Individual Follicles Research Articles

Associations of the levels of adipokines and cytokines in individual follicles with in vitro fertilization outcomes in women with different ovarian reserves

BackgroundTo a large extent, the ovarian reserve determines a woman’s reproductive potential. The etiological and pathological mechanisms of diminished ovarian reserve (DOR) remain unclear, and no reliable treatment is currently available for DOR. Adipokines and cytokines in follicular fluid (FF) play pivotal roles in follicular development and maturation. The concentrations of adipokines and cytokines in FF from individual follicles of women with DOR undergoing in vitro fertilization (IVF) were studied. In particular, we investigated the associations between the levels of adipokines and cytokines in individual FFs from women with different ovarian reserves and between the follicular levels of adipokines and cytokines and IVF outcomes in individual follicles.MethodsA total of 115 women who underwent IVF were recruited. Patients diagnosed with DOR, defined as a basal antral follicle count < 5 or an anti-Mullerian hormone concentration < 1.1 ng/mL, were assigned to the DOR group, while patients with a normal ovarian reserve (NOR) were assigned to the NOR group. FF was sampled from the first follicle with a diameter of approximately 18–20 mm from each patient, and the IVF outcome of the oocyte from the corresponding follicle was tracked. The levels of 5 adipokines (including visfatin-1, monocyte chemoattractant protein-1 [MCP-1], resistin, leptin, and chemerin) and 3 cytokines (including interleukin [IL]-6, IL-12p70, and tumor necrosis factor [TNF]-α) in FF were determined by Luminex technology.ResultsThe follicular levels of TNF-α, IL-6, visafatin, MCP-1, IL-12, and chemerin were significantly lower in women with NOR than in those with DOR. The follicular level of IL-6 was negatively correlated with the quality of embryos according to the binary logistic regression analysis, while the follicular levels of adipokines and other cytokines did not correlate with IVF outcomes regardless of the woman’s ovarian reserve.ConclusionsOur study demonstrated that the levels of adipokines and cytokines in individual follicles in women with DOR were different from those in women with NOR, indicating that increased intrafollicular inflammation might be related to DOR. Moreover, a high follicular level of IL-6 might negatively impact embryo quality.

Development of a Deep Learning Tool to Support the Assessment of Thyroid Follicular Cell Hypertrophy in the Rat.

Thyroid tissue is sensitive to the effects of endocrine disrupting substances, and this represents a significant health concern. Histopathological analysis of tissue sections of the rat thyroid gland remains the gold standard for the evaluation for agrochemical effects on the thyroid. However, there is a high degree of variability in the appearance of the rat thyroid gland, and toxicologic pathologists often struggle to decide on and consistently apply a threshold for recording low-grade thyroid follicular hypertrophy. This research project developed a deep learning image analysis solution that provides a quantitative score based on the morphological measurements of individual follicles that can be integrated into the standard pathology workflow. To achieve this, a U-Net convolutional deep learning neural network was used that not just identifies the various tissue components but also delineates individual follicles. Further steps to process the raw individual follicle data were developed using empirical models optimized to produce thyroid activity scores that were shown to be superior to the mean epithelial area approach when compared with pathologists' scores. These scores can be used for pathologist decision support using appropriate statistical methods to assess the presence or absence of low-grade thyroid hypertrophy at the group level.

Luteinizing hormone receptor deficiency in immature cumulus-oocyte complexes retrieved for assisted reproduction.

This study investigated whether luteinizing hormone receptor (LHR) expression varies in the granulosa cells of individual follicles according to the maturation stage of the oocytes harvested for assisted reproductive technology treatment. We observed minimal to no LHR messenger ribonucleic acid and protein expression in cumulus cells surrounding oocytes arrested in the germinal vesicle stage. Interestingly, their ability to mature was confirmed by rescue invitro maturation, suggesting somatic cell LHR deficiency as a key factor for the retrieval of germinal vesicle oocytes in assisted reproductive technology procedures.

A Review of Mathematical Modelling Methods for Bovine Hormone Dynamics.

This paper explores various approaches to modeling the bovine estrus cycle, focusing on improving estrus detection to enhance reproductive management in dairy cattle. The review examines a range of mathematical models, including hormonal models based on ordinary differential equations (ODEs) that simulate hormone dynamics, as well as follicular competition models that track the maturation of individual follicles. In addition to these, Precision Dairy Monitoring Technologies (PDMT) are discussed, which utilize real-time physiological and behavioral data to provide predictive insights into estrus. The paper assesses the effectiveness of both traditional modeling approaches and PDMT in improving breeding efficiency, minimizing labor, and optimising herd management practices.

