Lung cancer remains one of the major human malignancies affecting both men and women worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent type. Multiple mechanisms have been identified that favor tumor growth as well as impede the efficacy of therapeutic regimens in lung cancer patients. Among tumor suppressor genes that play critical roles in regulating cancer growth, the phosphatase and tensin homolog (PTEN) constitutes one of the important family members implicated in controlling various functional activities of tumor cells, including cell proliferation, apoptosis, angiogenesis, and metastasis. Notably, clinical studies have also documented that lung tumors having an impaired, mutated, or loss of PTEN are associated with low survival or high tumor recurrence rates. To that end, PTEN has been explored as a promising target for anti-cancer agents. Importantly, the ability of PTEN to crosstalk with several signaling pathways provides new approaches to devise effective treatment options for lung cancer treatment. The current review highlights the significance of PTEN and its implications in therapeutic approaches against NSCLC. Lung cancer remains a major cause of cancer-associated mortality in both men and women in the United States and around the world ,2,3,4,5.While the prognosis of lung cancer remains relatively poor, an initial evaluation can be conducted depending on the history of the patients, laboratory tests, and chest radiography ,16. FurtOf importance, targeted therapy is specifically designed to eradicate tumor cells that harbor activating mutations in any particular gene of a signaling cascade and is currently being implemented to treat advanced-stage or metastatic lung cancer patients ,30. For The oncogene-driven paradigm of cancer predominated from the 1970s through the 1980s, and it was proposed that the major driver of transformation is the overexpression or aberrant activation of certain genes ,37. HoweImportantly, Knudson’s two-hit theory was explicitly supported by the finding that both Rb alleles were lost . AccordiAs detailed above, among various cellular targets, the phosphatase and tensin homolog (PTEN) is one of the important family members of tumor-suppressive genes, which has been extensively studied in preclinical and clinical settings of lung cancer ,56,57. PHowever, an impaired and/or loss of the functional activity of PTEN is a major determinant in impacting tumor development across all cancer types ,62. To tOn the other hand, several strategies have been explored to induce PTEN expression/ activity with the overall goal of regulating the signaling pathways involved in cancer growth and resistance to therapeutic agents. To that end, the treatment with naturally occurring phytochemicals such as curcumin, indol-3-carbinol, genistein, resveratrol, and sulforaphane has been found to induce PTEN expression or activity ,71,72,73Several studies have used in vitro culture systems and mouse models to define the functional significance of PTEN in NSCLC. The summaries of the in vitro studies that included the overexpression of PTEN in NSCLC cell lines and in vivo studies are given in Moreover, the PTEN expression in lung cancer cells is regulated by deubiquitylase Ataxin-3 . For theIn addition, pemetrexed treatment for lung cancer cells has also been documented to inhibit the PI3K pathway. In a study, Li and colleagues demonstrated that PTEN overexpression can increase the efficacy of pemetrexed in the A549 NSCLC cell line . ImportaImportantly, Noro and colleagues determined how PTEN inactivation can affect tumor progression and drug resistance in lung cancer . By usinG12D activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter resulted in the development of both adenocarcinoma and squamous cell carcinoma in the lungs. The authors found that when PTEN deletion occurs in targeting basal cells, it can initiate both lung adenocarcinoma and squamous cell carcinoma formation. Although PTEN deletion is a weaker tumor initiator than KrasG12D, with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and more malignant than KrasG12D-initiated tumors. When genetic modifications were focused on a particular cell, the fact that PTEN loss did not enhance lung SCC development in comparison to KrasG12D activation implies that the initiating genetic event does not determine tumor histology. These findings also indicated that conducting airway basal cells can give rise to a variety of NSCLC tumors [As PTEN deletion has been shown to occur within the tumor cells residing in the airway basal cells, Malkoski and colleagues performed a study using a mouse model to examine if the loss of PTEN in airway basal cells can initiate tumor formation or increase squamous cell carcinoma formation . The autC tumors .Along similar lines, Yu and colleagues determined the mechanisms