Single Follicle Research Articles

Comprehensive analysis of mRNAs and miRNAs in the ovarian follicles of uniparous and multiple goats at estrus phase

BackgroundFertility is an important economic trait in the production of meat goat, and follicular development plays an important role in fertility. Although many mRNAs and microRNAs (miRNAs) have been found to play critical roles in ovarian biological processes, the interaction between mRNAs and miRNAs in follicular development is not yet completely understood. In addition, less attention has been given to the study of single follicle (dominant or atretic follicle) in goats. This study aimed to identify mRNAs, miRNAs, and signaling pathways as well as their interaction networks in the ovarian follicles (large follicles and small follicles) of uniparous and multiple Chuanzhong black goats at estrus phase using RNA-sequencing (RNA-seq) technique.ResultsThe results showed that there was a significant difference in the number of large follicles between uniparous and multiple goats (P < 0.05), but no difference in the number of small follicles was observed (P > 0.05). For the small follicles of uniparous and multiple goats at estrus phase, 289 differentially expressed mRNAs (DEmRNAs) and 16 DEmiRNAs were identified; and for the large follicles, 195 DEmRNAs and 7 DEmiRNAs were identified. The functional enrichment analysis showed that DE genes in small follicles were significantly enriched in ovarian steroidogenesis and steroid hormone biosynthesis, while in large follicles were significantly enriched in ABC transporters and steroid hormone biosynthesis. The results of quantitative real-time polymerase chain reaction were consistent with those of RNA-seq. Analysis of the mRNA-miRNA interaction network suggested that CD36 (miR-122, miR-200a, miR-141), TNFAIP6 (miR-141, miR-200a, miR-182), CYP11A1 (miR-122), SERPINA5 (miR-1, miR-206, miR-133a-3p, miR-133b), and PTGFR (miR-182, miR-122) might be related to fertility, but requires further research on follicular somatic cells.ConclusionsThis study was used for the first time to reveal the DEmRNAs and DEmiRNAs as well as their interaction in the follicles of uniparous and multiple goats at estrus phase using RNA-seq technology. Our findings provide new clues to uncover the molecular mechanisms and signaling networks of goat reproduction that could be potentially used to increase ovulation rate and kidding rate in goat.

Quantification of viable granulosa cells in murine ovarian follicles

ABSTRACT Ovarian follicle growth and oocyte maturation depend on the viability of granulosa cells (GC). We quantified GC in whole mouse follicles. Single follicles were isolated from adult mouse ovaries and stained with DAPI or Live-Dead stain before fixation. An objective image analysis protocol for counting fluorescent labeled GC was developed that used Image J software to measure GC cytoplasmic and nuclear areas. These data were compared to the number of GC obtained by disaggregating 96 follicles with enzymes to produce a suspension of GC, which then was stained with trypan blue and assessed using a hemocytometer. We found a linear relation between GC/follicle and follicle diameter. Viability of GC/follicle ranged from 40 ± 11 to 72 ± 7%. The coefficient of variation for image analysis of DAPI stained GC by different assessors was 4%, but the number of GC obtained from image analysis was approximately 50% less than from disaggregated follicles. The number of GC in intact mouse follicles was greater than the number reported earlier for fixed ovarian sections. We found that the number of GC was less in fluorescence labeled follicles; it is possible that the three-dimensional structure of the intact follicles obscured the fluorescent signal. Direct quantification of viable GC isolated from follicles appears to be the most accurate method.

Warty dyskeratoma; A rare entity with involvement of multiple adjoining follicles

Warty dyskeratoma is a rare epidermal tumor which occurs in middle aged or older persons, clinically manifests as papule or nodule and involves head and neck region in most of the cases. 65y F presented to the OPD with complaint of nodular swelling over the right cheek with itching for 15 years. On clinical examination patient had 3x1.8 cm nodule, firm in consistency, mobile, with ulceration over tumor. Routine investigations were within normal limits and Fine Needle Aspiration Cytology was reported as suspicious of malignancy. Histopathology of excised mass revealed hyperkeratotic, parakeratotic and acanthotic epidermis show large multiple invaginations comprising of lacunae along with villi formation. Stratum corneum and upper malpighi layer shows abundant dyskeratotic acantholytic cells. As per literature these changes are usually noticed in single hair follicle. Here we describe a rare case of warty dyskeratoma with multiple adjacent hair follicles involvement.

