Comparing the efficacy and pregnancy outcome of intrauterine balloon and intrauterine contraceptive device in the prevention of adhesion reformation after hysteroscopic adhesiolysis in infertile women: a prospective, randomized, controlled trial study.

STUDY OBJECTIVE: To evaluate the efficacy and pregnancy outcomes of intrauterine balloon and intrauterine contraceptive devices in the prevention of adhesion reformation following hysteroscopic adhesiolysis in infertile women with moderate to severe intrauterine adhesion. DESIGN: A prospective, randomized, controlled trial study. SETTING: A tertiary university hospital. PATIENTS: A total of 130 patients with moderate (American Fertility Society [AFS] score of 5-8) and severe (AFS score of 9-12) intrauterine adhesions were recruited. INTERVENTIONS: 86 patients were evenly allocated to group treated with an IUD for 1 month and group treated with an IUD for 2 months. 44 patients were allocated to group treated with a Foley catheter balloon.(IUD: Yuangong IUD). MEASUREMENTS AND MAIN RESULTS: The primary outcome measures were the AFS score, endometrial thickness, and pregnancy outcome. After hysteroscopy, the AFS score was significantly decreased(P<0.05), whereas endometrial thickness was significantly increased across the three groups(P<0.001). Notably, the decline in the AFS score in the balloon group was greater than that in the IUD-1-month group and IUD-2-month group(P<0.01), with no significant difference between the IUD groups(P = 0.298). Lastly, In addition, the extent of the increase in endometrial thickness(P = 0.502) and the pregnancy outcomes(P = 0.803) in the three groups were not significantly different. CONCLUSION: Inserting a balloon or placing an IUD for one or two months can effectively lower the risk of adhesion recurrence and restore the shape of the uterine cavity. While the therapeutic effect of the balloon was superior to that of the IUD, no significant differences were observed in the one-month and two-month IUD groups. TRIAL REGISTRATION: This research was registered in the Chinese Clinical Trial Registry ( http://www.chictr.org.cn/enIndex.aspx ); Clinical trial registry identification number: ChiCTR-IOR-17,011,943 ( http://www.chictr.org.cn/showprojen.aspx?proj=17979 ). Date of trial registration: July 11, 2017.

Spatiotemporal patterns of the pregnancy microbiome and links to reproductive disorders.

The microbiome of females undergoes extensive remodeling during pregnancy, which is likely to have an impact on the health of both mothers and offspring. Nevertheless, large-scale integrated investigations characterizing microbiome dynamics across key body habitats are lacking. Here, we performed an extensive meta-analysis that compiles and analyzes microbiome profiles from  >10,000 samples across the gut, vagina, and oral cavity of pregnant women from diverse geographical regions. We have unveiled unexpected variations in the taxonomic, functional, and ecological characteristics of microbial communities throughout the course of pregnancy. The gut microbiota showed distinct trajectories between Western and non-Western populations. The vagina microbiota exhibited fluctuating transitions at the genus level across gestation, while the oral microbiota remained relatively stable. We also identified distinctive microbial signatures associated with prevalent pregnancy-related disorders, including opposite variations in the oral and gut microbiota of patients with gestational diabetes and disrupted microbial networks in preterm birth. This study establishes a comprehensive atlas of the pregnancy microbiome by integrating multidimensional datasets and offers foundational insights into the intricate interplay between microbes and host factors that underlie reproductive health.

Research on a New Intelligent and Rapid Screening Method for Depression Risk in Young People Based on Eye Tracking Technology.

Depression is a prevalent mental disorder, with young people being particularly vulnerable to it. Therefore, we propose a new intelligent and rapid screening method for depression risk in young people based on eye tracking technology. We hypothesized that the "emotional perception of eye movement" could characterize defects in emotional perception, recognition, processing, and regulation in young people at high risk for depression. Based on this hypothesis, we designed the "eye movement emotional perception evaluation paradigm" and extracted digital biomarkers that could objectively and accurately evaluate "facial feature perception" and "facial emotional perception" characteristics of young people at high risk of depression. Using stepwise regression analysis, we identified seven digital biomarkers that could characterize emotional perception, recognition, processing, and regulation deficiencies in young people at high risk for depression. The combined effectiveness of an early warning can reach 0.974. Our proposed technique for rapid screening has significant advantages, including high speed, high early warning efficiency, low cost, and high intelligence. This new method provides a new approach to help effectively screen high-risk individuals for depression.