and functional significance of the abnormal expression of PTEN using various in vitro and in vivo models of NSCLC . The autNotably, protein ubiquitination plays a dynamic role in modulating protein stability, including in PTEN, and it requires ubiquitin-specific proteases (USPs) or deubiquitinases (Dubs) to hydrolyze the ubiquitin molecules from their substrates. In a study, He and colleagues determined the effect and mechanism of USP10 as a tumor suppressor Dub of PTEN . The autOf importance, the role and mechanisms of the RNA-binding motif protein 10 (RBM10), an alternative splicing regulator that plays an important role in regulating proliferation and apoptosis, was determined in NSCLC . An analV12. Importantly, high expression of CK1α, PTEN, and Atg7 in human NSCLC tissues was found to be associated with increased overall survival and vice versa. Overall, these findings indicated that PTEN interacts with CK1α, which maintains PTEN stability and activity to induce autophagy in NSCLC.Along similar lines, studies by Cai and colleagues demonstrated that casein kinase 1 alpha 1 (CK1α), a negative regulator of the Wnt pathway, induces PTEN stabilization and activity by preventing PTEN and NEDD4-1 binding and inhibiting PTEN polyubiquitination and phosphorylation . This efIn one study, He and colleagues determined the role and mechanism of protein tyrosine phosphatase interacting protein 51 (PTPIP51) as a tumor suppressor and its interaction with PTEN in NSCLC . Using aSeveral studies have investigated the impacts of somatic mutations in PTEN in NSCLC patients. The summaries of the clinical studies are given in In addition, Sos and colleagues used a vast collection of genomically defined NSCLC cell lines to identify genomic traits that distinguish EGFR-dependent from EGFR-independent EGFR-mutant lung tumor cells. The authors used a combination of computational, biochemical, and cellular methods to discover new mechanisms that uncouple EGFR-dependent tumors from their downstream signaling. The computational genomics analyses found PTEN homozygous deletion to be a candidate for EGFR inhibitor resistance. The functional studies revealed that PTEN loss causes a considerable decrease in apoptosis sensitivity in EGFR-mutant cells by activating AKT and EGFR, and it was hypothesized that ERK activation in PTEN-deficient cells leads to the increased transcription of EGFR ligands, such as amphiregulin .In another study, Yoo and colleagues evaluated whether surgically resected primary NSCLC and the loss of PTEN expression correlates with clinicopathological parameters or is related to EGFR gene status in NSCLC . The autOf importance, Cumberbatch and colleagues found that clinical SCC specimens had a significantly increased expression of PI3Kβ and reduced/loss of PTEN expression compared to adenocarcinoma specimens . DetaileAlong similar lines, Panagiotou and colleagues conducted a study to quantitatively analyze the PTEN protein expression in NSCLC. A sample of 61 NSCLC cases was used in the study; about 80% of the cases were males, while 20% of the cases were females. A total of 57% of patients had adenocarcinoma, and 42% had squamous cell carcinoma. Digital image analysis was conducted to analyze the PTEN protein quantitively, and it was found that patients who developed a progressive loss of PTEN expression appeared to have a higher rate of metastases than those with PTEN overexpression. Therefore, in NSCLC, PTEN deregulation was associated with an aggressive phenotype, and this is an important factor in determining a tailored targeted therapy regimen, especially when combined with HER2/PI3K inhibitors .While there are therapeutic potentials to modulate PTEN, it still is an undruggable target. Thus, PI3K/AKT/mTOR inhibitors have been used in clinical settings to treat human malignancies, including lung cancer. A list of ongoing and recently completed clinical trials of PI3K/AKT/mTOR inhibitors is given in Several studies have confirmed the potential implications of PTEN in NSCLC.While the primary mechanism of its tumor suppressor activity is mediated via its ability to dephosphorylate PIP3 into PIP2, resulting in antagonizing PI3K/AKT signaling and inducing tumor cell death, the detailed mechanisms  are yet to be fully determined. Given that a reduced or loss of tumoral PTEN expression has been associated with the overall low survival of NSCLC patients, approaches to overcome this effect would aid in the ongoing challenges for the treatment of NSCLC patients. Of significance, given the importance of predictive biomarkers in devising promising strategies to decrease the risk and/or improve the overall therapeutic outcomes in NSCLC patients, several studies have supported the applicability of the PTEN status to be explored as a prognostic marker against various malignancies, including NSCLC.


