinary incontinence and pelvic organ prolapse in the country. As Hungary is a new affiliated partner of the European Urogynecological Association, we hope that this goal can be achieved soon. In addition, a curriculum for urogynecological specialization is needed. Orv Hetil. 2022; 163(52): 2072-2078.

The possibilities of intrapartum fetal monitorization in obese pregnant women / A magzatok intrapartum monitorizálásának lehetoségei elhízott várandósokban

Obesity, as an epidemic, is one of the major public health challenges of the 21st century. It affects more and more people annually, and through its numerous negative psychological and physical effects as a risk factor it increases the morbidity and mortality of several diseases. It has a major impact on pregnancy; it is well established that both infertility and abortion is more common among obese women. During pregnancy, obesity increases the risk of intrapartum complications, affects the way of delivery, and has an impact on fetal development. Obesity puts an extra burden on the already overloaded maternal circulation, leading to electrophysiological deviations. Increased maternal fat tissue limits the assessment of fetal well-being during labor, which is external fetal monitoring (cardiotocography, CTG) nowadays. It has the advantage of being non-invasive, although subjectivity in the interpretation of the CTG trace and discrepancies in the analysis decreases the sensitivity and the reliability of the method. The current review aims to summarize what kind of intrapartum methods are currently available to have a better assessment of the fetal well-being, and to achieve better perinatal outcomes in the case of obese mothers.

Preeclampsia subtypes: Clinical aspects regarding pathogenesis, signs, and management with special attention to diuretic administration.

During normal pregnancy, blood volume increases by nearly two liters. Distinctively, the absence coupled with the extreme extent regarding the volume expansion, are likely accompanied with pathological conditions. Undoubtedly, preeclampsia, defined as the appearance of hypertension and organ deficiency, such as proteinuria during the second half of pregnancy, is not a homogenous disease. Clinically speaking, two main types of preeclampsia can be distinguished, in which a marked difference between them is vascular condition, and consequently, the blood volume. The "classic" preeclampsia, as a two-phase disease, described in the first, latent phase, in which, placenta development is diminished. Agents from this malperfused placenta generate a maternal disease, the second phase, in which endothelial damage leads to hypertension and organ damage due to vasoconstriction and thrombotic microangiopathy. In this hypovolemia-associated condition, decreasing platelet count, signs of hemolysis, renal and liver involvement are characteristic findings; proteinuria is marked and increasing. In the terminal phase, visible edema develops due to increasing capillary transparency, augmenting end-organ damages. "Classic" preeclampsia is a severe and quickly progressing condition with placental insufficiency and consequent fetal growth restriction and oligohydramnios. The outcome of this condition often leads to fetal hypoxia, eclampsia or placental abruption. The management is limited to a diligent prolongation of pregnancy to accomplish improved neonatal pulmonary function, careful diminishing high blood pressure, and delivery induction in due time. The other subtype, associated with relaxed vasculature and high cardiac output, is a maternal disease, in which obesity is an important risk factor since predisposes to enhanced water retention, hypertension, and a weakened endothelial dysfunction. Initially, enhanced water retention leads to lowered extremity edema, which oftentimes progresses to a generalized form and hypertension. In several cases, proteinuria appears most likely due to tissue edema. This condition already fully meets preeclampsia criteria. Laboratory alterations, including proteinuria, are modest and platelet count remains within the normal range. Fetal weight is also normal or frequently over average due to enhanced placental blood supply. It is very likely, further water retention leads to venous congestion, a parenchyma stasis, responsible for ascites, eclampsia, or placental abruption. During the management of this hypervolemia-associated preeclampsia, the administration of diuretic furosemide treatment seemingly offers promise.

Tissue Sodium Accumulation: Pathophysiology and Clinical Implications.

Excessive sodium intake has been well established as a risk factor for the development and progression of cardiovascular and renal diseases. Its adverse effects are achieved by renal sodium retention and related volume expansion and by inducing low-grade inflammation and oxidative stress (OS) in the target tissues. This review presents the recent concept of nonosmotic sodium storage in the skin interstitium, the subsequent dissociation of sodium and volume homeostasis, and the cellular response to the increased tissue sodium concentration. Furthermore, data are shown on the sodium barrier and buffering potential of the endothelial glycocalyx that may protect the functional integrity of the endothelium when it is challenged by an increased sodium load. Finally, examples will be given of the involvement of oxygen free radicals (OFR) in sodium-induced tissue damage, and some clinical entities will be mentioned that are causally associated with sodium/volume retention and OS.

Assessment of the potential impacts of the Chernobyl nuclear disaster on maternal and fetal health in Hungary.