LncRNA DANCR counteracts premature ovarian insufficiency by regulating the senescence process of granulosa cells through stabilizing the interaction between p53 and hNRNPC.

BACKGROUND: Premature ovarian insufficiency (POI) is one of the common women reproductive endocrine diseases which adversely impacts female fertility, but the etiology and pathogenesis still remain elusive. Recently increasing researches focus on the roles of lncRNA in POI. LncRNA DANCR was involved in cell differentiation and multiple cancers. It's highly expressed in ovary while the role of DANCR in POI is still unknown. RESULTS: Here, we identify a new POI related lncRNA DANCR, which negatively contributes to ovarian granulosa cells aging and follicular atresia. DANCR is proved to be decreasingly expressed in POI patients' granulosa cells. Additionally, Dancr knockout (Dancr-/-) mice were constructed and characterized with POI phenotypes and fertility decline, compared with Dancr+/+ mice. Further, in vitro experiments indicated that DANCR knockdown in granulosa cells led to cell aging and series of aging-related changes including proliferation inhibition, cell cycle G1 arrest and DNA damage. Mechanism research revealed DANCR binds with hNRNPC and p53, while DANCR knockdown attenuates the binding of hNRNPC and p53, thus enhancing protein level of p53 and promoting granulosa cells aging significantly. CONCLUSION: The newly identified lncRNA DANCR inhibits p53-dependent granulosa cells aging by regulating hNRNPC-p53 interaction, and eventually counteracting POI. This provides new insights into the pathogenesis of POI and provides a potential target for future diagnosis and treatment.

Diagnostic value of non-coding RNAs in ovarian cancer.

The type of primary tumour of the ovary ranks first among all organs in the body. Although the incidence of malignant ovarian tumour ranks third among gynaecological malignancies, it is the most fatal type. A lack of effective diagnostic methods for early ovarian cancer remains, and the efficacy of advanced ovarian cancer is often unsatisfactory; the five-year survival rate of stage III-IV is less than 30%. Non-coding RNA is RNA that does not have protein-coding potential and was once considered as 'junk DNA'. However, increasing number of studies have shown that the disorder of non-coding RNA is related to a variety of diseases, including the occurrence and development of tumours. We summarised the dysregulated non-coding RNAs (miRNAs, circRNAs, and lncRNAs) reported currently in ovarian cancer and their functional mechanisms, and the clinical value of different types of ncRNAs as diagnostic or predictive markers for ovarian cancer, providing further evidence for non-coding RNAs to be considered as biomarkers of ovarian cancer.

Fertility Intention Among Chinese Reproductive Couples During the COVID-19 Outbreak: A Cross-Sectional Study.

A decline in the fertility rate has been observed worldwide, which hampers social development severely. Given the impacts of COVID-19 on individuals and society, it is of great significance to investigate the fertility intention of reproductive couples under COVID-19. The convenience sampling method was used to obtain our study sample. The self-administered questionnaire included the following components: sociodemographic characteristics (age, residence, education, occupation, characteristics of the couples, and annual household income), reproductive history (parity, number of children, child gender, and duration of preparing pregnancy), and attitudes toward COVID-19, was distributed online via an applet of WeChat. The results showed that among 4,133 valid questionnaires, 1,091 had fertility intention before COVID-19, whereas 3,042 did not, indicating a fertility intention rate of 26.4% among participating couples. Of the 1,091 couples who had fertility intention before COVID-19, 520 (47.7%) were affected by the outbreak, whereas 571 (52.3%) were not. By multivariable logistic regression analysis, we further found that couples living in Hubei Province, the epicenter in China (OR 2.20, 95% CI 1.35-3.60), and couples who prepared for pregnancy longer before COVID-19 (OR 1.19, 95% CI 1.06-1.33) were more likely to change their fertility intention under the pandemic. In addition, most of the participants reported their fertility intention was affected by the inconvenience of seeking medical service under COVID-19. Therefore, more forms of medical services to provide convenience for patients might be effective ways to reverse the declined fertility intention rate in facing COVID-19.