Follicle-intrinsic and spatially distinct molecular programs drive follicle rupture and luteinization during ex vivo mammalian ovulation

During ovulation, the apical wall of the preovulatory follicle breaks down to facilitate gamete release. In parallel, the residual follicle wall differentiates into a progesterone-producing corpus luteum. Disruption of ovulation, whether through contraceptive intervention or infertility, has implications for women’s health. In this study, we harness the power of an ex vivo ovulation model and machine-learning guided microdissection to identify differences between the ruptured and unruptured sides of the follicle wall. We demonstrate that the unruptured side exhibits clear markers of luteinization after ovulation while the ruptured side exhibits cell death signals. RNA-sequencing of individual follicle sides reveals 2099 differentially expressed genes (DEGs) between follicle sides without ovulation induction, and 1673 DEGs 12 h after induction of ovulation. Our model validates molecular patterns consistent with known ovulation biology even though this process occurs in the absence of the ovarian stroma, vasculature, and immune cells. We further identify previously unappreciated pathways including amino acid transport and Jag-Notch signaling on the ruptured side and glycolysis, metal ion processing, and IL-11 signaling on the unruptured side of the follicle. This study yields key insights into follicle-inherent, spatially-defined pathways that underlie follicle rupture, which may further understanding of ovulation physiology and advance women’s health.

Secondary growth ovarian follicles of the pigmented sterlet sturgeon Acipenser ruthenus L. 1758 (Acipenseriformes, Chondrostei, Actinopterygii, Osteichthyes) – Microscopic study of oocytes, egg envelope and diversification of follicular cells

The individual ovarian follicle of sturgeons (Acipenseriformes, Acipenseridae) contains an oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. The late stages of the secondary growth of follicles (mid- and advanced vitellogenic) are not fully explained in Acipenseriformes. To explore and discuss the ultrastructure of oocytes, FCs, an egg envelope, and explain how micropylar cells differentiate and the canals of a multiple micropyle are formed, the samples of ovaries of the mature sterlet sturgeon Acipenser ruthenus were examined. The oocytes are polarized, the nucleus is located in the animal hemisphere, contains lampbrush chromosomes and multiple nucleoli. In the ooplasm three regions are present: a perinuclear (contains the mitochondria), an endoplasm (contains the lipid droplets and yolk platelets), and a periplasm (contains the cortical granules, melanosomes, endocytotic and exocytotic vesicles). The melanosomes in animal hemisphere form two concentric rings separated by a lighter region between them. The FCs are differentiated into bright and dark cells that are both translationally and secretory active. Diversification of FCs involves repeated and cytoskeleton-dependent change of shape. In the advanced follicles the FCs are diversified into micropylar, the animal and vegetal regions cells, and the cells that delaminated from the epithelium in the animal region. The egg envelope is present in the perioocytic space and consists of three layers: (1) an inner layer or vitelline envelope, (2) a middle layer, and (3) an outer layer. The inner layer consists of four sublayers: (a) a filamentous sublayer composed of filaments released from the oocytes, (b) a trabecular 1 sublayer and (c) a trabecular 2 sublayer named due to the sequence of the deposition, and composed of filaments, fibres and trabecules, (d) a homogeneous sublayer located between the trabecular 1 and trabecular 2 sublayers composed of filaments that adhere to each other closely. The middle layer contains two sublayers: a porous 1 and a porous 2 (composed of granular material) which are released by the oocyte and FCs. The outer layer consists of fibrillar material released by the FCs. The egg envelope is pierced by radial canals formed around the microvilli of the oocyte and the microvilli-like processes of FCs. A micropylar field in the egg envelope that covers the animal pole of the oocyte contains 1 – 4 micropylar canals. Micropylar cells are involved in their formation. The shape of these cells is icicle-like and the cytoplasm is differentiated into two regions (a basal and apical bearing a projection) equipped with different sets of organelles.