PIK3CA mutations have been reported in macrodactyly, a rare condition related to LPN, and in other localized lipomatous overgrowth syndromes. In this retrospective study, we report 6 cases of FPN involving the median nerve . All cases were diagnosed via biopsy or resection supplemented by MRI. Patients were 4 males and 2 females aged 23 to 60 years (mean 38 years). One patient with bilateral lesions had in addition extensive angiomatosis of the paravertebral region. Histological examination showed an abnormal amount of mature fatty tissue containing disordered fibrous bands, entrapping normal-looking nerve fibers with prominent perineurial and endoneurial fibrosis. Genetic analysis using snapshot assay constructed to detect hotspots mutations in PIK3CA revealed similar PIK3CA mutations  in 5/6 cases (83.3%). Our study represents a further contribution to the literature on LPN and highlights the diagnostic value of PIK3CA mutation testing as surrogate tool in equivocal cases and in those lesions without associated macrodactyly, especially as the biopsy findings of this lesion are essentially nonspecific.Lipomatosis of peripheral nerves  is a very rare, benign, intraneural, tumorous lesion that predominantly involves the median nerve but may rarely affect any peripheral nerve. Recently,  PIK3CA mutations has been largely limited to series addressing macrodactyly, and the association of PIK3CA mutations with LPN has not been much emphasized in the pathology literature. In this retrospective study, we present our experience with LPN of the median nerve with particular focus on PIK3CA mutation testing as surrogate for diagnosis.Lipomatosis of peripheral nerves  is a very rare, benign, intraneural tumor consisting of abnormal amount of mature fibrofatty tissue , 2. Its Our institutional archive was searched for cases of carpal tunnel syndrome and for LPN of the median nerve. Clinical examination, including detailed physical examination, was performed, and the clinical histories were extensively reviewed. Additionally, MRI-imaging data were carefully evaluated, and all available pathology specimens were re-reviewed. In addition, cases seen by one of the authors (AA) in the setting of histopathological consultation were included as well. The tissue specimens were fixed in formalin and processed routinely for histopathology. Hematoxylin and eosin (H&E) slides were reviewed retrospectively to exclude lipomatous neoplasms.PIK3CA gene  [For DNA extraction, the whole specimen was marked on an H&E-stained slide in 5 of the cases. In one case, the neural tissue and the fibrolipomatous component were marked for manual microdissection to test the two components separately. DNA was isolated using the Blood DNA Preparation Kit  according to the manufacturer’s protocols. A previously described highly sensitive SNaPshot© multiplex assay, based on the SNaPshot Multiplex System assay , was used to screen for three mutation hotspot regions in the PIK3CA mutation) was excluded to maintain homogeneity of the series.During the period 2012 to 2022, a total of 570 patients underwent surgical decompression of the carpal tunnel at our center based on a clinical diagnosis of carpal tunnel syndrome. Among them, we identified four patients suffering from carpal tunnel syndrome caused by a compressive neuropathy of the median nerve due to LPN (0.7%). In addition to these four in-house patients, two cases were retrieved from the histopathological consultation files of one of the authors (AA). One consultation case originating in the lower limb  or during surgical intervention. One patient presented intraoperatively with a constriction of the median nerve due to a thickened epineurium and perineurium. Therefore, neurolysis was performed and the median nerve was covered with a fascial sheet to prevent postoperative scarring. One patient (case 4) underwent initial decompression of the carpal tunnel in an external center without preoperative MRI-Imaging. Intraoperatively, a gigantic median nerve was found. After initial surgery, the patient reported improvement of the numbness of the thumb and index and middle fingers but was still suffering from paresthesia. The patient was referred to our center for further diagnostic workup. After MRI-imaging, the patient underwent surgical therapy at our center with neurolysis of the enlarged median nerve. Additionally, biopsies were taken for histopathological analysis. The two consultation cases were treated by unspecified surgical resection of the mass as noted from the submitted specimens.Post-surgery, all patients reported initial improvement of symptoms. However, one patient reported persistent pain, edematous swelling of the right hand 4 weeks after surgery, and a complex regional pain syndrome (CRPS), which was treated by methylprednisolone, pregabalin, and vitamin C as well as DMSO/ambroxol cream, resulting into recovery.One patient presented with recurrence of symptoms and a slowly growing palpable mass at the wrist 10 years after