Effect of endometrial biopsy method on ribonucleic acid quality and gene expression analysis in patients with leiomyoma.

Objective: To compare ribonucleic acid (RNA) quantity and purity in tissue collected with different endometrial sampling methods to establish the optimal tool for use in endometrial gene expression studies. Design: Observational study. Setting: University hospital. Patients: Fourteen patients with submucosal leiomyomas. Interventions: Unguided biopsies were obtained using a low-pressure suction device before hysteroscopy from 14 patients with submucosal leiomyomas followed by guided biopsy with a resectoscope loop. Fifty-seven samples were collected: 25 obtained using a suction device and 32 with a loop. Main Outcome Measures: Total biopsy weight, RNA purity, and RNA yield for each collection method. After complementary deoxyribonucleic acid synthesis, HOXA10 expression was measured by quantitative polymerase chain reaction in the endometrium overlying and remote from the leiomyoma, as similar expression throughout the cavity was a prerequisite for the use of unguided biopsy method. Results: The median weight of the samples was significantly larger when obtained with the low-pressure suction device than with the resectoscope loop (153 vs. 20 mg). The RNA yield was similar (suction curette, 1,625 ng/mg; resectoscope loop, 1,779 ng/mg). The A260-to-A280 ratio was satisfactory for 94.7 % of the samples, with no difference between the groups. The endometrial expression of HOXA10 was similar in areas overlying the leiomyoma compared with that in remote endometrial sites (2-ΔCt = 0.0224 vs. 0.0225). Conclusions: Low-pressure endometrial suction devices provide tissue samples with acceptable RNA purity and quantity for gene expression studies. The expression of HOXA10 did not differ between endometrial sampling sites even in the presence of leiomyomas.

Single-cell m6A mapping in vivo using picoMeRIP-seq.

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

Endometrial cytokines in patients with and without endometriosis evaluated for infertility.

OBJECTIVE: To evaluate whether endometrial molecular profiles distinguish subsets of patients according to clinical characteristics, and to infer dysregulated immune networks, by measuring cytokines, chemokines, and growth factors in endometrial biopsy specimens from a cohort of infertile women with a high incidence of endometriosis. DESIGN: Prospective cohort study. SETTING: Department of Gynecology at a university hospital. PATIENT(S): Patients undergoing laparoscopy for infertility assessment (n = 103). INTERVENTION(S): Endometrial biopsies were performed during surgery. Fertility outcome and clinical parameters were registered preoperatively and after 6 months. MAIN OUTCOME MEASURE(S): The concentrations of 48 factors in endometrial biopsy specimens were analyzed with respect to clinical status in univariate and multivariate frameworks. RESULT(S): The concentrations of 44 factors from endometrial tissues of 74 patients were suitable for analysis. Although the tissue concentrations of interleukin (IL)15, IL-7, and interferon γ-induced protein (IP)-10 were individually lower in patients with endometriosis than in those without endometriosis, the differences were not significant after multiple comparison. However, multivariate modeling incorporating covariation showed separation between subsets of endometriotic and nonendometriotic patients, based predominantly on IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18; this result was independent of cycle and fertility status. Analysis restricted to endometrial tissues from the secretory phase separated endometriotic and nonendometriotic patients by a combination of IL-15, IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18. This combination suggests a uterine natural killer cell defect. We found no significant correlations between endometrial cytokines and fertility outcome. CONCLUSION(S): A molecular signature in endometrial tissue was able to distinguish endometriotic from nonendometriotic patients, implicating uterine natural killer cells in endometriosis.

Can time to menopause be predicted?

Menopause represents the definite end of a woman's reproductive life and the onset of a persistent hypoestrogenic state. This postmenopausal period will for most women last several decades. Although mean menopausal age seems to have increased somewhat during the last century, there is a significant individual variation in age at natural menopause. With efficient contraception, women of reproductive age can now, to some extent, choose when they want to have children. As a consequence of this and other sociodemographic changes, age at first birth has increased significantly over the last 50 years. It is well documented that long before a woman enters the menopausal transition and subsequent menopause, fertility declines and finally ceases. Being able to predict when a woman will enter menopause would therefore, from a reproductive perspective, be of major interest. Several sociodemographic, morphometric, and endocrine factors are associated with age at menopause or time to menopause. Unfortunately the sensitivity and specificity of these in predicting time to or age at menopause are low. Therefore, with the exception of anti-Müllerian hormone measurements, either alone or in combination with chronological age close to menopause, there are as of now no reliable ways of predicting when a woman will enter menopause.

Spatial and temporal changes in follicle distribution in the human ovarian cortex.