From follicle to blastocyst: microRNA-34c from follicular fluid-derived extracellular vesicles modulates blastocyst quality

BackgroundWithin the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent).MethodsBovine antral follicles were dissected, categorized as small (2–4 mm) or large (5–8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM).ResultsWe identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect.ConclusionThis study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

P-700 Modelling individual follicle growth rates during ovarian stimulation in medically assisted reproduction cycles to enable prediction of final follicle profiles

Abstract Study question Can the growth of individual follicles during ovarian stimulation in medically assisted reproduction (MAR) cycles be modelled to enable prediction of final follicle profiles? Summary answer Mean follicle growth rate is 1.35 mm per day. Our model reliably forecasts follicle size profiles on the final scan within ±2mm with 75.1% accuracy. What is known already Follicle growth rates during ovarian stimulation are believed to be linear with various mean growth rates reported between 1 to 4 mm per day. Follicles are believed to grow faster during ovarian stimulation than in the natural menstrual cycle. The change in mean follicle size was 1.69 mm per day with ovarian stimulation in 131 MAR cycles, and 1.4 mm per day in 50 natural cycles. However, there are only very limited data using individual follicle sizes to model follicle growth to enable prediction of future follicle size profiles. Study design, size, duration We conducted a retrospective cohort study of 12,950 patients from 11 European clinics (2005-2023). First IVF/ICSI cycles with at least three follicles and two scans were analysed. Using 8,564 cycles with scans one or two days apart, 98,029 follicles were modelled to estimate follicle growth rates per day. Stratification by age, weight, total antral follicle count (AFC), initial FSH dose, and suppressant protocol was performed. Predictive modelling was conducted using 39,698 scans including 434,082 follicles. Participants/materials, setting, methods Assessment of Follicle Growth: Follicles were ranked and the difference in corresponding follicle sizes over consecutive scans estimated, and the impact of baseline characteristics evaluated. Prediction of follicle size profile on final scan: A kernel density estimator and random forest model were developed using the same training data. Both approaches estimated future follicle growth from the initial scan. The two predictions were combined using maximum-likelihood adjustments. Generalisation error was estimated using independent test data. Main results and the role of chance The mean growth rate of individual follicles was 1.350 mm per day (95% CI 1.346 – 1.353 mm per day), which was lower than the previously reported mean follicle growth rate during ovarian stimulation (1.7 mm per day). The large number of patient-cycles and follicles assessed enabled a precise estimate of follicle growth per day during ovarian stimulation. Age, total AFC, initial FSH dose, bodyweight, and suppressant protocol had no statistically significant effect on the estimated mean growth rate. Using only the follicle sizes from the initial scan during ovarian stimulation, our model had 75.1% accuracy for predicting follicle sizes on the final scan within ±2mm. Including follicle size data from the first two scans improved the accuracy of the model to predict the follicle sizes on the final scan to 80.4%. In a confirmatory analysis, we cross-validated our findings across each of 11 individual clinics, and similar estimates for the generalisation error as for the standard test/train split were maintained, suggesting suitable generalisability of these findings. Limitations, reasons for caution A key simplifying assumption in this study is that follicles demonstrate monotonic growth patterns. Specifically, the relative ordering of follicle sizes is preserved over the entire cycle. This assumption enables straightforward extraction of growth statistics from the observed data but assumes that follicles do not regress in size. Wider implications of the findings We have used a large high-quality dataset, allied to advanced modern machine learning techniques, to precisely estimate follicle growth rates during ovarian stimulation, and use this to reliably predict future follicle size profiles during ovarian stimulation. This supports the design, personalisation, streamlining and success of future assisted conception cycles. Trial registration number None

P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with &amp;gt;1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p &amp;lt; 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p &amp;lt; 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable

P-620 Oocyte competence in Vitro Fertilization: relation between serum anti-Müllerian hormone (AMH) concentrations and complement C4 protein expression in follicular fluid

