Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHRN316S mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHRN316S mice exhibited reduced numbers of corpora lutea and ovulation. The LHRN316S mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHRN316S female mice with ICR wild type male mice revealed that the infertility rate of LHRN316S mice was 21.4% (3/14). Litter sizes from LHRN316S mice were smaller than those from control wild type female mice. The oocytes from LHRN316S mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHRN316S mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.

During the development of animal organs, various adverse stimuli or toxic environments can induce oxidative stress and delay ovarian development. Paeoniflorin (PF), the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., has protective effects on various diseases by preventing oxidative stress. However, the mechanism by which PF attenuates oxidative damage in mouse ovaries remains unclear. We evaluated the protective effects of PF on ovaries in an H2O2-induced mouse oxidative stress model. The H2O2-induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development. Histology and follicular development were observed. We then detected related indicators of cell apoptosis, oxidative stress, and autophagy in mouse ovaries. We found that PF inhibited H2O2-induced ovarian cell apoptosis and ferroptosis and promoted granulosa cell proliferation. PF prevented oxidative stress by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels. In addition, the autophagic flux of ovarian cells was activated and was accompanied by increased lysosomal biogenesis. Moreover, PF-mediated autophagy was involved in clearing mitochondria damaged by H2O2. Importantly, PF administration significantly increased the number of primordial follicles, primary follicles, secondary follicles, and antral follicles. PF administration improved ovarian sizes compared with the H2O2 group. The present study suggested that PF administration reversed H2O2-induced ovarian developmental delay and promoted follicle development. PF-activated mitophagy is crucial for preventing oxidative stress and improving mitochondrial quality.

OBJECTIVE: This study aimed to assess differences in various scalp parameters between patients with androgenetic alopecia (AGA) and healthy volunteers using 22 MHz ultrasound.
METHODS: Thirty patients with AGA (AGA group) and 30 healthy volunteers (control group) who visited the Department of Dermatology at the Second Affiliated Hospital of Soochow University from September 2021 to June 2022 were randomly selected. The patients with AGA met the diagnostic criteria outlined in the Chinese Guidelines for the Diagnosis and Treatment of Androgenetic Alopecia. The severity of alopecia was assessed for males between grades 2 and 4 on the Norwood-Hamilton scale, and for females between stages 2 and 3 on the Ludwig scale. No artificial interventions were conducted at the vertex, and all examination conditions remained consistent. Ultrasound examinations at 22 MHz were performed on the scalp at the vertex in both the AGA and control groups. Seven parameters were measured, namely, epidermis + dermis thickness, entire scalp thickness, subcutaneous tissue thickness, average follicle width, average follicle length, follicle count, and the presence of color flow signals in the subcutaneous tissue. The differences in these parameters were then compared.
RESULTS: The AGA group showed reduced thickness of the entire scalp and subcutaneous tissue, narrower average follicle width, shorter average follicle length, lower hair follicle count, and fewer instances of color flow signals in the subcutaneous tissue at the vertex area (p < 0.05).
CONCLUSIONS: High-frequency (22 MHz) ultrasonography can be employed to visualize the entrance echo, dermis, subcutaneous tissue, and hair follicles of the scalp, thereby providing imaging for the clinical assessment of hair loss.