initial surgical decompression. Revision surgery revealed significant scarring of the former carpal ligament. Consequently, after decompression, the enlarged median nerve was secondarily covered with a fascial sheet to reduce the recurrence of scarring. Although details regarding other associated genetic disorders are not complete, bilateral LPN of the median nerve as possible feature of a genetic predisposition was observed in one patient (1/6 = 16.7%). The same patient presented with histologically verified extensive capillary-venous-type angiomatosis in the paravertebral soft tissue.Histologically, all cases were similar, being composed of an abnormal amount of normal-looking mature fatty tissue containing variable amount of disordered fibrous tissue forming linear bands and small aggregates. Based on the type of biopsy and tissue amount, variable numbers of small or thick nerve fascicles were seen entrapped within the fibrolipomatous background, associated with a variable degree of perineurial and endoneurial fibrosis Fig. . The resPIK3CA revealed an identical pathogenic PIK3CA mutation  in 5 of 6 cases (83.3%). In one patient, we tried separate manual microdissection of the nerves and the hamartomatous tissue; we detected the same mutation in both samples. However, due to the manual nature of microdissection, we cannot verify the presence of the mutation in the nerves with certainty as contamination cannot be reliably ruled out. Molecular testing of the angiomatosis lesion of soft tissue in case 2 failed probably due to the decalcification of the resection specimen. No clinicopathological differences were observed regarding the single PIK3CA wild-type case compared to the mutated cases.Genetic analysis using snapshot assay constructed to detect hotspots mutations in Lipomatosis of peripheral nerves (LPN) is a distinctive entity first described by Silverman and Enzinger in 1985 as fibrolipomatous hamartoma of peripheral nerves . AssociaThe clinical diagnosis of LPN can be challenging. If there is no obvious mass lesion, symptoms such as numbness or pain can be nonspecific, as they can also be associated with trauma, other neural tumor entities, or just a result of overtraining –10. CertAlthough morphologically being a simple bland lesion, histopathological diagnosis of LPN is often a challenge due to two major reasons: 1) the entity is very rare; hence, majority of pathologists (except for soft tissue pathologists) might not be aware of it and 2) the histological biopsy findings are nonspecific and practically indistinguishable from normal tissue, resulting frequently in descriptive diagnoses of “normal fibrofatty tissue or negative for pathological findings or neoplasms. This is further enhanced by the fact that only small tissue fragments are usually submitted in an attempt to avoid scarifying the nerves, so the mass-forming appearance of the lesion is only rarely seen by pathologists . In linePIK3CA gene mutations in LPN with or without associated macrodactyly/overgrowth, leading to an establishment of the concept of PIK3CA-related “overgrowth syndromes” [PIK3CA mutations in the majority of cases, independent of absence of macrodactyly. Accordingly, we advocate the use of PIK3CA mutation testing as a valuable surrogate for confirming diagnosis in equivocal cases on limited biopsies. Clustering of the mutations at codon 3410 points to the reliable rational use of hotspots testing methods to recognize most of the mutations expected in these lesions.Recent exome sequencing studies reported somatic activating ndromes” , 15. OurPIK3CA mutations in the pathogenesis of diverse localized or diffuse overgrowth syndromes has been increasingly recognized. One of our cases presented with bilateral LPN involving the median nerve in addition to soft tissue angiomatosis of the paravertebral region, suggesting either an inherited or a mosaic disorder, in line with the recently reported PIK3CA-related vascular anomalies [PIK3CA-associated vascular lesions were clinically isolated and were not associated with other disorders or with LPN as in one of our cases. Moreover, PIK3CA mutations were also reported in more extensive phenotypes in the disorders collectively termed PIK3CA-related “overgrowth spectrum=PROS” including Klippel–Trenaunay syndrome [PIK3CA, but no case had reported vascular anomalies. Accordingly, our case 2 represents a novel addition to the clinicopathological associations of LPN.The role of nomalies . Notablysyndrome . In one Surgical treatment of LPN of the median nerve should be initiated individually. Our approaches vary depending on the symptoms, radiologic, and clinical as well as on intraoperative findings. A conservative treatment regimen is possible if the patient does not suffer from pain or neurological impairment. In all other cases, a surgical procedure should be indicated to resolve the symptoms and to avoid permanent nerve damage. The first step of the surgical treatment is an open carpal tunnel decompression. Moreover, taking