Abstract Study question To investigate if serum AMH concentration variations are correlated with protein expression in follicular microenvironment affecting IVF outcomes. Summary answer Serum AMH concentrations were associated with altered levels of intrafollicular protein involvement of the complement pathway in immunity response in follicular fluid (FF). What is known already Anti-Müllerian hormone (AMH) plays a crucial role in regulating growth and development of ovarian follicles. The follicular fluid is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. AMH concentration in follicular fluid can be a marker of ovarian reserve status and oocyte competence. AMH elevated levels may indicate a higher ovarian reserve and are sometimes associated with a greater ovarian response to ovarian stimulation used in IVF. However, the exact relationship between AMH levels in follicular fluid and IVF outcomes may vary among women and depend on various factors. Study design, size, duration The study included FF collected from 75 patients underwent IVF from December 2018 to June 2019.All patients enrolled underwent GnRH antagonist stimulation protocol with r-FSH according to clinical/ovarian features.Patients were sorted into three groups according to serum AMH concentrations: AMH≤1.0 ng/ml, n = 25 (A group); AMH=1.1-2.0 ng/ml, n = 29 (B Group); AMH&amp;gt;2.0 ng/ml, n = 21 (C group).Cutoffs for defining low(A), average(B), and high(C) AMH serum concentrations corresponded to round values of 30th and 70th centiles of each measurement. Participants/materials, setting, methods Exclusion criteria: endometriosis, metabolic/endocrinology diseases and oligoasthenoteratozoospermia. FF aspirates from individual follicles (diameter ≥18 mm) were collected on the day of oocyte retrieval. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). Oocyte number and quality, fertilization rate are IVF outcome measures. Data were analyzed using the unpaired Student’s t-test by IBM SPSS-STATISTICS versions 20.0 and considered significant if p-value ≤ 0.05. Main results and the role of chance 180 protein spots were highlighted in three groups: 80% is represented in all the samples analyzed, whereas 19 protein spots out of 180 are group-specific. Five unique upregulated proteins were identified in the A group and five in C group respect to control group (B). These were identified IG, CO4, ALBU, TRFE, IGLC in A group, while A1AT, ANT3 A2HS, HPT and CO4 in C group. A and C group showed worse oocyte quality associated with a lower fertilization rate than B group. On the basis of the proteome analysis, the component of the complement cascade C4 was found more plentiful in patient with low and high AMH serum concentration compared to average AMH serum concentration ones. The presence of high levels of the unprocessed precursor C4 correlated to serum AMH fluctuations is an indication of non-activation of the complement pathways fundamental for follicle maturation and ovulation compromising IVF outcome. Limitations, reasons for caution Limited number of patients. Age and lifestyle factors (i.e. smoking, diet) are not considered. Wider implications of the findings Results showed an increase of CO4 in FF of patients with low and high AMH serum concentrations. CO4 FF high levels could be indicative of defective complement activation during ovulation with consequent impaired processes and lower standard oocyte competence. Thus, CO4 could be identify as biomarker to predict IVF success. Trial registration number Not Applicable

A Dose-Response Study on Functional and Transcriptomic Effects of FSH on Ex Vivo Mouse Folliculogenesis.

Follicle-stimulating hormone (FSH) binds to its membrane receptor (FSHR) in granulosa cells to activate various signal transduction pathways and drive the gonadotropin-dependent phase of folliculogenesis. Both FSH insufficiency (due to genetic or nongenetic factors) and FSH excess (as encountered with ovarian stimulation in assisted reproductive technology [ART]) can cause poor female reproductive outcomes, but the underlying molecular mechanisms remain elusive. Herein, we conducted single-follicle and single-oocyte RNA sequencing analysis along with other approaches in an ex vivo mouse folliculogenesis and oogenesis system to investigate the effects of different concentrations of FSH on key follicular events. Our study revealed that a minimum FSH threshold is required for follicle maturation into the high estradiol-secreting preovulatory stage, and such threshold is moderately variable among individual follicles between 5 and 10 mIU/mL. FSH at 5, 10, 20, and 30 mIU/mL induced distinct expression patterns of follicle maturation-related genes, follicular transcriptomics, and follicular cAMP levels. RNA sequencing analysis identified FSH-stimulated activation of G proteins and downstream canonical and novel signaling pathways that may critically regulate follicle maturation, including the cAMP/PKA/CREB, PI3K/AKT/FOXO1, and glycolysis pathways. High FSH at 20 and 30 mIU/mL resulted in noncanonical FSH responses, including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, this study improves our understanding of gonadotropin-dependent folliculogenesis and provides crucial insights into how high doses of FSH used in ART may impact follicular health, oocyte quality, pregnancy outcome, and systemic health.

AKT, p-AKT, ERK1/2 and p-ERK1/2 in Mural Granulosa Cells Are Not Correlated to Different Ovarian Stimulation Protocols in Patients Undergoing Assisted Reproductive Treatment.

(1) Background: In this paper we aim to study the relationship between the expression levels of molecules involved in apoptotic/survival pathways, considered as molecular markers of oocyte competence (i.e., AKT, p-AKT, ERK1/2, and p-ERK1/2) in mural granulosa cells (MGCs) and the administration of r-FSH alone or combined with exogenous r-LH, in ovarian stimulation protocol. Moreover, we aim to evaluate oocyte competence by comparing normally cleaved embryos that were transferred in the uterus, with embryos that were arrested during in vitro culture. (2) Methods: The study included 34 normo-responder women undergoing ICSI procedures. All subjects were divided into two groups. Group A consisted of 18 women stimulated with r-FSH and used as a control group; Group B consisted of 14 women stimulated with r-FSH combined with r-LH. The MGCs were obtained from individual follicles. Immunoblot analyses were carried out to analyze the AKT, p-AKT, ERK1/2, and p-ERK1/2 levels in MGCs and to correlate them with the ovarian stimulation protocol. Furthermore, the oocyte competence was evaluated, for each follicle, according to the development of the embryo during in vitro culture and the pregnancy outcome. (3) Results: We found no significant difference in the levels of molecules in isolated MGCs between groups A and B. These results, in light of our previous research, suggest for the first time, to our knowledge, that cumulus cells and mural granulosa cells in the same follicle show different expression levels of molecules involved in the apoptotic mechanism. (4) Conclusions: Our results could clarify some controversial data in the literature where cumulative cell pools of cumulus and granulosa were analyzed, described as ovarian follicle cells, and used as markers of oocyte competence. In this paper, we found evidence that cumulus and granulosa cells need to be analyzed separately.