OBJECTIVE: Today, there is only limited knowledge of the spatial organization of hair chemistry. Infrared microspectroscopy is a well-established tool to provide such information and has significantly contributed to this field. In this study, we present new results combining multiple infrared microspectroscopy methods at different length scales to create a better chemical histology of human hair, including the hair follicle, hair shaft, hair medulla and hair cuticle.
METHODS: We used hyperspectral IR imaging & spectroscopy (HIRIS) and synchrotron-radiation FTIR microspectroscopy (SR-μFTIR) to measure transversal hair sections and SR-μFTIR to obtain high-resolution maps of longitudinal sections from the hair shaft and from the hair follicle. We used optical photothermal IR microspectroscopy (OPTIR) to analyse the cuticle surface of intact hairs.
RESULTS: By mapping longitudinal sections of the human hair follicle with confocal SR-μFTIR, we report the first demonstration of glycogen presence in the outer root sheath of the hair follicle by spectroscopy, and its quantification at the micron scale. Spectral maps, combined with machine learning-based analysis, enabled us to differentiate the various layers of the hair follicle and provided insights into the chemical changes that occur during hair formation in the follicle. Using HIRIS and SR-μFTIR to analyse the hair medulla in transversal sections of human hairs, we report here, for the first time by vibrational spectroscopy methods, the detection of unsaturated lipids at very low concentrations in the medulla. By analysing longitudinal sections of the hair shaft with SR-μFTIR, we found that calcium carboxylates are present in large regions of the hair cuticle, and not just in small focal areas as previously thought. We then use OPTIR to analyse the hair cuticle of intact hairs at submicron resolution without sectioning and report the distribution of calcium carboxylates at the surface of intact hair for the first time.
CONCLUSIONS: These new findings illustrate the potential of infrared microspectroscopy for imaging the chemical composition of human hair and may have implications for biomedical research or cosmetology.
OBJECTIVE: Aujourd\'hui, les connaissances sur l\'organisation spatiale de la chimie capillaire sont limitées. La microspectroscopie infrarouge est un outil bien établi pour fournir de telles informations et a largement contribué à ce domaine. Dans cette étude, nous présentons de nouveaux résultats combinant plusieurs méthodes de microspectroscopie infrarouge à différentes échelles de longueur pour créer une meilleure histologie chimique des cheveux humains, y compris le follicule pileux, la tige pilaire, la moelle pilaire et la cuticule pilaire. MÉTHODES: Nous avons utilisé l\'imagerie et la spectroscopie hyperspectrales infrarouges (Hyperspectral IR Imaging & Spectroscopy, HIRIS) et la microspectroscopie IRTF par rayonnement synchrotron (synchrotron‐radiation FTIR microspectroscopy, SR‐μFTIR) pour mesurer des coupes transversales de cheveux, et la SR‐μFTIR pour obtenir des cartes à haute résolution des coupes longitudinales de la tige pilaire et du follicule pileux. Nous avons utilisé la microspectroscopie photothermique infrarouge optique (Optical Photothermal IR microspectroscopy, OPTIR) pour analyser la surface des cuticules de cheveux intacts. RÉSULTATS: En cartographiant les coupes longitudinales du follicule pileux humain avec la SR‐μFTIR confocale, nous rapportons la première démonstration par spectroscopie de la présence de glycogène dans la gaine de la racine externe du follicule pileux, et sa quantification à l\'échelle du micron. Les cartes spectrales, combinées à une analyse basée sur l\'apprentissage automatisé, nous ont permis de différencier les différentes couches du follicule pileux et de mieux comprendre les changements chimiques qui surviennent pendant la formation des cheveux dans le follicule. En utilisant la méthode HIRIS et la SR‐μFTIR pour analyser la moelle pilaire dans les coupes transversales des cheveux humains, nous rapportons ici, pour la première fois par des méthodes de spectroscopie vibrationnelle, la détection de lipides insaturés à de très faibles concentrations dans la moelle. En analysant les coupes longitudinales de la tige pilaire par SR‐μFTIR, nous avons constaté que les carboxylates de calcium sont présents dans de vastes régions de la cuticule pilaire, et pas seulement dans de petites zones focales comme on le pensait auparavant. Nous utilisons ensuite la méthode OPTIR pour analyser la cuticule pilaire de cheveux intacts à une résolution inférieure au micron sans sectionner les cheveux et rapportons pour la première fois la distribution des carboxylates de calcium à la surface des cheveux intacts.
CONCLUSIONS: Ces nouveaux résultats illustrent le potentiel de la microspectroscopie infrarouge pour l\'imagerie de la composition chimique des cheveux humains et peuvent avoir des implications pour la recherche biomédicale ou la cosmétologie.