RESEARCH QUESTION: How does follicle distribution evolve in the human ovarian cortex between the ages of 20 and 35 years? DESIGN: Fragments of ovarian cortex from women undergoing unilateral oophorectomy for fertility preservation were obtained for quantitative histological assessment, including recording the two-dimensional coordinates of the follicles. Data were analysed using spatial statistical methods. RESULTS: A total of 53 ovarian cortex tissue samples, containing 1-803 follicles each, were obtained from 14 women aged 20-35 years. Primordial and transitory follicles lay in a clustered manner in the human ovarian cortex, with an average cluster radius of around 270 µm (95% confidence interval 154-377 µm; n = 49). Follicle density declined with age (P = 0.006, n = 13), and the distance from the nearest neighbouring follicle increased (P = 0.004, n = 13). Cluster radius decreased with age (P = 0.02, n = 13), but the degree of clustering tended to increase (P = 0.11, n = 13). In the majority of the samples, follicles at different stages lay in different clusters (P < 0.05, n = 13). CONCLUSIONS: This study shows that primordial and transitory follicles lie in different clusters in the human ovarian cortex. Spatio-temporal computer simulation suggests that interfollicular signals may hinder follicle loss and may therefore drive clustered follicle distribution. In clinical practice, the woman's age should be taken into account when assessing follicle density, as follicle distribution is increasingly clustered with advancing age.

Ovulation induction in polycystic ovary syndrome.

The objective of this narrative review was to suggest a rational order of treatment choices in anovulatory women with polycystic ovary syndrome (PCOS), for whom a multitude of treatment options exist. In obese/overweight women with PCOS the importance of weight reduction should be stressed. Inositol, a dietary supplement with a documented effect on ovulation and without adverse effects in the doses recommended, may be suggested. Additional first-line medical alternatives include insulin sensitizers, selective estrogen receptor modulators, and aromatase inhibitors. Of these, the aromatase inhibitor letrozole and the combination of clomiphene citrate and metformin have the highest rates of ovulation and live birth. Second-line treatments are ovarian electrocautery and low-dose follicle-stimulating hormone stimulation. Controlled ovarian stimulation with in vitro fertilization, should be considered the last option as it carries a significant risk of ovarian hyperstimulation syndrome in patients with PCOS.

The prevalence of Type 2 diabetes is not increased in normal-weight women with PCOS.

STUDY QUESTION: Is oral glucose tolerance test (OGTT) needed in all women with polycystic ovary syndrome (PCOS)? SUMMARY QNSWER: OGTT is not routinely needed in women with PCOS and BMI < 25 kg/m2. WHAT IS KNOWN ALREADY: PCOS is associated with insulin resistance and increased prevalence of prediabetes and Type 2 diabetes (T2D) which is closely linked to obesity and possibly age, ethnicity and PCOS phenotype. Several guidelines recommend OGTT upon diagnosis of PCOS and during follow-up. STUDY DESIGN, SIZE, DURATION: A Nordic cross-sectional study including 876 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 876 Nordic women with PCOS, aged 14-57 years, were examined for T2D and prediabetes (impaired glucose tolerance [IGT] or impaired fasting glucose (IFG) by OGTT. MAIN RESULT AND THE ROLE OF CHANCE: Of all study subjects 3% (23/876) had T2D, 23% (204/876) prediabetes and 74% (649/876) had normal glucose tolerance (NGT). Increased BMI and waist circumference were significantly (P < 0.001) associated with prevalence of prediabetes and T2D. No normal-weight woman (BMI < 25 kg/m2) was diagnosed with T2D. The prevalence of BMI ≥ 25 kg/m2 was 66% (578/ 876). 91% of women (21/23) with T2D had BMI ≥ 30 kg/m2. Testosterone levels and PCOS phenotype did not predict 2-h glucose levels during OGTT after adjustment for BMI and age. LIMITATIONS, REASONS FOR CAUTION: The present study included cross-sectional data and prospective studies are needed to confirm our results. These results may not apply to populations of other ethnic origin. WIDER IMPLICATIONS OF THE FINDINGS: Routine OGTT may not be indicated in normal-weight women with PCOS. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Genome-wide profiling of DNA 5-hydroxymethylcytosine during rat Sertoli cell maturation.

Sertoli cells have dual roles during the cells' lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression.

Peritoneal fluid cytokines related to endometriosis in patients evaluated for infertility.