Karyotype of the Blastocoel Fluid Derived by Laser-Assisted Hatching Demonstrates a Low Agreement With the Trophectoderm.

Objective: The aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst. Methods: Fifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS). Results: Fifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23). Conclusion: Blastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised.

Effect of new biological patch in repairing intrauterine adhesion and improving clinical pregnancy outcome in infertile women: study protocol for a randomized controlled trial.

BACKGROUND: Endometrial fibrosis caused by intrauterine adhesion (IUA) can lead to hypomenorrhea, amenorrhea, and even infertility and abortion. The postoperative recurrence rate of severe IUA remains high, giving rise to low pregnancy rates. An extracellular matrix (ECM) scaffold, a new biological material that can promote cell proliferation and differentiation at lesions, has been widely used in general surgery and neurosurgery. The present study applied ECM scaffolds in obstetrics and gynecology for the first time to improve endometrial fibrosis, repair severe IUA, and improve pregnancy outcomes for infertile patients. METHODS: This paper presents a prospective randomized single-blind controlled superiority study of infertile women aged ≤40 years with IUA. According to the scoring criteria for IUA established by the American Fertility Society, patients with moderate or severe IUA were randomized into two groups at a ratio of 1:1; patients in the experimental group were treated with an ECM scaffold (small intestinal submucosa [SIS]) + intrauterine balloon, while patients in the control group were treated with an intrauterine balloon only. A hysteroscopic examination of adhesion repair was performed again after 2 months of postoperative hormone replacement therapy. Endometrial tissue was sampled during the two operations, and immunohistochemistry was used to observe endometrial and microvascular proliferation. After thawing and resuscitation, a postoperative frozen embryo transfer was performed on the participants in both groups, and their endometrial thickness, intrauterine volume, endometrial vascularization flow index, endometrial flow index, and uterine artery blood flow resistance were evaluated by 3D ultrasonography. The rates of embryo implantation, clinical pregnancy, and early spontaneous abortion were observed. DISCUSSION: The ECM scaffold (SIS) + intrauterine balloon method was able to repair endometrial fibrosis and improve IUA. This new technique represents a novel treatment method for improving the pregnancy outcome of infertile patients with moderate/severe IUA. TRIAL REGISTRATION: Chinese Clinical Trial Register ChiCTR2100052027 . Registered on October 14, 2021.

The Value of Circulating microRNAs for Diagnosis and Prediction of Preeclampsia: a Meta-analysis and Systematic Review.

Preeclampsia (PE) is one of the main causes of maternal death worldwide, but our understanding of the molecular characteristics of disease progression is limited. In this meta-analysis, we aimed to assess the value of peripheral blood microRNAs (miRNAs) as diagnostic and predictive markers of PE. We screened PubMed, Web of Science, and Embase databases; searched articles about "miRNAs and PE" up to November 30, 2020; and conducted biological information and subgroup analysis. We used QUADAS-2 (quality assessment of diagnostic accuracy studies-2) to evaluate the included articles by two independent reviewers, calculated the combined diagnostic and predictive indicators using the random effects model, explored the sources of potential heterogeneity through subgroup analysis, and evaluated publication bias using Deeks' funnel plot asymmetry test using Stata 14.0 and Review Manager 5.3 software. Forty-three miRNAs from 15 studies, including 2042 healthy controls and 2685 PE patients, had a pooled sensitivity of 0.86 (95% CI: 0.81-0.90), specificity of 0.89 (95% CI: 0.85-0.92), and an AUC of 0.94 (95% CI: 0.91-0.96). Moreover, before 20 weeks of gestation, the combined sensitivity was 0.86 (95% CI: 0.75-0.92), and the specificity was 0.90 (95% CI: 0.83-0.95), which indicated that some of the circulating miRNAs had changed significantly before the clinical symptoms appeared in PE patients. Circulating miRNAs have high diagnostic and predictive accuracy and may be used as non-invasive biomarkers for the diagnosis and prediction of PE. However, a large sample prospective study is still needed.