Intra- and inter-individual variability of fatty acid composition of the follicular fluid in a cohort of 23 women undergoing assisted reproductive treatment.

To examine the intra- and inter-individual variability in fatty acid composition of follicular fluid (FF) of 23 patients undergoing assisted reproductive treatment. The average coefficient of variation within each patient (CVw) and intra-class correlation coefficient (ICC) values of FF fatty acid composition as well as correlation between the fatty acid composition of individual, pooled or first-punctured follicles, were assessed. The proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 20:5n-3, total MUFA and n-3 PUFA showed good reproducibility (CVw < 10%). Although CVw values of 18:3n-3 and 20:3n-6 exceeded 10%, variation between patients exceeded intra-individual variation as indicated by elevated ICC values (0.61 and 0.66, respectively). Nevertheless, 20:4n-6 and 22:6n-3 showed non-negligible intra-patient variation. With the exception of some minor fatty acids (< 0.30g/100g), strong relationships were demonstrated between the average proportion in individually analysed follicles and the proportion determined in pooled samples and in the first, largest follicle. The CVw and ICC values of proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 18:3n-3, 20:5n-3, total MUFA and n-3 PUFA showed limited intra-individual variation and moderate to good reliability. However, this is not the case for some other PUFA, such as 20:4n-6 and 22:6n-3. Nevertheless, for all of these fatty acid(s) (groups), calculated average fatty acid proportions were highly correlated with proportions determined in pooled samples and in the first, largest follicle. This implies that single or pooled follicle aspiration suffices to assess intra-individual variation in the FF of these fatty acids.

Trichoscopic Patterns of Nonscarring Alopecia's.

Background:Hair loss (alopecia) is a common problem and is a major cause of psychological stress and anxiety among affected individuals. It is of utmost importance to diagnose these cases at the earliest and treat them accordingly. Trichoscopy provides a noninvasive option that can be used for early diagnosis and monitoring the progression of the hair disorders.Aims and Objectives:To perform trichoscopy and document the findings in patients with nonscarring alopecia's.Materials and Methods:A total of 100 cases satisfying the inclusion criteria were screened for general physical examination and scalp examination including hair shaft and root, and tests for hair anchorage and fragility were also done. The lesions were examined through dermoscope, photographs were taken, and findings were documented.Results:Among the total of 100 cases screened, 57 were female and 43 were male. The mean age of the study group was 26 ± 14.8 years. Females were affected by alopecia areata (AA) and female pattern hair loss (29.8%) equally, whereas males were most commonly affected by AA (41.8%). The common trichoscopic follicular features noted were broken hair (48%), black dots (48%), single hair follicle unit (45%), short vellus hair (44%), upright hair (41%), and yellow dots (40%). The common interfollicular features seen were honeycomb pigmentation (26%) and arborizing red lines (12%).Conclusion:The emergence of newer hair signs on trichoscopic studies aids in identification and has a definitive role in the diagnosis of clinically difficult cases, so it is recommended to use trichoscopy in the routine examination of nonscarring alopecia's.

Oocyte matched follicular fluid anti-Müllerian hormone is an excellent predictor of live birth after fresh single embryo transfer.

