A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Single-Incision Laparoscopic Surgery for Ovarian Tissue Cryopreservation.

STUDY OBJECTIVE: To present single-incision laparoscopic surgery (SILS) as an alternative to standard multiport laparoscopic surgery (MPLS) for ovarian tissue cryopreservation. DESIGN: Retrospective cohort study (Canadian Task Force classification II-2). SETTING: Tertiary medical center. PATIENTS: Two hundred thirty-one patients referred for oncologic fertility preservation. INTERVENTION: Non-inferiority comparison of SILS with MPLS for ovarian cryopreservation for future transplantation. MEASUREMENTS AND MAIN RESULTS: We compared duration of the procedure, intra- and postoperative complications, hospital stay, and time (days) from surgery to chemotherapy. We additionally compared number of ampules (each ampule contains 10 slivers of ovarian cortex tissue) preserved and number of oocytes retrieved during the preparation process. Adjustments for age, previous chemotherapy, and partial versus complete oophorectomy ratio were performed. MPLS was performed in 163 patients (71.2%) and SILS in 66 patients (28.8%). Ten patients (15.2%) in the SILS group and 32 (19.8%) in the MPLS group were prepubertal. Malignant conditions distribution was similar. Procedure duration and overall complication rates were similar. Number of ampules extracted from the preserved tissue was somewhat higher in the SILS group as compared with the MPLS group (14.7 vs 10.6, respectively; p < .01). CONCLUSION: Our findings suggest that SILS is an interesting alternative to MPLS. Future prospective trials may prove some benefit in ovarian tissue volume or time until chemotherapy initiation.

A Case Report of Familial Mayer-Rokitansky-Küster-Hauser Syndrome as Part of the Phenotypic Spectrum of the 2q37 Deletion.

We performed a genetic investigation into the case of an inherited Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Our patients were an adolescent and her mother, both with MRKH syndrome. The delivery of a biological offspring was achieved via a gestational carrier. Karyotype and exome sequencing were used to complete a three-generation genetic analysis of the family. Both the mother and her daughter harbored a deletion of 4 Mb at the locus of 2q37, a syndrome rarely described in association with MRKH. No pathogenic single-nucleotide variant relevant to the phenotype was found. The deletion was not inherited from either parent of the mother. In addition, some physical findings suggesting 2q37 deletion syndrome were found in our patients. We conclude that when combined with the use of a gestational carrier or uterine transplantation, the identification of a genetic cause for MRKH may enable the application of preimplantation genetic testing on embryos, thus potentially averting the transmission of the genetic anomaly to subsequent generations.

Impact of obesity on female puberty and pubertal disorders.

The worldwide epidemic of obesity appears to be one of the crucial health problems. One-third of children and adolescents in the United States are classified as either overweight or obese and 6% of adolescents are severely obese. With the development of high technology, children, and adolescents, spend more time in sedentary life. Together with the availability of consuming fast caloric food, it is almost unavoidable to get an imbalance between caloric intake and caloric expenditure of energy, resulting in the accumulation of fat-energy stores. Obesity has an enormous impact on physical and psychological health and is associated with more than 29 adverse health consequences previously identified in the adult population only, such as type 2 diabetes mellitus, hypertension, metabolic syndrome, postmenopausal breast cancer, and others. In addition, excess adiposity may exert a harmful effect on the reproductive system, resulting in precocious puberty, irregular menstrual cycle, polycystic ovary syndrome, and high-risk sexual behavior. Intervention programs for healthy lifestyle modification, consisting of caloric restriction together with physical activity did not gain the expected efficacy. Other approaches as medical and surgical therapies are currently not evidence-based for the young population. The obesity crisis in children and adolescents requires a better understanding of etiology, pathophysiology, and management of obesity in this unique population. Researchers and physicians must "turn over every stone" to find a solution for deviating obesity ascent.

Fertility preservation in pediatric, adolescent and young adult female cancer patients.

Advancements in chemotherapy and radiation therapies have increased survival rates in cancer patients. Late effects of therapy, including infertility, become increasingly more important with increased survival. High dose alkylating agents, total body irradiation and radiation to the gonads have the greatest effect on fertility. Fertility preservation therapies improve the reproductive potential of adolescent and young adult cancer patients receiving cancer therapies. Gonadal shielding, ovarian transposition and embryo cryopreservation are standard fertility preserving therapies. Oocyte and ovarian tissue cryopreservation have proven successful and the latter may be implemented under IRB approval. Gonadal suppression with gonadotropin releasing hormone agonist (GnRH) therapy has yielded conflicting results and larger prospective trials in both adolescents and adults are necessary. A team approach involving oncology, gynecology and reproductive endocrinology and infertility provides the best outcome for patients.