Analysis of the effect of phloroglucinol on pregnancy outcomes involving frozen embryo transfers in patients with endometriosis: A retrospective case-control study.

Objective: Abnormal contraction of uterus and vascular smooth muscle lead to the formation of hypoxia environment in uterus. Abnormal contraction may be the basis of dysmenorrhea, endometriosis, infertility and other diseases. Phloroglucinol is a non-atropine and non-papaverine smooth muscle spasmolytic agent, which can reduce the abnormal contraction of uterine smooth muscle. This study investigated the effect of phloroglucinol on frozen embryo transfer in patients with endometriosis. Methods: The data of patients with endometriosis who underwent a frozen embryo transfer in Shanghai Changzheng Hospital from August 2018 to August 2021, comprising a total of 453 cycles, were retrospectively analyzed. The patients for whom phloroglucinol was included over 217 cycles were administered intramuscully 40 mg phloroglucinol starting on the day of progesterone administration, then once daily up to day 7 after the embryo transfer. Those for whom phloroglucinol was not administered over 236 cycles were used as the control group. The age of 35 years was used as a boundary in this study to observe the pregnancy outcomes of patients in the two different age groups. Results: The biochemical pregnancy rate (63.13% vs. 51.27%), embryo implantation rate (44.64% vs. 33.60%), clinical pregnancy rate (59.64% vs. 48.30%), and live birth rate (52.99% vs. 36.86%) after the administration of phloroglucinol were higher than for patients in the control group, and the early abortion rate (7.75% vs. 20.18%) was also lower. The differences were statistically significant (P < 0.05). In particular, in the age group <35 years old, the embryo implantation rate (51.81% vs. 39.38%), clinical pregnancy rate (69.34% vs. 57.55%), and the live birth rate (63.50% vs. 44.60%) after phloroglucinol intervention rose significantly, and the abortion rate dropped (6.32% vs. 17.5%), indicating a statistically significant difference (P < 0.05). However, pregnancy outcomes showed no difference in the age group ≥35 years old (P > 0.05). Conclusion: Continuous low-dose phloroglucinol pretreatment before and after frozen embryo transfer can improve both the clinical pregnancy and live birth rates and reduce the risk of abortion in younger infertile patients with endometriosis.

Novel LAT Pathogenic Variants in a POI Family and Its Role in the Ovary.

Premature ovarian insufficiency (POI) affects about 1% of women under 40 years and leads most often to definitive infertility with adverse health outcomes. Genetic factor has been reported to play an important role in POI. However, the genetic etiology remains unknown in the majority of the POI patients. Whole-exome sequencing and variant analysis were carried out in a POI pedigree. In vitro studies of the wild-type and mutant proteins were conducted in primary granulosa cells (GCs) and granulosa cell line. The result showed that the patients carried compound heterozygous nonsynonymous mutations (c.245C > T and c.181C > G) in LAT gene, which were identified to be transmitted from their parents. The two variants were assessed to affect residues that were conserved across different species examined, and were predicted to be deleterious by software predictions. Protein structure predicting result indicated that the two variants could alter their interactions with surrounding residues, which may change the internal structure of the LAT protein. Moreover, LAT protein expression in GCs was demonstrated for the first time, and further functional assays suggested that this mutation could reduce LAT expression and influence GC survival, which may contribute to the etiology of POI. In summary, we detect novel LAT pathogenic variants in a POI pedigree and report for the first time that LAT is present and functional in the GCs of the ovary. Our findings not only shed new light on the role of LAT in GCs, but also broaden the spectrum of genetic causes of POI.

Comprehensive Characterization of Microbial Community in the Female Genital Tract of Reproductive-Aged Women in China.