Adaptive feature selection for active trachoma image classification

Trachoma is a neglected tropical eye disease caused by ocular strains of Chlamydia trachomatis, which affects millions of people worldwide. To examine the eye for signs of active trachoma, healthcare providers typically look for clusters of five or more follicles on the conjunctiva of the upper eyelid for the follicular inflammatory trachoma stage. However, it is also possible to find individual follicles scattered throughout the conjunctiva, particularly in mild or early-stage trachoma cases. Additionally, the datasets are photographic images collected in the field that can be high-dimensional and may contain large amounts of redundant information. We propose integrating novel attention-based feature extraction and feature selection techniques to address these challenges. First, we present the Lambda layer within the Convolutional Block Attention Module (L-CBAM) to normalize attention weights and improve the feature extraction process. Second, we introduce an adaptive mechanism, Adaptive Beta Hill Climbing (AβHC) with Social Ski-Driver (SSD), which adjusts the exploration-exploitation trade-off during the search process, allowing for better exploration of the search space and more efficient convergence toward an optimal feature subset. We then use the multilayer perceptron (MLP) classifier to produce final classification results using selected subsets. We evaluated the proposed approach on active trachoma inverted eyelid images and obtained accuracy scores of 93.3% with only 19.7% of the selected features, surpassing many of the algorithms used for comparison. Our proposed method has demonstrated excellent performance compared to recent works utilizing the same datasets. The source code of this work is available at https://github.com/mshitie2/Active_Trachoma.

A Chitosan-Based Biomaterial Combined with Mesenchymal Stem Cell-Conditioned Medium for Wound Healing and Skin Regeneration.

The aim of this study was to provide a beneficial treatment effect of novel chitosan bio-polymeric material enriched with mesenchymal stem cell products derived from the canine adipose tissue (AT-MSC) on the artificial skin defect in a rabbit model. For the objectivity of the regeneration evaluation, we used histological analysis and a scoring system created by us, taking into account all the attributes of regeneration, such as inflammatory reaction, necrosis, granulation, formation of individual skin layers and hair follicles. We observed an acceleration and improvement in the healing of an artificially created skin defect after eight and ten weeks in comparison with negative control (spontaneous healing without biomaterial). Moreover, we were able to described hair follicles and epidermis layer in histological skin samples treated with a chitosan-based biomaterial on the eighth week after grafting.

Significantly shortened telomere length and altered androgen receptor level in cumulus cells from women with polycystic ovary syndrome

ObjectiveThe aim of this study was to investigate the correlation between hormone receptor levels and telomere length (TL) in infertile women with and without polycystic ovary syndrome (PCOS). Materials and methodsThis prospective cohort study recruited a total of 431 cumulus oocyte complex (COC) from 88 infertile women between July 2012 and June 2014. The participants were divided into three groups: young age (<38 years, n = 42 and 227 COC), advanced age (≥38 years, n = 33 and 107 COC) and PCOS patients (n = 13 and 97 COC). Cumulus cells were collected from individual follicle during oocyte pick-up, and the mRNA levels of hormone receptors and TL were measured using real-time PCR. ResultsThe cumulus cells of PCOS patients demonstrated lower mRNA levels of LH receptor (75.57 ± 138.10 vs. 171.07 ± 317.68; p < 0.01) and androgen receptor (1.13 ± 1.52 vs. 4.08 ± 9.57; p < 0.01), as well as a shorter TL (2.39 ± 2.58 vs. 3.96 ± 4.72; p < 0.01) compared to those of the young age group. In the young age group, only androgen receptor mRNA level showed a significant association with TL (rho = 0.148, p = 0.026), while FSH receptor mRNA level was the only factor associated with TL (rho = 0.247, p = 0.015) in PCOS patients. For advanced-aged patients, no significant relationship was observed between hormone receptor mRNA levels and TL. Alternative splicing of androgen receptors was identified in some PCOS patients but not in young age controls. ConclusionThe findings suggest that the androgen receptor level and function may be altered in the cumulus cells of PCOS patients, leading to a shorter TL in cumulus cells in PCOS patients.