Outpatient hysteroscopic removal of intrauterine devices in early pregnancy: feasibility and outcomes.

OBJECTIVE: To describe our experience with outpatient hysteroscopy for removal of intrauterine devices (IUDs) in pregnant patients, along with the pregnancy-related outcomes. DESIGN: Retrospectively and prospectively collected data between January 2015 and April 2021. SETTING: Hysteroscopic outpatient clinic (See and Treat Clinic) at a university affiliated, tertiary medical center. PATIENT(S): Forty-one patients with an inadvertent but desired pregnancy up to 12 weeks + 6 days gestational age with an IUD in situ, with documented failed attempts of IUD removal. INTERVENTION(S): After ultrasonographic confirmation of IUD location, gestational age, and viability, a small-caliber hysteroscope was introduced via a vaginoscopic approach. The uterus was distended using 0.9% normal saline until a clear view was achieved. On visualization, the IUD was grasped by its strings, tail, or lateral arm using a semirigid hysteroscopic grasper. Oral antibiotic prophylaxis was prescribed in all cases. MAIN OUTCOME MEASURE(S): The primary outcome of interest was term delivery, from 37 weeks of gestation. Secondary outcomes included patient-reported tolerability and satisfaction, and procedure-related and pregnancy related complications. RESULT(S): The procedure was completed successfully in all 41 patients. Thirty-three patients continued their pregnancies, resulting in 32 full-term singleton deliveries and 1 singleton preterm delivery at 33 + 4 weeks. Median gestational age at delivery was 39 weeks, with a median birthweight of 3,450 grams. Eight patients (19.5%) miscarried, 4 of these within a week of the procedure. CONCLUSION(S): In cases of desired pregnancy with an IUD, outpatient hysteroscopic removal of the IUD is a safe and effective management option. Broader uptake of outpatient hysteroscopy and development of hysteroscopic skills will allow more clinicians to offer patients this effective solution.

Reduced Activity and Quality of Life in Women Soldiers with Heavy Menstrual Bleeding and Dysmenorrhea.

STUDY OBJECTIVE: The aim of the study was to examine the effect of heavy menstrual bleeding (HMB) and dysmenorrhea on daily activity and quality of life (QoL) in young women engaged in demanding activities. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 422 Israeli women soldiers in combat and non-combat roles. INTERVENTIONS: Participants were asked to provide consent and to complete study questionnaires. MAIN OUTCOME MEASURES: A pictorial bleeding assessment chart (PBAC), visual analog scale (VAS), verbal multidimensional scoring system for assessment of dysmenorrhea, and approved Hebrew-translated age-appropriate Pediatric Quality of Life Inventory (PedsQL). RESULTS: HMB (PBAC >100) and severe HMB (PBAC >185) were demonstrated in 181 (50%) and 96 (26%) participants, respectively. A high PBAC score was recorded in 20% who answered "no" or "unknown" in the questionnaire on having HMB. Mild, moderate, and severe dysmenorrhea were demonstrated in 80 (21.5%), 115 (31%), and 142 (38%) participants, respectively. The prevalence of HMB and dysmenorrhea was similar in soldiers in combat and non-combat roles. Diagnosis of HMB was related to the lower fitness-for-service score, history of bleeding, and dysmenorrhea. Daily activity and QoL were both affected by the severity of HMB and dysmenorrhea. CONCLUSION: Underdiagnosis of HMB and dysmenorrhea results from a combination of unawareness from the women's side and inattention from the system. In an era of female empowerment, each woman should be at the optimal physiological and psychological level to start her career; thus, addressing the menstrual burden and providing effective treatment is needed in the military scenario and other settings with demanding activities.

Pre-pubertal oocytes harbor altered histone modifications and chromatin configuration.

Pre-pubertal oocytes are still dormant. They are arrested in a GV state and do not undergo meiotic divisions naturally. A multitude of molecular pathways are changed and triggered upon initiation of puberty. It is not yet clear which epigenetic events occur in oocytes upon pubertal transition, and how significant these epigenetic events may be. We evaluated epigenetic marker levels in mouse pre-pubertal and post-pubertal female oocytes. In addition, we evaluated H3K9me2 levels in human oocytes collected from fertility preservation patients, comparing the levels between pre-pubertal patients and post-pubertal patients. The chromatin structure shows a lower number of chromocenters in mouse post-pubertal oocytes in comparison to pre-pubertal oocytes. All heterochromatin marker levels checked (H3K9me2, H3K27me3, H4K20me1) significantly rise across the pubertal transition. Euchromatin markers vary in their behavior. While H3K4me3 levels rise with the pubertal transition, H3K27Ac levels decrease with the pubertal transition. Treatment with SRT1720 [histone deacetylase (HDAC) activator] or overexpression of heterochromatin factors does not lead to increased heterochromatin in pre-pubertal oocytes. However, treatment of pre-pubertal oocytes with follicle-stimulating hormone (FSH) for 24 h - changes their chromatin structure to a post-pubertal configuration, lowers the number of chromocenters and elevates their histone methylation levels, showing that hormones play a key role in chromatin regulation of pubertal transition. Our work shows that pubertal transition leads to reorganization of oocyte chromatin and elevation of histone methylation levels, thus advancing oocyte developmental phenotype. These results provide the basis for finding conditions for in-vitro maturation of pre-pubertal oocytes, mainly needed to artificially mature oocytes of young cancer survivors for fertility preservation purposes.