biopsies is recommended to rule out malignancies. A resection of the mass is impossible without causing permanent damage to the affected nerve and should not be performed.In some cases, as described in the first patient, no further surgical therapy is necessary . However, the third case shows that if the surgeon encounters a constriction of the median nerve due to a thickened epineurium and perineurium, we suggest performing a neurolysis. Additionally, the nerve can be covered with a fascial sheet to prevent scarring and improve gliding , 16. AnoPIK3CA mutations in these rare but diagnostically and therapeutically challenging lesions represent a valuable surrogate molecular marker for confirming diagnosis in limited biopsies or clinically ambiguous cases. Moreover, the role of these mutations as a potential therapeutic target using mTOR inhibitors like Everolimus and other substances remains to be verified in future studies.Lipomatosis of the median nerve is a very rare cause of carpal tunnel syndrome. Early surgical decompression of the carpal tunnel as well as biopsies of the mass is mandatory to achieve improvement of symptoms and to avoid long-term damage of the nerve as well as to rule out malignancies. Recurrent


Vulvodynia is a debilitating condition characterized by painful sensitivity to touch and pressure in the vestibular tissue surrounding the vaginal opening. It is often a “diagnosis of exclusion” of idiopathic pain made in the absence of visible inflammation or injury. However, the association between increased vulvodynia risk and a history of yeast infections and skin allergies has led researchers to explore whether immune mechanisms of dysregulated inflammation might underlie the pathophysiology of this chronic pain condition. Here we synthesize epidemiological investigations, clinical biopsies and primary cell culture studies, and mechanistic insights from several pre-clinical models of vulvar pain. Taken together, these findings suggest that altered inflammatory responses of tissue fibroblasts, and other immune changes in the genital tissues, potentially driven by the accumulation of mast cells may be key to the development of chronic vulvar pain. The association of increased numbers and function of mast cells with a wide variety of chronic pain conditions lends credence to their involvement in vulvodynia pathology and underscores their potential as an immune biomarker for chronic pain. Alongside mast cells, neutrophils, macrophages, and numerous inflammatory cytokines and mediators are associated with chronic pain suggesting immune-targeted approaches including the therapeutic administration of endogenous anti-inflammatory compounds could provide much needed new ways to treat, manage, and control the growing global pandemic of chronic pain. De Medicina by Roman encyclopedist Aulus Celsus) and is key to both healing, and withdrawal from harmful exposures. However, long-lasting inflammation can lead to dysfunctional tissue remodeling and/or central nervous sensitization producing unresolved pathological pain  as vulvar pain lasting at least 3 months without readily detectable accompanying inflammation or infection . While Lcal pain . Fibroblin vitro . Repeatein vitro  and multin vitro  or a comin vitro  producedin vitro, as well as a correlation of increased interleukin (IL-6) and prostaglandin E2 (PGE2) levels with pain sensitivity  activation  as well in vitro .Candida albicans via Dectin-1 and release cytokines and other granule contents during the early immune response to fungal infection  daily for 10 days on the labial skin of previously sensitized female ND4 mice provoked tactile peri-vaginal pain responses that lasted days to weeks after overt inflammation resolved . Accumulbiopsies .+ regulatory CD4+ T cells and IFN-γ producing memory CD8+ T cells in the affected tissues. Notably, therapeutic local mast cell depletion in both oxazolone and DNFB-driven pain by injections of mast cell degranulating compound 48/80 (c48/80) and topical application of tetrahydrocannabinol (THC) respectively reduced pain responses strengthening the case for mast cell-driven immune changes in perpetuating long-term maladaptive vulvar sensitivity.To demonstrate that these findings were not oxazolone-specific, we used a different hapten, dinitrofluorobenzene (DNFB), that we applied daily for 10 days to the vaginal canal of previously sensitized mice  and reprReed and colleagues found a positive association between vulvodynia and the reported history of exposures to a variety of household and work-related environmental toxins . Isothiain vitro, mirroring the behavior of fibroblasts derived from vulvodynia patients , IL-1 β, and IL-6 via central sensitization mechanisms in a rat model of migraine pain . Mast ceProstatic secretions from patients with chronic pelvic pain syndrome had increased numbers of degranulated mast cells and higher NGF levels; mast cell-deficient mice had a higher threshold for lower abdominal pain driven by