What is the relationship between the anti-Müllerian hormone (AMH), gonadotropin and androgen concentrations within a single follicle and live birth after ICSI and a transfer of an embryo developed from the matched oocyte? Among the analysed markers on the day of oocyte retrieval, AMH concentration in follicular fluid (FF) is a predictor of live birth after single embryo transfer (SET). High serum concentrations of AMH and low FSH concentrations have been associated with a high chance of pregnancy after ART. Whether there are differences in the hormonal milieu for individual follicles and whether this impacts the laboratory and clinical outcomes for the individual oocyte developing within that follicle are unknown. This prospective cohort study included 322 individual FF samples from 199 infertile women scheduled for ICSI/SET over an 18-month period. Of these women, 76 provided a single FF sample, while 123 women contributed two FF samples taken from two different follicles. The first follicle aspirated in each ovary on the day of oocyte retrieval had the FF aspirated; the individual cumulus-oocyte complex (COC) was tracked, and the associated FF was stored at -80°C. FF AMH, FSH, LH, testosterone (T) and androstenedione (A2) levels were measured by mass spectrometry (androgens) and immunoassays. The laboratory and clinical outcomes for each individual oocyte were related to their unique follicle hormone concentrations. Of the 322 oocytes with paired FF samples, 70 (21.7%) oocytes did not fertilise. From the remaining 252 2PN embryos, 88 (34.9%) were transferred as single embryos on Day 3; of the remaining 164, 78 developed into blastocysts, and 18 single blastocyst transfers were performed. Thus, a total of 106 transferred embryos had matching FF samples. An analysis of these individual FF concentrations revealed that AMH concentrations were higher in follicles in which the oocyte developed into a top quality (TQ) blastocyst (6.33 ± 5.52ng/ml) and whose transfer led to live birth (7.49 ± 5.03ng/ml) than those in which there was a failure of fertilisation (3.34 ± 2.21ng/ml). In contrast, follicular FSH concentrations were the lower for oocytes that resulted in a TQ blastocyst (5.36 ± 2.20 mIU/ml) and live birth (5.60 ± 1.41 mIU/ml) than for oocytes that failed to fertilise (9.06 ± 3.36 mIU/ml). FF AMH was the only studied marker that increased the chance of live birth (odds ratio: 1.93 [95% CI: 1.40-2.67], P < 0.001). The receiver operating characteristic analysis showed that FF AMH levels predicted live birth with a very high sensitivity (91.2%), specificity (91.7%) and an excellent AUC value of 0.954, whereas serum AMH level only had a fair (AUC = 0.711) significance as a predictor for live birth after ICSI/SET. The predictive capabilities of the interfollicular markers were not limited to the TQ embryos or blastocysts; they applied to all SET cycles. Whether an altered intrafollicular hormonal environment reflects the developmental capacity of the oocyte or defines cannot be determined from this cross-sectional analysis. Inclusion of 21 subjects with polycystic ovary syndrome (PCOS) may have biased the findings due to a unique intrafollicular milieu associated with PCOS. Our results suggest that highly competent human oocytes have an FF composition of AMH, FSH, T and A2 that is close to that in a natural cycle. Also, the relationships between intrafollicular AMH, gonadotropin and androgen levels in the same follicle support the hypothesis that FF AMH concentration may reflect granulosa cell proliferation during gonadotropin-stimulated follicle growth. Finally, the serum AMH concentration is markedly lower than the FF AMH concentration, with a moderate correlation between serum and FF AMH, implying ovarian follicle autonomy with regards to its secretory products. The National Science Centre of Poland supported this work (grant number: N N407 217 040). The authors declare that there is no conflict of interest regarding the publication of this article.

Effect of single dose follicle stimulating hormone on follicular aspiration, in vitro fertilization and pregnancy rate

The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in vitro fertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p&lt;0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p&gt;0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p&gt;0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.

Hormonal mechanisms regulating follicular wave dynamics II: Progesterone decreases diameter at follicle selection regardless of whether circulating FSH or LH are decreased or elevated

Selection of a single dominant follicle is morphologically manifested by diameter deviation between the future dominant follicle (F1) and the future largest subordinate follicle (F2). Conventional deviation is defined as F2≥7 mm when F1 reaches ∼8.5 mm whereas, undersized deviation is if F2<7 mm when F1 reaches ∼8.5 mm. Greater frequency of undersized deviation has been temporally associated with greater circulating progesterone (P4) and greater FSH but reduced LH in observational studies. Experiment 1 was conducted to directly test if elevating P4 increased the likelihood of undersized deviation and altered circulating concentrations of LH and FSH. Experiment 2 was conducted to test if increasing LH action by treatment with exogenous porcine LH or human chorionic gonadotropin (hCG) in the presence of elevated P4, would stimulate growth of F2 and increase the likelihood of conventional deviation. Ovaries were evaluated by ultrasound and blood samples collected every 12 h after development of a new wave following follicle ablation on D6 (D0 = ovulation). Data were normalized to F1≥7.5 mm and compared using SAS software. In experiment 1 (n = 20), the CL was regressed by prostaglandin F2α treatment and heifers were randomized on D6 into control (no P4 treatment) or P4 treatment (75 mg every 12 h for 5.5 d) beginning when F1 reached ∼3 mm (P4-3 mm group) or ∼6 mm (P4-6 mm group). The P4 treatment significantly increased the frequency of undersized deviation from 0% (controls) to 54%, decreased LH by 44%, and increased FSH by 32%. In experiment 2 (n = 27) heifers were randomized on D6 into control (saline) or treatment with the LH analogs - pLH (1.25 mg porcine LH/12 h) or hCG (160 IU initially and subsequently 96 IU/24 h).Treatment with LH analogs significantly increased P4 (control, 4.6 ± 0.3 ng/mL; pLH, 6.6 ± 0.4 ng/mL; and hCG, 8.9 ± 0.4 ng/mL) and decreased FSH (control, 0.46 ± 0.03 ng/mL; combined-pLH/hCG, 0.34 ± 0.02 ng/mL). However, F1 and F2 diameter and frequency of conventional (37%) and undersized (48%) deviations were similar between the control and combined-pLH/hCG groups. In conclusion, elevated P4 was directly linked to undersized deviation but the P4 effect on decreasing F2 diameter occurred independently of the P4 effects on FSH and LH concentrations.