FRI427 Hypocaloric Dietary Intervention Reduces Excessive Follicle Recruitment In Women With PCOS

Abstract Disclosure: F. Carter: None. N. Lee: None. A. Reich: None. B.Y. Jarrett: None. H. Vanden Brink: None. A. Oldfield: None. M.E. Lujan: None. Introduction: Disordered antral follicle development in polycystic ovary syndrome (PCOS) is characterized by excessive recruitment, early or absence of follicle selection and decreased incidence of dominance and ovulation. Whether lifestyle interventions aimed at weight loss improve defects in antral folliculogenesis to promote ovulation in women with PCOS and comorbid obesity is unknown. Objective: To test the hypothesis that a hypocaloric dietary intervention improves antral follicle development in a subset of women with PCOS and obesity as evidenced by decreased recruitment, and increased selection, dominance, and ovulation. Methods: Preliminary analysis of women with PCOS and obesity (7 out of 20) who participated in a hypocaloric dietary intervention was conducted. The study protocol consisted of every-other-day visits to a clinical research center for ovarian ultrasonography and venipuncture over a 4-week baseline period (pre-intervention), followed by a 12-week commercial hypocaloric dietary meal plan intervention aimed at achieving 1-2 lbs of weight loss per week. Anthropometry, DEXA, hirsutism scoring, and oral glucose tolerance testing were conducted at baseline and during the final month of the intervention in the follicular phase. The number and diameter of all follicles ≥ 2 mm at each visit were recorded offline, and individual follicles ≥ 7 mm were tracked using the Identity Method throughout the 16-weeks. Clinical, morphologic, and endocrine markers were compared using paired t-tests pre- vs. post-intervention, and linear mixed models assessed changes in recruitment, selection, dominance, and ovulation. Results: The hypocaloric dietary intervention resulted in mean weight loss of 8.8 ± 2.9% and significant reductions in waist (-13 ± 9 cm) and hips (-10 ± 3 cm) circumference, body mass index (-3.4 ± 1.4 kg/m2), and percent total body fat (-1.5 ± 1.3%; all p&amp;lt;0.02). Glucoregulatory status, menstrual cycle lengths, reproductive hormone (LH, FSH, E2, P4, TT, AMH) concentrations, and hirsutism scores did not differ pre- vs. post-intervention. The number of follicle recruitment events decreased with percent weight loss during the intervention (p=0.01), as did the average number of follicles recruited per cohort (p=0.01). By contrast, the frequency of follicle selection, dominance, and ovulation did not change with weight loss (p&amp;gt;0.05). Higher pre-intervention LH was associated with fewer follicles recruited per cohort during the intervention (p=0.03). No other metabolic or endocrine marker was correlated with changes in follicular events. Conclusion: Clinically meaningful weight loss induced by a hypocaloric dietary intervention resulted in improvements in early stages of antral follicle development in women with PCOS, albeit these changes did not affect ovulatory follicle dynamics or the frequency of ovulation Presentation: Friday, June 16, 2023

Functional anatomy of mystacial active sensing in rats.

Rats' whisking motion and objects' palpation produce tactile signals sensed by mechanoreceptors at the vibrissal follicles. Rats adjust their whisking patterns to target information type, flow, and resolution, adapting to their behavioral needs and the changing environment. This coordination requires control over the activity of the mystacial pad's intrinsic and extrinsic muscles. Studies have relied on muscle recording and stimulation techniques to describe the roles of individual muscles. However, these methods lack the resolution to isolate the mystacial pad's small and compactly arranged muscles. Thus, we propose functional anatomy as a complementary approach for studying the individual and coordinated effects of the mystacial pad muscles on vibrissae movements. Our functional analysis addresses the kinematic measurements of whisking motion patterns recorded in freely exploring rats. Combined with anatomical descriptions of muscles and fascia elements of the mystacial pad in situ, we found: (1) the contributions of individual mystacial pad muscles to the different whisking motion patterns; (2) active touch by microvibrissae, and its underlying mechanism; and (3) dynamic position changes of the vibrissae pivot point, as determined by the movements of the corium and subcapsular fibrous mat. Finally, we hypothesize that each of the rat mystacial pad muscles is specialized for a particular function in a way that matches the architecture of the fascial structures. Consistent with biotensegrity principles, the muscles and fascia form a network of structural support and continuous tension that determine the arrangement and motion of the embedded individual follicles.