Age-Dependent in vitro Maturation Efficacy of Human Oocytes - Is There an Optimal Age?

In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over ovarian tissue cryopreservation (OTC), as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that in vitro maturation (IVM) performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. We conducted a retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM. A total of 133 chemotherapy naïve patients aged 1-35 years were included in the study. The primary outcome was IVM rate in the different age groups - pre-menarche (1-5 and ≥6 years), post-menarche (menarche-17 years), young adults (18-24 years) and adults (25-29 and 30-35 years). We demonstrate a gradual increase in mean IVM rate in the age groups from 1 to 25 years [4.6% (1-5 years), 23.8% (6 years to menarche), and 28.4% (menarche to 17 years)], with a peak of 38.3% in the 18-24 years group, followed by a decrease in the 25-29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30-35 years group. A significant difference in IVM rates was noted between the age extremes - the very young (1-5 years) and the oldest (30-35 years) groups, as compared with the 18-24-year group (p < 0.001). Importantly, number of oocytes matured, percent of patients with matured oocytes, and overall maturation rate differed significantly (p < 0.001). Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggests that oocytes retrieved during OTC prior to chemotherapy have an optimal window of age that shows higher success rates, suggesting that oocytes may have an inherent tendency toward better maturation in those age groups.

Gonadotropin releasing hormone analogue treatment of central precocious puberty is not associated with altered prevalence of polycystic ovary syndrome: a single center cohort study.

BACKGROUND: There is conflicting evidence regarding an association between gonadotropin releasing hormone analogue (GnRHa) therapy and polycystic ovary syndrome (PCOS). This study aimed to compare the prevalence of endocrine disorders, primarily PCOS, between women who had been treated with GnRHa for central precocious puberty (CPP) and those who were not treated. METHODS: This was a retrospective cohort study, including women diagnosed with central precocious puberty between 1989 and 2011 in a university affiliated tertiary medical center. Data collected included demographic data, medical background, clinical presentation at diagnosis and duration of treatment (zero for non-treated). Gynecologic and endocrine long-term outcomes were compared by treatment group. RESULTS: Fifty-one women were included in the study, 27/51 had been treated with gonadotropin releasing hormone analogue (GnRHa). Overall prevalence of PCOS was 19.6%. No statistically significant difference in prevalence of PCOS was demonstrated between the treated and non-treated groups. Similarly, overall prevalence of either clinical or laboratory hyper-androgenism, was 29.4% and 33.3%, for the treatment and non-treatment groups respectively (p = non-significant). CONCLUSIONS: GnRHa treatment for precocious puberty is not associated with increased risk of polycystic ovary syndrome.

Standardizing care to manage bleeding disorders in adolescents with heavy menses-A joint project from the ISTH pediatric/neonatal and women's health SSCs.

BACKGROUND: Bleeding disorders (BD) are under-recognized in adolescents with heavy menstrual bleeding (HMB). OBJECTIVES: The lack of clinical guidelines and variable symptomatic management of HMB created the imperative to standardize HMB care to identify and manage BD in adolescents. METHODS: We convened an international working group (WG), utilized the results of a literature review to define knowledge gaps in HMB care, and used the collective clinical experience of the WG to develop care considerations for adolescents with BD and HMB. We then solicited input on the appropriateness of HMB care considerations from expert stakeholders representing hematology, adolescent medicine, and obstetrics-gynecology. We conducted an expert panel online, using the ExpertLens platform. During a three-round online modified-Delphi process, the expert panel rated the appropriateness of 21 care considerations using a 9-point scale to designate care as appropriate (7-9), uncertain (4-6), or inappropriate (1-3) covering screening for BD, the laboratory work-up, and management of adolescents with BD that present with HMB. We used the RAND/UCLA appropriateness method to determine the existence of consensus among the interdisciplinary panel of experts. RESULTS: Thirty-nine experts participated in the panel. The experts rated fifteen HMB care considerations as appropriate, six as uncertain, and none as inappropriate. CONCLUSIONS: The HMB care statements represent the first set of HMB care considerations in adolescents with BD, developed with broad expert input on appropriateness. Although likely to be of interest to a range of clinicians who routinely manage adolescents with HMB, additional research is required in many key areas.