Prematurely ruptured dominant follicles often retain competent oocytes in infertile women

Ovulation consists of a follicle’s rupture and subsequent oocyte extrusion, although there is a paucity of evidence regarding whether every follicle’s rupture is associated with extrusion of its oocyte. We examined this issue in a large-scale window-of-opportunity study by attempting aspiration of single dominant follicles that were found to have ruptured before a scheduled oocyte retrieval during in vitro fertilisation and embryo transfer treatment of infertile women. We were able to aspirate 587 of 1,071 ultrasonographically confirmed post-rupture dominant follicles from 1,071 women (i.e. one dominant follicle per woman) and retrieved 225 oocytes (oocyte recovery ratio: 43.4% of aspirated follicles), which yielded 28 live births (live birth ratio: 11.0% of retrieved oocytes). Interestingly, the live birth ratio for post-rupture dominant follicles was not statistically different from that achieved using regular pre-rupture aspiration of dominant follicles (1,085/8,977, 12.1%). These findings suggest that oocyte extrusion frequently does not occur after follicle rupture in infertile women undergoing in vitro fertilisation treatment, although the oocyte retained in the follicle can remain competent for use during that treatment.

Chemotherapy causes primordial follicle death in the human ovary via multiple apoptotic pathways and not by “burn out”

It has been proposed that gonadotoxic chemotherapy results in the “burn out” of primordial follicle reserve by activating PI3K/PTEN/Akt signaling pathway. Others have challenged this concept and put forward DNA damage and apoptosis as the main mechanism of follicle loss. We conducted this study to answer this controversy and conclusively determine the mechanism of chemotherapy-induced damage to ovarian reserve in women. Ovarian cortical pieces from organ donors aged ≤32 years were xenografted subcutaneously to SCID mice (n=12 mice/ tissue from 4 donors each). After 10 days, the mice were given an injection of cyclophosphamide (75mg/kg) or the vehicle. The tissues were recovered 12 hours later. The recovered xenografts were assessed for apoptosis by anti-caspase-3 (AC3) and DNA double strand breaks by γ-H2AX immunostaining as well as follicle growth initiation rate (FGIR) by primary/primordial follicle ratios. Single primordial follicle oocytes were laser captured for RNA sequencing and single cell quantitative real time PCR (qRT-PCR) to determine the signaling pathways activated in response to chemotherapy exposure. Immunofluorescence microscopy and image analysis were used to assess phosphorylation of PI3K/PTEN/Akt pathway and Bad-Bcl2 co-localization. There was no difference in the primordial follicle growth initiation rate (FGIR) between the vehicle and the cyclophosphamide-treated xenografts (p=0.35). However, cyclophosphamide caused a significant increase in the percentage of apoptotic (65.02±3.55% vs. 28.27±5.77%; p=0.001) and DNA-damaged (82± 2.7% vs. 23.82±8.66%;p=0.0001) primordial follicles . Ingenuity Pathway Analysis (IPA) of the RNA sequencing data from laser captured primordial follicles showed significant decrease in the expression of PECAM (p=0.00009), IKBKE (P=0.0001), and ANGPT1 (p=0.003) after cyclophosphamide treatment. These are positive regulators of the Akt pathway and hence results indicate that cyclophosphamide suppresses rather than activating Akt. In addition, IPA predicted that suppression of these pathways favor apoptosis in single primordial follicles. By qRT-PCR analyses, there was no change in the expression of Akt (p=0.9). Further, cyclophosphamide treatment did change the expression of the phospho-Akt (P=0.14), phospho-Foxo3a (p=0.17), or phospho-rpS6 (0.48) compared to the vehicle. By qRT-PCR, chemotherapy increased the expression of the anti-apoptotic Bcl2 (p=0.0003) accompanied by enhanced colocalization of the pro-apoptotic BAD-Bcl2 complex (p=0.006) in the primordial follicles, confirming that cyclophosphamide induces follicle death via apoptosis. This single cell transcriptomic and immunohistochemical analysis of human primordial and primary follicles prove that gonadotoxic chemotherapy agents do not cause follicle activation; they rather create a pro-apoptotic state resulting in massive loss of ovarian reserve. Future research on pharmacological fertility preservation should target preventing DNA damage and apoptosis rather than follicle activation.