P-429 Transcriptomic heterogeneity within cortical ovarian follicle pool in child and adult

Abstract Study question What are the transcriptomic differences between child and adult ovarian follicles in the human cortex? Summary answer Our results suggest that human ovarian cortical follicles show marked differences before and after puberty, with the greatest differences being present in the secondary stage. What is known already The ovaries and their main functional structures, follicles, are important for determining the reproductive health. Follicle growth and maturation are controlled by multiple mechanisms, including the hypothalamus pituitary ovarian axis and local paracrine signalling. Although dormant primordial follicles are activated to grow even in infancy, all growing follicles before the first menarche are bound to undergo atresia. Follicle growth activation in infancy and adulthood remains poorly understood. Additionally, child and adult follicles behave differently during in vitro culture suggesting differing biology, raising many questions e.g., about their suitability for fertility preservation. Study design, size, duration Ovarian cortical tissues from children were collected through fertility preservation program at the Helsinki Children’s hospital (Finland). Families of underage patients signed an informed written consent. Adult ovarian tissue was collected from gender reassignment patients at the Helsinki University hospital and Karolinska university hospital after informed written consent. Follicles at different maturation stages were isolated from ovarian cortex for single follicle transcriptomic analysis. Freshly-fixed cortical biopsies (3x3x1 mm3) were used for validation of transcriptomic analysis. Participants/materials, setting, methods Single viable follicles (n = 120, 60/age group) were mechanically and enzymatically isolated from frozen-thawed ovarian tissue from four adults (age &amp;gt;18 yrs) and five children (age 1-11 yrs) and processed for RNA-sequencing. Individual follicles were lysed, prepared for RNA libraries following Smart-Seq2 protocol and sequenced using NextSeq 500 platform. After quality assessment, 109 follicles were analysed (Adult n = 54, Child n = 55). DDX4, AMH, FOXL2 and DAZL transcripts and proteins localization were evaluated via immunodetection and RNA-FISH assay. Main results and the role of chance Principal component analysis (PCA) of all sequenced follicles demonstrated an expected timely progression of their stages based on maturation, and an overall similarity between child and adult. Interestingly, PCA displayed two distinct clusters of follicles in both adult and child samples. The first group contained only growing follicles and, after differentially expressed gene (DEG) analysis, demonstrated lower expression of traditional oocyte markers (group 1). The second group consisted of primordial and growing follicles and had lower expression of traditional granulosa marker (group 2). Group-related differential expression of main oocyte (DDX4, DAZL) and granulosa (FOXL2, AMH) marker genes were validated in tissue sections with RNA-FISH. Downstream analyses focused on group 2 follicles. When child and adult follicles were compared to each other, the highest number of DEGs were found in the secondary stage (primordial n = 32, primary n = 14, secondary n = 387). Pathway analysis of DEG displays enrichment of oestrogen-related related pathways in adult secondary follicles. Furthermore, we analysed DEGs between the follicular stages per age group, finding high number of DEGs between primordial and primary follicles (Adult n = 1663, Child n = 1914). The majority of those genes are unique to the age group (common=512). Limitations, reasons for caution Adult ovarian tissue is obtained from gender reassignment patients, who receive androgen treatments prior to surgery. Child ovarian tissue was collected from fertility preservation programme where some patients were exposed to chemotherapy prior to tissue collection. Wider implications of the findings The differences between child and adult follicles encourage further investigation of the suitability of child ovarian tissue in current fertility preservation protocols. Understanding transcriptomic changes during early folliculogenesis and reasons for follicle degeneration can help tailor protocols for in vitro growth of follicles. Trial registration number not applicable

P-142 Alterations in mitochondrial DNA levels in luteal granulosa cells may affect the morphology of mature oocytes and subsequently their fertilization potential: a retrospective multicenter study