UNASSIGNED: Our objective was to investigate the efficacy of letrozole co-treatment in an antagonist protocol for infertile women with polycystic ovary syndrome (PCOS).
UNASSIGNED: This retrospective cohort study included infertile women with PCOS undergoing IVF/ICSI with and without letrozole co-treatment in an antagonist protocol from 2007-2021 at Shanghai Ninth People\'s Hospital (Shanghai, China). A total of 1559 participants were enrolled, with 1227 women in the antagonist group and 332 women in the letrozole co-treatment group. Propensity score-based patient-matching model was conducted to balance covariates between the groups. The primary outcome was the number of retrieved oocytes, with secondary outcomes including endocrine parameters, ovarian stimulation outcomes, pregnancy outcomes, and obstetrical and neonatal complications.
UNASSIGNED: Letrozole co-treatment induced significant changes in hormonal regulation, increased the percentage of large follicles, and resulted in fewer retrieved oocytes (P < 0.05). However, there was no negative impact on the number of usable embryos or good-quality embryos (P > 0.05). The live birth rates following fresh embryo transfer were comparable between the letrozole and control groups (single embryo transfer: 28.9% vs 29.7%, P > 0.05; double embryo transfer: 37.3% vs 45.6%, P > 0.05). Additionally, there were no significant differences between the two groups in the live birth rate per patient after frozen embryo transfer and the cumulative live birth rate (P > 0.05). No significant differences in obstetrical and neonatal complications were observed between the groups (P > 0.05).
UNASSIGNED: The addition of letrozole to the antagonist protocol for women with PCOS undergoing IVF induces a higher percentage of large follicles during oocyte retrieval, while reducing the overall number of retrieved oocytes. Moreover, the use of letrozole demonstrates comparable clinical outcomes following embryo transfers. These findings highlight the potential application of letrozole in an antagonist protocol for women with PCOS.

Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered \"immune-privileged\" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. Here we analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post-ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC-layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post-ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)-cAMP-PKA-CREB signaling cascade, is necessary for mGC-layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of \"mGC-layer stiffening\", shedding light on the mechanism that prevents mGC escape from the post-ovulatory follicle.

Previous in vitro studies have suggested that SLIT ligands could play roles in regulating ovarian granulosa cell proliferation and gene expression, as well as luteolysis. However, no in vivo study of Slit gene function has been conducted to date. Here we investigated the potential role of Slit1 in ovarian biology using a Slit1-null mouse model. Female Slit1-null mice were found to produce larger litters than their wild-type counterparts due to increased ovulation rates. Increased ovarian weights in Slit1-null animals were found to be due to the presence of greater numbers of healthy antral follicles with similar numbers of atretic ones, suggesting both an increased rate of follicle recruitment and a decreased rate of atresia. Consistent with this, treatment of cultured granulosa cells with exogenous SLIT1 induced apoptosis in presence or absence of FSH, but had no effect on cell proliferation. Although few alterations in the mRNA levels of FSH-responsive genes were noted in granulosa cells of Slit1-null mice, LH target gene mRNA levels were greatly increased. Finally, increased phospho-AKT levels were found in granulosa cells isolated from Slit1-null mice, and SLIT1 pretreatment of cultured granulosa cells inhibited the ability of both FSH and LH to increase AKT phosphorylation, suggesting a mechanism whereby SLIT1 could antagonize gonadotropin signaling. These findings therefore represent the first evidence for a physiological role of a SLIT ligand in the ovary, and define Slit1 as a novel autocrine/paracrine regulator of follicle development.

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Continuous ovarian imaging has been proven to be a method for monitoring the development of follicles in vivo. The aim of this study was to evaluate the efficacy of combining ultrasound bio-microscopy (UBM) with an intravital window for follicle imaging in rabbits and to monitor the ovarian dynamic processes. New Zealand White female rabbits (n = 10) received ovarian translocation to a subcutaneous position. The ovarian tissue was sutured onto the abdominal muscles and covered with an intravital window for the continuous monitoring of the follicles using UBM. Results show that physiological changes (red blood cell and white blood cell counts, feed intake, and body weight change) in rabbits induced by surgery returned to normal physiological levels in one week. Furthermore, UBM could provide high-resolution imaging of follicles through the intravital window. Daily monitoring of ovarian dynamic processes for 6 days displayed variabilities in follicle counts and size. Collectively, these results provide a relatively new method to monitor ovarian dynamic processes and to understand the reproductive physiology of female rabbits.