OBJECTIVE: Our aim was to characterize peritoneal cytokine profiles in patients with infertility, with and without endometriosis, to illuminate potential differences in immune profiles that may reflect mechanistic differences between these two patient populations. DESIGN: Cross-sectional study. SETTING: University hospital and research center. PATIENT(S): Women undergoing laparoscopy for infertility investigation (n = 107). INTERVENTION(S): Peritoneal fluid was collected during surgery. Clinical characteristics were registered preoperatively. MAIN OUTCOME MEASURE(S): We determined the concentration of 48 different cytokines from the peritoneal fluid with multiplex immunoassays. Associations between cytokines and clinical findings were assessed with logistic regression and partial least squares discriminant analyses (PLS-DA). RESULT(S): Concentrations of SCGF-ß, IL-8, HGF, and MCP-1 were significantly higher, while IL-13 was significantly lower in the endometriosis group compared with the group without endometriosis. Multiple stepwise logistic regression identified a combination of SCGF-ß, IL-13, and G-CSF concentrations that predicted the presence of endometriosis with 86% sensitivity and 67% specificity. PLS-DA identified a class of 11 cytokines (SCGF-ß, HGF, IL-13, MCP-1, CTACK, MCP-3, M-CSF, LIF, IL-5, IL-9, and IFN-a2) that were more characteristic of endometriosis than nonendometriosis patients. CONCLUSION(S): By combining univariate and multivariate analyses, profiles of cytokines more likely to be enriched or depleted in infertility patients with endometriosis compared with those without endometriosis were identified. These findings may inform future analyses of pathophysiological mechanisms of endometriosis in infertile patients, including dysregulated Th1/Th2 response and mobilization and proliferation of hematopoietic stem cells.

Endometriosis-associated infertility: aspects of pathophysiological mechanisms and treatment options.

Endometriosis is a common condition in women of reproductive age. In addition to pain, endometriosis may also reduce fertility. The causes of infertility in women with endometriosis may range from anatomical distortions due to adhesions and fibrosis to endocrine abnormalities and immunological disturbances. In some cases, the various pathophysiological disturbances seem to interact through mechanisms so far not fully understood. Whether surgery should be offered as a treatment option in endometriosis-associated infertility has become controversial, partly due to its modest or undocumented effect. Medical or hormonal treatment alone has little or no effect and should only be used in conjunction with assisted reproductive technology (ART). Of the various methods of ART, intrauterine insemination, due to its simplicity, can be recommended in women with minimal or mild peritoneal endometriosis, even though insemination may yield a lower success rate than in women without endometriosis. In vitro fertilization (IVF) is an effective treatment option in less-advanced disease stages, and the success rates are similar to the results in other causes of infertility. However, women with more advanced stages of endometriosis have lower success rates with IVF.

Reversible RNA modifications in meiosis and pluripotency.

Post-transcriptional RNA modifications were discovered several decades ago, but the reversible nature of RNA modifications has only recently been discovered. Owing to technological advances, knowledge of epitranscriptomic marks and their writers, readers and erasers has recently advanced tremendously. Here we focus on the roles of the dynamic methylation and demethylation of internal adenosines in mRNA in germ cells and pluripotent stem cells.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Sequencing of FTO and ALKBH5 in men undergoing infertility work-up identifies an infertility-associated variant and two missense mutations.

OBJECTIVE: To assess whether men with reduced semen quality exhibit genetic variants in the genes coding for the messenger RNA methylation erasers FTO and ALKBH5. DESIGN: DNA of men undergoing infertility work-up was extracted and the FTO and ALKBH5 genes were sequenced. Statistical analysis was used to study the correlation between the identified ALKBH5 and FTO variants and sperm quality. SETTING: University hospital infertility clinic. PATIENT(S): Semen samples from 77 unselected men that had been referred to Oslo University Hospital for routine semen analysis as part of infertility work-up. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Immunohistochemistry and Western blot were used to confirm the presence of ALKBH5 and FTO in human testis. DNA extraction from samples was followed by Illumina MiSeq amplicon high throughput sequencing and sequence alignment. Variant calling was carried out using GATK's UnifiedGenotyper. Standard semen parameter analysis was performed according to World Health Organization guidelines. RESULT(S): We found an FTO genetic variant to be associated with reduced semen quality. We also identified two FTO missense variants, one mutation (p.Cys326Ser) was located in the important linker between the two protein domains; the other mutation (p.Ser256Asn) was situated in a flexible loop able to interact with other molecules. CONCLUSION(S): The discovery of two missense mutations with potentially detrimental effect on the functionality of the methylation eraser protein FTO, as well as a genetic variant of the same protein that is associated with altered semen quality could suggest that aberrant demethylation of messenger RNA is a factor involved in reduced male fertility.

Autotransplantation of cryopreserved ovarian tissue after treatment for malignant disease - the first Norwegian results.