Therapeutic effects of dimethyldiguanide combined with clomifene citrate in the treatment of polycystic ovary syndrome.

In view of the high incidence of polycystic ovary syndrome (PCOS) and the unsatisfactory therapeutic effects of dimethyldiguanide or clomifene citrate alone, our study aimed to investigate the therapeutic effects of dimethyldiguanide combined with clomifene citrate in the treatment of PCOS. A total of 79 patients with POCS and 35 healthy females were included, and endometrial biopsies were obtained. The sterol regulatory element-binding protein-1 (SREBP1) expression in endometrial tissues was detected by qRT-PCR. POC patients were randomly divided into group A (n=40) and group B (n=39). Patients in group A were treated with dimethyldiguanide combined with clomifene citrate, while patients in group B were treated with clomifene citrate alone. The number of mature follicles and cervical mucus score, follicular development rate and single follicle ovulation rate, cycle pregnancy rate, early miscarriage rate, ovulation rate, endometrial thickness, positive rate of three lines sign, follicle stimulating hormone level and luteinizing hormone level were compared between the two groups. The expression level of SREBP1 was higher in PCOS patients than that in the healthy control. SREBP1 expression was inhibited after treatment, while the inhibitory effects of combined treatment were stronger than those of clomifene citrate alone. Compared with clomifene citrate alone, the combined treatment improved cervical mucus score, follicle development rate, single follicle ovulation rate, endometrial thickness, positive rate of three lines sign, and follicle-stimulating hormone level. The therapeutic effect of combined treatment is better than clomifene citrate alone in the treatment of PCOS.

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows.

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

Microanatomy of the female reproductive system of the viviparous freshwater whipray Fluvitrygon signifer (Elasmobranchii: Myliobatiformes: Dasyatidae). I. The ovary

The microanatomy of the Fluvitrygon signifer ovary was investigated. The females had a single left ovary containing various stages of germ cells and follicles. Both immature and mature female ovaries contained oogonia initially observable in the germinal epithelium as solitary cells or oogonial nests. Herein, three developmental pathways for the oogonia to become primary oocytes are proposed. (1) The single oogonia developed into individual primary oocytes and, in association with follicular cells, became follicles. (2) Oogonial nests constituted sex cords, which released single follicles. (3) The nests detached from the germinal epithelium as isolated nests, which transformed into nests of primary oocytes and subsequently nests of previtellogenic follicles. We point out a possible mechanism for de novo formation of nests of primary follicles from a single follicle. The presence of follicle nests in adults appeared to be a specific characteristic of F. signifer. Atretic preovulatory follicles were divided into atretic previtellogenic and atretic vitellogenic follicles; the latter were subcategorized into four stages and postovulatory follicles were classified into six stages. In general, F. signifer revealed similar microscopic structures of the ovary to those in other elasmobranchs, with the exception of specific features during early folliculogenesis and potential indeterminate oogenesis.

Putative role of cocaine- and amphetamine-regulated transcript (CARTPT) in dominant follicle selection in cattle

The mechanisms regulating development of a single (dominant) follicle capable of ovulation during each follicular wave in cattle and atresia of remaining follicles (dominant follicle selection) are not well understood. FSH and IGF1 are known regulators of follicle growth and granulosa cell estradiol production during follicular waves. Recent evidence indicates cocaine and amphetamine regulated transcript (CARTPT), with intraovarian expression only in single-ovulating species, is a novel regulator of follicular development. The mature CARTPT peptide (CART) is a potent negative regulator of FSH and IGF1 action on granulosa cells in vitro and can inhibit follicular estradiol production in vivo. Follicular fluid CART concentrations in healthy follicles decrease after dominant follicle selection and CARTPT mRNA is lower in healthy versus atretic follicles collected prior to and early after initiation of follicle dominance, suggestive of a regulatory role in the selection process. The inhibitory actions of CART on FSH signaling and estradiol production are dependent on the Go/i-subclass of inhibitory G proteins and linked to multiple components of the FSH signal transduction pathway resulting in reduced CYP19A1 mRNA and estradiol production. Evidence to date supports a potential important functional role for CART in regulation of dominant follicle selection and the species-specific ovulatory quota in monotocous species.