Objective: Radiation exposure is known to be mutagenic and teratogenic. The aim of this study was to analyze the effects of the increased ionizing radiation emitted by the Chernobyl nuclear disaster on maternal and fetal outcomes in Hungary.Methods: A retrospective analysis of abortion, stillbirth, and congenital anomaly data for pregnancies in Hungary between 1 January 1981 and 31 December 1991 was conducted.Results: Trend analysis revealed increasing trends in spontaneous and voluntary abortion rates in Hungary during the study time period, while late pregnancy losses showed a decreasing trend. Overall, there were generally decreasing incidence rates for birth defects throughout the 1980s. Increased voluntary abortions over the study period might reflect, at least in part, maternal anxiety in the post-Chernobyl years. Decreased late pregnancy loss over the same period may be attributable to improvements in prenatal diagnostics. A notable weakness of this study is that missing data could not be complemented due to the decades that have passed since the incident.Conclusions: In conclusion, the present data suggest that the nuclear catastrophe in 1986 did not cause a significant increase in pregnancy loss or congenital malformations in Hungary.

Radiation exposure in Hungary caused by the Chernobyl nuclear power plant disaster did not results in increases in fetal death or congenital anomaly rates.

Key to Life: Physiological Role and Clinical Implications of Progesterone.

The most recent studies of progesterone research provide remarkable insights into the physiological role and clinical importance of this hormone. Although the name progesterone itself means "promoting gestation", this steroid hormone is far more than a gestational agent. Progesterone is recognized as a key physiological component of not only the menstrual cycle and pregnancy but also as an essential steroidogenic precursor of other gonadal and non-gonadal hormones such as aldosterone, cortisol, estradiol, and testosterone. Based on current findings, progesterone and novel progesterone-based drugs have many important functions, including contraception, treatment of dysfunctional uterine bleeding, immune response, and prevention of cancer. Considering the above, reproduction and life are not possible without progesterone; thus, a better understanding of this essential molecule could enable safe and effective use of this hormone in many clinical conditions.

Towards a Koopman theory for dynamical systems on completely regular spaces.

The Koopman linearization of measure-preserving systems or topological dynamical systems on compact spaces has proven to be extremely useful. In this article, we look at dynamics given by continuous semiflows on completely regular spaces, which arise naturally from solutions of PDEs. We introduce Koopman semigroups for these semiflows on spaces of bounded continuous functions. As a first step we study their continuity properties as well as their infinitesimal generators. We then characterize them algebraically (via derivations) and lattice theoretically (via Kato's equality). Finally, we demonstrate-using the example of attractors-how this Koopman approach can be used to examine properties of dynamical systems. This article is part of the theme issue 'Semigroup applications everywhere'.

Can we define excess vaginal space? - Genital hiatus size and prolapse severity are correlated with cube pessary size.

OBJECTIVE: Prior studies demonstrated a positive association between increased genital hiatus (GH), advanced prolapse stage and levator ani muscle injury. Moreover wide GH is an established risk factor for recurrent pelvic organ prolapse (POP). Since excess vaginal space is not yet a dimension to estimate in Pelvic Organ Prolapse, we hypothesized that excess vaginal space has a positive correlation with increased GH and could be a new aspect for the assessment of the severity of POP and underlying pelvic muscle damage. We attempted to quantify excess vaginal space by different volumes of different cube pessary sizes. STUDY DESIGN: In a prospective study, 716 symptomatic POP patients without any prior operations were enrolled from January 2011 to December 2017. All patients suffered from stage 2 POP or greater, where either the anterior, middle or posterior compartments or combinations of these were affected. As a conservative self-therapy, space-filling (Dr. Arabin®) cube pessaries were fitted. The size of each was individually adapted for each woman. For data analysis we used Spearman correlation test and Nonparametric statistical test. RESULTS: All patients included in the study were asymptomatic one week after fitting the pessary. We revealed a positive significant correlation between the genital hiatus (GH) and the size of the cube pessary (ƍ = 0.777,p ≤ 0.001). We also found a positive significant correlation between the size of the cube pessaries and the POP-Q stage. We also managed to find significant differences between cube pessary sizes and corresponding GH values. CONCLUSIONS: Cube pessary sizes may indicate the amount of excess vaginal space. Since excess vaginal space significantly correlates with the increase of the genital hiatus, it could be consider - as well as GH - as a marker for advanced prolapse stage, and a risk factor for the recurrence of pelvic organ prolapse.More studies are needed to identify factors related to excess vaginal space.

Comparative analysis of abdominal fluid cytokine levels in ovarian hyperstimulation syndrome (OHSS).