The microbiota in the human body play critical roles in many physiological and pathological processes. However, the diversity and dynamics of the female genital tract (FGT) microbiota have not been fully unveiled. In this study, we characterized the microbiome variations in reproductive-aged Chinese women, and we revealed that the cervicovaginal microbiota were dominated by Lactobacillus. Overall, the composition of microbiota in the uterine cavity was more diverse than that in the vagina and cervix. A positive correlation between Lactobacillus iners and Lactobacillus crispatus was observed in both the vagina and the cervix, suggesting that these two species might have a symbiotic relationship in the cervicovaginal microbiota. Moreover, we, for the first time, stratified the reproductive-aged Chinese women into subgroups, based on their microbiome profiles. Furthermore, we identified the bacteria whose abundance changed in the uterine cavity of infertile patients when compared with healthy controls, such as L. iners and L. crispatus. Functionally, the metabolism-related pathways, neurotrophin signaling pathway, and adipocytokine signaling pathway were predominantly dysregulated in the uterine cavity of infertile patients. In conclusion, we characterized a comprehensive microbial landscape in FGT, as well as their functional roles in female infertility of the Chinese population.

Roles of noncoding RNAs in preeclampsia.

Preeclampsia (PE) is an idiopathic disease that occurs during pregnancy. It comprises multiple organ and system damage, and can seriously threaten the safety of the mother and infant throughout the perinatal period. As the pathogenesis of PE is unclear, there are few specific remedies. Currently, the only way to eliminate the clinical symptoms is to terminate the pregnancy. Although noncoding RNA (ncRNA) was once thought to be the "junk" of gene transcription, it is now known to be widely involved in pathological and physiological processes, including pregnancy-related disorders. Moreover, there is growing evidence that the unbalanced expression of specific ncRNA is involved in the pathogenesis of PE. In the present review, we summarize the expression patterns of ncRNAs, i.e., microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), and the functional mechanisms by which they affect the development of PE, and examine the clinical significance of ncRNAs as biomarkers for the diagnosis of PE. We also discuss the contributions made by genetic polymorphisms and epigenetic ncRNA regulation to PE. In the present review, we wish to explore and reinforce the clinical value of ncRNAs as noninvasive biomarkers of PE.

The m6A mRNA demethylase FTO in granulosa cells retards FOS-dependent ovarian aging.

Multifunctional N6-methyladenosine (m6A) has been revealed to be an important epigenetic component in various physiological and pathological processes, but its role in female ovarian aging remains unclear. Thus, we demonstrated m6A demethylase FTO downregulation and the ensuing increased m6A in granulosa cells (GCs) of human aged ovaries, while FTO-knockdown GCs showed faster aging-related phenotypes mediated. Using the m6A-RNA-sequence technique (m6A-seq), increased m6A was found in the FOS-mRNA-3'UTR, which is suggested to be an erasing target of FTO that slows the degradation of FOS-mRNA to upregulate FOS expression in GCs, eventually resulting in GC-mediated ovarian aging. FTO acts as a senescence-retarding protein via m6A, and FOS knockdown significantly alleviates the aging of FTO-knockdown GCs. Altogether, the abovementioned results indicate that FTO in GCs retards FOS-dependent ovarian aging, which is a potential diagnostic and therapeutic target against ovarian aging and age-related reproductive diseases.

The psychosocial impact of premature ovarian insufficiency on male partners and their perceptions of the disease.

Premature ovarian insufficiency (POI) is affecting about 1% women of reproductive age. However, current studies have primarily focused on women with the views of male partners greatly absent from the literature. We conduct this research to investigate the psychosocial effect of POI on male partners and their perceptions of the disease.52 male partners of POI patient (experiment group) and 52 controls (control group) were available for analysis. Anxiety, depression, and marital relationship were assessed for male partners in both groups. A questionnaire about perceptions of POI was completed by the experiment group. Male partners of POI patient experienced greater levels of anxiety (10.96 versus 4.88; P < 0.01) and depression (12.23 versus 5.19; P < 0.01) compared with controls. In addition, they experienced worse marital relationship in several aspects than their counterparts. The findings also demonstrate that most POI patient male partners had inadequate and inaccurate knowledge about their partners' disease, which may be the results of insufficient professional counseling from health-care practitioners. Moreover, their understanding level of the disease was correlated to anxiety (r = -0.64; P < 0.01), depression (r = -0.38; P < 0.01), and communication (r = 0.28; P < 0.05).The study highlights the need for health-care services, as well as support and professional information resources aimed at POI patients' male partners.