Intrinsic activation of cell growth and differentiation in ex vivo cultured human hair follicles by a transient endogenous production of ROS

The emerging variety of signalling roles for ROS in eukaryotic cells and tissues is currently a matter of intense research. Here we make use of ex vivo cultured single human hair follicles as an experimental model to demonstrate that a transient production of non-lethal endogenous ROS levels in these mini-organs promotes efficiently the entry into the growth phase (anagen). The stimulatory process implicates the specific activation of the hair follicle stem cell niche, encompassing the induction of stem cell differentiation markers (Ck15), overall cell proliferation and sustained growth of the tissue associated with expression of gen targets (Ccnd1) concomitant with the inhibition of Wnt signaling antagonists and repressors (Dkk1, Gsk3β) of Wnt signaling. As a whole, this observation indicates that, once activated, ROS signalling is an intrinsic mechanism regulating the hair follicle stem cell niche independently of any external signal.

Abstract P4-14-13: Tamoxifen induced ovarian hyperstimulation during hormonal therapy for breast cancer

Abstract Introduction Adjuvant endocrine therapy is an integral compo-nent of care for endocrine-dependent breast cancer. To date, international consensus statements recommend tamoxifen (20 mg/day) for five years as the standard adjuvant endocrine therapy for premenopausal women. Tamoxifen is a potent inducer of ovarian function and consequent hyper-estrogenism in premenopausal women. However, the incidence rate and risk factors associated this phenomenone were not clarified. Methods Among consecutive patients who were operated under diagnosis of breast cancer from March 2012 to December 2016 in Chung-Ang university hospital, patients who received post-operative tamoxifen therapy for endocrine-dependent breast cancer (stage 0-III) at age under 60 were selected and retrospectively analysed. Serial data on serum estradiol and follicular stimulating hormone(FSH) were collected. When the serum concentration of estradiol was higher than 400 pg/mL, which exceeds the normal estradiol production by a single preovulatory follicle, we classified them as tamoxifen induced ovarian hyperstimulation group. Clinicopathologic factors were analyzed between ovarian hyperstimulation group and non-hyperstimulation group by x2 and student t-test. Results Among 205 patients, 19 patients(9.3%) showed high values of serum estradiol during tamoxifen therapy. They showed 44 times of high estradiol level during follow up period. The serum concentrations of estradiol and FSH were 1047.97638.8pg/mL and 11.57.3 mIu/mL, respectively. The mean duration from the start of the single administration of tamoxifen to the initial detection of a high concentration of estradiol was 666.4+433.1 days. Univariate and multivariate analysis between ovarian hyperstimulation and non-hyperstimulation groups showed younger age(&amp;lt;40years) and only endocrine therapy without chemotherapy were related to higher prevalence of ovarian hyperstimulation significantly. (p &amp;lt;0.001, =0.031 each) Pathologic stages and progesterone receptor expressions on breast tumor were not related to manifestation of ovarian hyperstimulation. Conclusions The Incidence rate and occurrence time of ovarian hyperstimulation associated with adjuvant tamoxifen treatment in breast cancer patients under age 60 were 9.3% and around 2-year after treatment with tamoxifen. Young age under 40 years old and endocrine treatment without chemotherapy were risk factors predicting occurrence of ovarian hyperstimulation during tamoxifen treatment. It should be noted that tamoxifen is a potent inducer of ovarian function and close monitoring of the endocrine parameters during treatment with tamoxifen would be essential. Citation Format: Kim MK, Shin H-c. Tamoxifen induced ovarian hyperstimulation during hormonal therapy for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-14-13.

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish.