In vitro maturation rates in young premenarche patients.

OBJECTIVE: To evaluate in vitro maturation (IVM) efficacy and oocyte retrieval rates after ovarian tissue cryopreservation in young premenarche girls facing chemo- and radiotherapy. DESIGN: A retrospective cohort study. SETTING: University-affiliated tertiary medical center. PATIENT(S): A total of 84 chemotherapy-naïve patients ages 0-18 years referred for fertility preservation between 2004 and 2017: 33 premenarche and 51 postmenarche patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): IVM in the pre- and postmenarche groups and in the subgroups of very young (up to age 5 years) and older (5-10 years) premenarche girls. RESULTS: The number of oocytes retrieved did not significantly differ between the postmenarche and premenarche groups (10.8 ± 8.5 and 8.1 ± 6.8, respectively). However, the overall IVM rate was significantly higher in the postmenarche group (28.2% vs. 15.5%, respectively; odds ratio = 0.47). A separate analysis for patients up to 5 years of age demonstrated significantly lower oocyte yield compared with the older (5-10 years) premenarche girls (4.7 ± 5.2 vs.10.3 ± 7.0 oocytes, respectively) and much lower IVM rates (4.9% and 18.2%, respectively). Correlation of age with number of retrieved and matured oocytes showed a positive significant correlation (r = 0.45 and r = 0.64, respectively). CONCLUSIONS: IVM performed after ovarian tissue cryopreservation in premenarche girls and specifically in very young girls (4 years and younger) yields substantially decreased maturation rates compared with postmenarche patients, raising a question as to the utility of current IVM technique in this age group. Further studies are required to assess modification of the IVM technique for young girls.

Gynaecological issues affecting the obese adolescent.

The implications of obesity in childhood and adolescence resonate into adulthood and have implications at different levels that include psychosocial and health issues that impact beyond reproductive performance. This chapter explores the various facets and consequences on gynaecological issues of increased Body Mass Index in childhood, including the link with puberty, pubertal menorrhagia (also affecting children with complex needs) and the all too common problems surrounding hyperandrogenism, insulin resistance and the polycystic ovarian syndrome in particular which need to be seen in the specific context of the adolescent years. The wider ramifications of obesity on the psychosocial welfare of adolescents merits special attention. Finally management strategies are considered in the context of the needs of adolescents.

Two novel mutations identified in familial cases with Donohue syndrome.

Donohue syndrome (DS) is a rare and lethal autosomal recessive disease caused by mutations in the insulin receptor (INSR) gene, manifesting marked insulin resistance, severe growth retardation, hypertrichosis, and characteristic dysmorphic features. We report the clinical, molecular, and biochemical characterization of three new patients with DS, and address genotype-phenotype issues playing a role in the pathophysiology of DS. A female infant born to first-degree cousins Muslim Arab parents and two brothers born to first-degree cousins Druze parents presented classical features of DS with hypertrophic cardiomyopathy and died in infancy. Each patient was found homozygous for one missense mutation within the extracellular domain of the INSR gene. Western blot analysis identified the proreceptor of INSR, but not its mature subunits alpha and beta. Of 95 healthy Muslims, no heterozygous was found and of 52 healthy Druze from the same village, one was heterozygous. This study presents two novel familial mutations in the alpha subunit of the INSR which appear to impair post-translational processing of the INSR, resulting loss of its function. Both mutations cause DS with hypertrophic cardiomyopathy and early death. Identification of the causative mutation enables prevention of this devastating disease.

Diagnostic methods in pediatric and adolescent gynecology.

The evolving role of imaging techniques and endoscopic tools used in gynecologic practice has become essential over the last several years. Pelvic organs of the young girl are deeply hidden in the abdomen and the vagina is narrow and atrophic. The presence of an intact hymen provides an additional obstacle to vaginal examination. Therefore, bimanual pelvic (vaginal/rectal) examination has a limited advantage in this population. It could cause unnecessary discomfort and hamper further 'patient-doctor' dialog. Imaging techniques are constantly improving in sensitivity, thus providing more accurate diagnoses. Ultrasonography which is a safe and available modality provides real-time images of multiple planes and therefore is ideal for pelvic evaluation. It should be an integral part of child and young adolescent examination. Additional modalities such as CT and MRI should be reserved for cases which are ultrasonography doubtful and be applied as a subsequent evaluation. Vaginoscopy and laparoscopy, although invasive, are used for final diagnosis and provision of treatment as 'see and treat' procedures. These main modalities used in the diagnosis of gynecologic disorders in the young are described in this chapter.