[Research progress of kisspeptin in female reproductive endocrine and assisted reproductive techniques].

In recent years, it has been found that kisspeptin plays some key roles in the physiological processes of the brain, such as gender differentiation, positive and negative feedback of sex hormones, onset of puberty, and transduction of energy signals in the body, which suggests that kisspeptin may be a key molecule for the maturation and regulation of female reproductive function. In addition to the systemic roles of the kisspeptin, its local roles in reproductive organs are constantly being discovered. With the discovery that kisspeptin is involved in the pathological process of reproductive endocrine diseases such as isolated hypogonadotropic hypogonadism (IHH), polycystic ovary syndrome (PCOS), premature ovarian failure (POF) and pathological hyperprolactinemia, exogenous application of kisspeptin to solve reproductive problems has become a new hot topic. The review focuses on the research progress of kisspeptin in the female reproductive system, especially on its application in assisted reproduction.

The Efficacy and Safety of the mTOR Signaling Pathway Activator, MHY1485, for in vitro Activation of Human Ovarian Tissue.

BACKGROUND: Premature ovarian insufficiency (POI) is characterized by abnormal ovarian function before the age of 40. POI showed that primordial follicles developed in disorder. mTOR signaling plays a vital role in the process of follicle development. It has been verified that the mTOR signaling pathway activator, MHY1485, can promote primordial follicle development in mice. We considered that MHY1485 would be a promising fertility preservation method for POI patients. METHODS: The fragmented ovarian tissues of normal woman was cultured with activator MHY1485 in vitro, and then the control and activated ovaries were transplanted into the kidney capsules of ovariectomized mice. We then used the Infinium Human Methylation EPIC BeadChip to verify the DNA methylation level of ovarian tissues, thus exploring the effectiveness of them. RESULTS: MHY1485 stimulated mTOR, S6K1, and rpS6 phosphorylation. Cultured with MHY1485, ovarian weights increased and endocrine function was restored. The number of growing follicles was increased. The in vitro activation process did not induce histological changes or abnormal DNA methylation occurrence. CONCLUSION: MHY1485 for in vitro activation (IVA) is effective for ovarian rejuvenation and is a potential therapeutic treatment for POI patients.

H3K4me3-Mediated Upregulation of LncRNA-HEIPP in Preeclampsia Placenta Affects Invasion of Trophoblast Cells.

Preeclampsia (PE) is a pregnancy-related disease defined as onset of hypertension and proteinuria after the 20th week of pregnancy, which causes most maternal and perinatal morbidity and mortality. Although placental dysfunction is considered as the main cause of PE, the exact pathogenesis of PE is not yet fully understood. Long non-coding RNAs (lncRNAs) are implicated in a broad range of physiological and pathological processes, including the occurrence of PE. In this study, we investigated the expression and functions of HIF-1α pathway-related lncRNA-HEIPP (high expression in PE placenta) in the pathogenesis of PE. The expression of lncRNA-HEIPP in the placenta from women who underwent PE was screened by lncRNA microarray and then verified using real-time polymerase chain reaction. Then, the methylation profile of the lncRNA-HEIPP promoter and the enrichment of H3K4me3 binding were assessed by bisulfite pyrosequencing and chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) assay, respectively. It was found that the level of lncRNA-HEIPP in the PE placenta was significantly higher than that in normal placenta and was increased in HTR-8/SVneo human trophoblast cells upon hypoxia treatment. Moreover, we reported that H3K4me3 manifested significantly higher promoter occupancy on lncRNA-HEIPP promoter in HTR-8/SVneo cells upon hypoxia treatment and found that the downregulation of lncRNA-HEIPP promoted trophoblast invasion. Our findings suggested that the hypoxia-induced expression of lncRNA-HEIPP mediated by H3K4me3 modification in trophoblast may contribute to the pathogenesis of PE.