Abstract Study question Do alterations in mitochondrial DNA levels in luteal granulosa cells (LGCs) affect the quality of mature oocytes in infertile women? Summary answer Alterations in the mitochondrial DNA levels in LGCs may modify the characteristics of the first polar body in oocytes and subsequently decrease their fertilization potential. What is known already Assisted reproductive treatment outcomes are closely linked to the oocyte quality as shown in several systematic reviews and meta-analyses. Particularly, the oocyte quality is highly affected by the surrounding luteal granulosa cells (LGCs). This is mainly due to the crucial role of the mitochondria in the LGCs in fueling the metabolic processes required for oocyte maturation. Therefore, modifications in the quality of LGCs may have direct effects on the developmental competence of oocytes. However, the exact mechanisms by which this could happen are not fully understood. Study design, size, duration A retrospective multicenter study was conducted on 303 mature oocytes retrieved from 51 women undergoing intracytoplasmic sperm injection (ICSI). It was conducted in Lebanon at Al Hadi IVF center and Azoury IVF clinic, between January 2019 and January 2020. G-Power 3.1 was used to determine the sample size for Generalized Linear Mixed Models through a one-sample t-test power analysis with alpha 0.05, power 0.8, medium effect size (f2 = 0.15), and 5 predictors. Resulting sample size: 43. Participants/materials, setting, methods This study excluded cases of premature ovarian failure, severe oligozoospermia (&amp;lt;2 x 102/ml), or cases using frozen gametes. Mature oocytes, from young women (&amp;lt; 36 years old), were injected and cultured in the Embryoscope where morphometric measurements were performed. LGCs vitality and mitochondrial DNA (mtDNA) levels were analyzed using trypan blue exclusion dye and next-generation sequencing, respectively. Possible associations between these independent variables and the size and integrity of the first polar body were evaluated. Main results and the role of chance The included women presented with primary infertility. Their mean age was 29.98 ± 5.5 years old and their mean body mass index was 23.33 ± 3.32 Kg/m2. 10% of the included women had polycystic ovary syndrome, and 48% were smokers. Regarding the LGCs parameters, a statistically significant negative correlation was found between the mtDNA levels in LGCs and their vitality percentage (r = −0.313, p = 0.029). Interestingly, the average size of the first polar body (PBI) was 346.67 µm. Here we found that the PBI size decreased by 15.05 units when the mtDNA level in LGCs increased by one unit above its average. In parallel, the median percentage of oocytes having a fragmented PBI was 33.33 (0-100). This percentage increased by 4.26% for every one unit increase in the mtDNA level in LGCs (p &amp;lt; 0.0001). In contrast, this percentage decreased by 0.86% for every unit increase in the percentage of LGCs vitality (p &amp;lt; 0.0001). Moreover, the mean percentage of fertilization rate was found to be 70.19 ± 26.7. A higher percentage of fragmented PBI (38.3%) was found among oocytes that did not fertilize compared to those that did successfully fertilize (24%) (p = 0.019). Limitations, reasons for caution This study only analyzed the effects of alterations in mtDNA levels in LGCs on oocytes. It would be worthwhile to also analyze the effects on foetal development and offspring health. Furthermore, it would be interesting to study the relationship between alterations in LGCs and oocyte quality within individual follicles. Wider implications of the findings Several studies have suggested that abnormal size and fragmentation of the PBI may impair embryo development. Therefore, treating factors that affect LGCs, such as obesity and polycystic ovary syndrome, may benefit infertile women. This highlights the need for new clinical trials in this context. Trial registration number Not applicable

MALDI mass spectrometry imaging shows a gradual change in the proteome landscape during mouse ovarian folliculogenesis.

Our knowledge regarding the role proteins play in the mutual relationship among oocytes, surrounding follicle cells, stroma, and the vascular network inside the ovary is still poor and obtaining insights into this context would significantly aid our understanding of folliculogenesis. Here, we describe a spatial proteomics approach to characterize the proteome of individual follicles at different growth stages in a whole prepubertal 25-day-old mouse ovary. A total of 401 proteins were identified by nano-scale liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS), 69 with a known function in ovary biology, as demonstrated by earlier proteomics studies. Enrichment analysis highlighted significant KEGG and Reactome pathways, with apoptosis, developmental biology, PI3K-Akt, epigenetic regulation of gene expression, and extracellular matrix organization being well represented. Then, correlating these data with the spatial information provided by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) on 276 follicles enabled the protein profiles of single follicle types to be mapped within their native context, highlighting 94 proteins that were detected throughout the secondary to the pre-ovulatory transition. Statistical analyses identified a group of 37 proteins that showed a gradual quantitative change during follicle differentiation, comprising 10 with a known role in follicle growth (NUMA1, TPM2), oocyte germinal vesicle-to-metaphase II transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2), and preimplantation development (TIF1B, KHDC3). The proteome landscape identified includes molecules of known function in the ovary, but also those whose specific role is emerging. Altogether, this work demonstrates the utility of performing spatial proteomics in the context of the ovary and offers sound bases for more in-depth investigations that aim to further unravel its spatial proteome.