The mechanisms that ensure fertilization of egg by a sperm are not fully understood. In all teleosts, a channel called the ‘micropyle’ is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the vitelline envelope by a single specialized follicle cell, the micropylar cell. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish (Danio rerio). Genome editing mutants affecting taz can grow to adults. However, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. We find that taz mutant eggs have no micropyle. Taz protein is specifically enriched in mid-oogenesis in the micropylar cell located at the animal pole of wild type oocyte, where it might regulate the cytoskeleton. Taz protein and micropylar cells are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

Evaluation of stemness marker expression in bovine ovarian granulosa cells.

The objective of this study was to assess the stemness marker expressions (Oct4, Nanog, and Sox2) of granulosa cells (GCs) collected from bovine ovarian follicles and in vitro expansion. The single bovine ovarian follicles were isolated and categorized into 4 groups according to their diameter including group A (<2 mm), group B (2-3 mm), group C (3-4 mm), and group D (>4 mm). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunostaining were applied to evaluate the stemness marker expression of bovine GCs from ovarian follicles. We also estimated the stemness marker transcript expressions of GCs during in vitro expression by qRT-PCR. qRT-PCR analysis demonstrated that fresh GCs from bovine ovarian follicles expressed the stemness markers (Oct4, Nanog, Sox2). These markers were down-regulated during antral stage follicular development. We also estimated stemness marker transcript expressions of GCs which were isolated and in vitro expanded from ovarian follicles of group A. The qRT-PCR results showed that Oct4 and Sox2 transcript expressions were reduced during in vitro expansion while Nanog transcript was not expressed.

Steroid profiles by liquid chromatography-mass spectrometry of matched serum and single dominant ovarian follicular fluid from women undergoing IVF.

Can IVF outcomes be predicted from the steroid profile generated by liquid chromatography-mass spectrometry (LC-MS/MS) from follicular fluid collected from a single dominant follicle and serum after ovarian stimulation. Prospective observational cohort study in which serum and follicular fluid were collected from women and used to generate steroid profiles by LC-MS/MS. A total of 93 consecutive women enrolled for IVF treatment were recruited at the Fertility Unit, Royal Prince Alfred Women and Babies Hospital, Sydney between September 2014 and July 2015. Baseline and serum levels at oocyte retrieval, as well as follicular fluid samples from the largest single antral follicle, were collected. All samples underwent steroid analysis within a single batch to measure progesterone (P4), oestradiol (E2), oestrone (E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and 3 α, 5α androstanediol (3α-diol) and 3β, 5α androstanediol (3β-diol). P4, E2, E1, A4, T, DHEA and A4 were detectable in all baseline serum levels, at oocyte retrieval and in follicular fluid samples, whereas DHT, 3α-diol and 3β-diol were only detectable in a minority of samples. The most consistent predictor of pre-transfer (number of follicles >14mm in diameter, oocytes retrieved or fertilized, day-5 blastocysts) outcomes was baseline serum anti-Müllerian hormone. In follicular fluid, E2 was a negative predictor of the number of oocytes retrieved and the number of day-5 blastocysts but no follicular fluid steroids predicted pregnancy outcome. None of the nine steroids measured in follicular fluid predicted pregnancy outcome in women undergoing IVF.

Extracellular microRNAs profile in human follicular fluid and IVF outcomes

Encapsulated microRNAs (i.e., miRNAs within the extracellular vesicles, i.e., EV-miRNAs) have been detected in follicular fluid in both animal and human studies and different profiles have been associated with IVF cycle characteristics. However, limited studies to date have investigated other IVF outcomes, including fertilization status and embryo quality on day three”. In this cohort, we performed a cross-sectional analysis on 126 women who contributed follicular fluid from a single follicle during a single IVF cycle. One hundred and ninety-two EV-miRNAs were assessed by univariable fold-change and multivariable logistic regression analyses. Hsa-miR-92a and hsa-miR-130b, were over-expressed in follicular fluid samples from oocytes that failed to fertilize compared to those that were normally fertilized. Additionally, hsa-miR-888 was over-expressed and hsa-miR-214 and hsa-miR-454 were under-expressed in samples that resulted in impaired day-3 embryo quality compared to top-quality day-3 embryos. After adjusting for confounders as BMI, smoking and total motile sperm, associations of these EV-miRNAs remained significant. In-silico KEGG pathway analyses assigned the identified EV-miRNAs to pathways of follicular growth and development, cellular signaling, oocyte meiosis, and ovarian function. Our findings suggest that EV-miRNAs may play a role in pathways of ovarian function and follicle development, which could be essential for understanding the molecular mechanisms that could lead to a successful pregnancy and birth.