INTRODUCTION: With increasing survival rates after treatment for cancer in prepubertal girls and women of reproductive age, an increasing focus on quality of life has emerged. Both irradiation and cytotoxic drugs can be detrimental to future fertility, consequently several treatment alternatives have been developed to spare or restore fertility in young females diagnosed with cancer. One of these options is cryopreservation of ovarian tissue before treatment and autotransplantation at a later time. MATERIAL AND METHODS: We present the Norwegian experience after 11 years of practice with ovarian tissue cryopreservation. A total of 164 patients have had ovarian tissue cryopreserved during the period 2004-2014. Fifteen patients died during the observation period. Six patients requested autotransplantation, which was performed in two women. RESULTS: Both patients conceived, one spontaneously and one after assisted reproduction due to a concomitant male factor. The pregnancies were uneventful and they each gave birth to a healthy child. CONCLUSIONS: Cryopreservation with later autotransplantation of ovarian tissue should be offered to a selected group of young women with cancer.

Fragmentation of human cleavage-stage embryos is related to the progression through meiotic and mitotic cell cycles.

OBJECTIVE: To study whether fragmentation of human embryos is related to the progression through meiotic and mitotic cell cycles. DESIGN: This report consists of two observational studies. SETTING: Not applicable. PATIENT(S): A total of 1,943 oocytes from 297 patients and 372 embryos from 100 patients were imaged in the Polscope instrument and monitored in the Embryoscope, respectively. INTERVENTION(S): Completion of the first meiotic division was determined by visualization of the meiotic metaphase II spindle in human oocytes, and the duration of the first three mitotic cell cycles was determined with time-lapse microscopy. The percentage of embryo fragmentation was recorded 42-45 hours after insemination. MAIN OUTCOME MEASURE(S): Appearance of the meiotic spindle; durations of the first, second, and third mitoses. RESULT(S): Human embryos with a low degree of fragmentation (<10%) at 42-45 hours after insemination originated from oocytes with an early appearance of the meiotic spindle (mean 35.5 hours after hCG injection), early first mitosis (28.2 hours after insemination), late start of the second mitosis (38.0 hours after insemination), and a shorter duration of the third mitosis (1.1 hours). Highly fragmented embryos (>50% fragmentation) originated from oocytes with a late-appearing meiotic spindle (36.5 hours after hCG injection), delayed initiation of the first mitosis (29.8 hours after insemination), early start of the second mitosis (36.4 hours after insemination), and a longer duration of the third mitotic cell cycle (4.1 hours). CONCLUSION(S): The observed associations suggest that the process of fragmentation of in vitro-derived embryos was related to the progress of the meiotic and the mitotic cell cycles.

Female reproductive decline is determined by remaining ovarian reserve and age.

The early decline and loss of female fertility in humans and other species represents an evolutionary paradox. Despite being born with a vast stock of oocytes, females encounter an exhaustion of ovarian reserve and sterility half way through their natural lives. Female reproductive ageing has been proposed to proceed as an ongoing decline in ovarian reserve, determined by remaining ovarian follicle number. However, despite extensive modelling, the respective contributions of intra-, inter-, and extra-ovarian signalling have not been fully characterised. It remains unclear whether reproductive ageing progresses simply as a pre-determined function of remaining ovarian follicles, or as an age-dependent process in humans. Here, we have analysed ovarian response to hormonal stimulation in women who have undergone surgical removal of a single ovary, in order to investigate the relative contributions of intra-, inter, and extra-ovarian signalling on reproductive ageing. Our data show that in unilaterally oophorectomised women, ovarian response to follicle stimulating hormone (FSH) declines beyond levels predicted by a total ovarian follicle pool model of reproductive ageing. Maintenance of ovarian function later in reproductive life, despite the removal of half of the total ovarian reserve, suggests a role for an extra-ovarian age-dependent regulation of reproductive decline. This highlights the need for further work to identify signalling factors that communicate age-related signals between the soma and the germline.

ALKBH4 depletion in mice leads to spermatogenic defects.

ALKBH4, an AlkB homologue in the 2-oxoglutarate and Fe2+ dependent hydroxylase family, has previously been shown to regulate the level of monomethylated lysine-84 in actin and thereby indirectly influences the ability of non-muscular myosin II to bind actin filaments. ALKBH4 modulates fundamental processes including cytokinesis and cell motility, and its depletion is lethal during early preimplantation embryo stage. The aim of this study was to investigate the effect of ALKBH4 deficiency in a physiological context, using inducible Alkbh4 knockout mice. Here, we report that ALKBH4 is essential for the development of spermatocytes during the prophase of meiosis, and that ALKBH4 depletion leads to insufficient establishment of the synaptonemal complex. We also show that ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia and primary spermatocytes.