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rare, yet severe, iatrogenic complication of ovulation induction therapy during assisted reproductive procedures. Our group previously detected atypical cells in the ascitic fluid of OHSS patients, although no malignancy developed during follow up. Here, the aim was to perform a comparative analysis of the cytokines present in the abdominal fluid of patients affected by OHSS versus patients with advanced ovarian cancer, a benign adnexal mass, or ovarian endometriosis. METHODS: This prospective, non-randomized study was conducted at the Clinical Center of the University of Pecs Department of Obstetrics and Gynecology/Reproductive Center between October 2016 and March 2018. Abdominal fluid samples were obtained from 76 patients and subjected to Luminex analysis. The samples were collected from patients with OHSS (OHSS; n = 16), advanced ovarian cancer (OC; n = 22), a benign adnexal mass (BAM; n = 21), or ovarian endometriosis (EM; n = 17). Data were subjected to the non-parametric Kruskal-Wallis test and Spearman's rank correlation coefficient to identify statistical differences between the four study groups. RESULTS: Leukocytosis and hemoconcentration were detected in the peripheral blood of OHSS patients. Abdominal fluid analysis further revealed significantly higher levels of interleukin (IL)-6, IL-8, IL-10, and transforming growth factor (TGF)-ß in both the OHSS and OC groups compared to the BAM and EM groups. The highest concentration of vascular endothelial growth factor (VEGF) was detected in the OC group, while a significantly lower level was detected in the OHSS group. Moreover, VEGF levels in OC and OHSS groups were significantly elevated compared to the levels in the BAM and EM groups. CONCLUSIONS: Vasoactive and hematogenic cytokines were present at higher levels in both the OHSS and OC abdominal fluid samples compared to the fluid samples obtained from the peritoneal cavity of the BAM patients. It is possible that these cytokines play an important role in the formation of ascites.

Validation and translation of the Hungarian version of the Female Sexual Function Index (FSFI-H).

INTRODUCTION AND HYPOTHESIS: The Female Sexual Function Index (FSFI) has been used for clinical and research purposes in many countries. The aim of this study was to translate, adapt and perform a psychometric validation of a Hungarian version of the FSFI. METHODS: The FSFI was translated into Hungarian, and its precision was ascertained through reverse translation by an expert team. As a first step, 40 volunteers participated in an evaluation of the test-retest reliability of the Hungarian version over a 2-week period. After that, 418 (331 control and 87 with pelvic organ prolapse) women who had been in a stable sexual relationship in the previous 4 weeks participated in the study. The data were summarized using descriptive statistics. The structure validity was examined by confirmatory factor analysis, with which we tested the hypothesized original factor structure, using maximum likelihood model estimation. We calculated the Comparative Fit Index (CFI), root mean square error of approximation (RMSEA), standardized root mean residual and Akaike information criterion (AIC). To test the internal consistency, Cronbach's alpha coefficients of the full scale were determined. Spearman's rank correlation was used for testing divergent validity and Mann-Whitney U-test for examining discriminant validity. RESULTS: The FSFI was easily understandable and acceptable as well as capable of adequately evaluating and measuring various aspects of female sexual functioning. A high degree of internal consistency was demonstrated by the Cronbach's alpha value (0.963). CONCLUSION: The FSFI Hungarian version is a valid tool that measures the same functioning as the original English questionnaire.

Female urethral dilatation (bougierung): a case report.

BACKGROUND: Primary bladder neck obstruction is a rare clinical entity, reported to be responsible for 2.7-8% of lower urinary tract symptoms. It can lead to various urinary storage and voiding symptoms. The mainstay of treatment of female urethral strictures is urethral dilatation. Despite the long history of this method, it is unclear how far the female urethra should be dilated in correlation with residual urine volume. CASE PRESENTATION: A 79-year-old Caucasian woman presented to our institute with urgency (12-15 times/day), nocturia (3 times/night), and reoccurring urinary tract infections. A physical examination revealed no anatomical malformation in her genital organs, 150 mL post-void urine retention, and a significant narrowing in the mid-segment of the urethra (4 mm). After informed consent, our patient underwent urethral dilatation ranging from Ch9 (3 mm) to Ch39 (13 mm), and reported no symptoms at the 4-week follow-up, with no post-void residual urine. CONCLUSIONS: The relatively low (around 50%) success rate of urethral dilatation might be improved by the utilization of wider dilatators, and the relaxation of the pubourethral ligament, achieved by a gentle downward saggital push during the intervention, although long-term studies with a large number of participants are necessary to prove our hypothesis.

The possible role of CD8+/Vα7.2+/CD161++ T (MAIT) and CD8+/Vα7.2+/CD161lo T (MAIT-like) cells in the pathogenesis of early-onset pre-eclampsia.

PROBLEM: The objective of this study was to compare the expressions of different immune-checkpoint molecules by MAIT and MAIT-like cells in healthy pregnancy and in early-onset pre-eclampsia. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMC) were stained with monoclonal antibodies to characterize MAIT and MAIT-like cells. Flow cytometric analyses were used to measure PD-1, TIM-3, activation markers, and intracellular perforin expression. RESULTS: We identified CD3+/CD8+/Vα7.2+/CD161++ MAIT cells and a minor cell population characterized by CD3+/CD8+/Vα7.2+/CD161lo surface markers. In measuring the expression of PD-1 receptor, we found a significantly lower expression by MAIT cells in women with early-onset pre-eclampsia. CD69 expression by MAIT cells was significantly elevated in early-onset pre-eclamptic patients. Intracellular perforin content by MAIT and PD-1+ MAIT cells was significantly increased in pre-eclamptic patients compared with healthy individuals. CONCLUSION: Altered frequency and reduced PD-1 expression combined together with the elevated perforin content of MAIT cells insinuate their potential roles in the pathogenesis of early-onset pre-eclampsia.