immune messengers CCL2 and CCL3 in a model of experimental autoimmune prostatitis . CCL2 meMast cells are important in tissue repair after injury or acute inflammatory events , and thiAs a cardinal sign of inflammation, pain is initially protective; it signals the detection of harmful stimuli and induces withdrawal and lack of use that allows healing . Innate Innate cells including neutrophils, mast cells, and macrophages can also regulate the transition of acute to chronic pain, and contribute to its maintenance via complex neuroimmune signaling  that is One mouse model of vaginal candidiasis-driven vulvar pain reported no increase in any immune cells in the affected tissue , in lineDespite these exceptions, as shown in multiple pre-clinical studies of vulvodynia discussed above , therapiThus, targeting specific, relevant, innate immune cellular and molecular components in ways that minimize immune suppression and maximize control of pathological inflammation can provide novel therapeutic approaches for vulvodynia and other chronic pain conditions.Vulvodynia affects quality of life in substantive and deleterious ways including sexual dysfunction, infertility, depression, and compromised mobility. A survey of 280 patients at University of California, San Francisco found that of all vulvar conditions included in the survey, a diagnosis of vulvodynia was the most strongly related to a poor quality of life, and effects on physical and social functions were worse than for other vulvar pathologies . DespiteGO, KT, and XMG reviewed the literature and drafted summaries. ET and DC wrote the manuscript. ET prepared the figure and edited the manuscript. DC conceived, designed, and supervised the manuscript writing. All authors contributed to the article and approved the submitted version.


Sinonasal and skull base malignancies are rare tumors which present adjacent to numerous critical anatomical structures. Despite the increased use of endoscopic techniques, many patients still require open surgery. Treatment with surgery requires careful pre-operative planning. Virtual surgical planning and three-dimensional, patient-specific models offer a range of benefits to clinicians in terms of planning tumor resection and reconstruction. In additional, these technologies improve communication with patients, trainee doctors and other colleagues. In this article, we explore the applications of virtual surgical planning in sinonasal and skull base surgery.Sinonasal and skull base malignancies represent a rare, heterogenous group of pathologies with an incidence of 0.556 per 100,000 persons in the population. Given the numerous critical anatomic structures located adjacent to the sinonasal cavity and skull base, surgery for tumors in this region requires careful pre-operative planning with the assistance of radiological imaging and intraoperative image guidance technologies to reduce the risk of complications. Virtual surgical planning (VSP) and three-dimensional models (3DMs) are adjunctive technologies which assist clinicians to better visualize patient anatomy using enhanced digital radiological images and physical stereolithographic models based on patients’ personal imaging. This review summarizes our institutional experience with VSP and 3DMs in sinonasal and skull base surgical oncology. A clinical case series is used to thematically illustrate the application of VSP and 3DMs in surgical ablation, reconstruction, patient communication, medical education, and interdisciplinary teamwork in sinonasal and skull base surgery. Sinonasal and skull base malignancies represent a rare, heterogenous group of pathologies with an incidence of 0.556 per 100,000 persons in the population . DespiteVirtual surgical planning (VSP) and three-dimensional models (3DMs) are adjunctive technologies which assist clinicians to better visualize patient anatomy using enhanced digital radiological images and physical stereolithographic models based on patients’ personal imaging . Three-dWhile the use of VSP and 3DMs has been explored in detail in maxillofacial, orthopedic, and craniofacial surgery, fewer reports have described its application in open or endoscopic sinonasal and skull base surgery. In addition, discussion of its role in guiding ablation in sinonasal and skull base surgery and patient communication for these diseases is more limited. This review summarizes our institutional experience with VSP and 3DMs in sinonasal and skull base surgical oncology. A clinical case series is used to thematically illustrate the application of VSP and 3DMs in surgical ablation, reconstruction, patient communication, medical education, and interdisciplinary teamwork in sinonasal and skull base surgery.n = 280). Cases were excluded where radiological imaging, VSP digital plans, or adequate clinical information were not available. Institutional Review Board approval was granted under an existing protocol to complete this study and written consent was obtained from all patient participants. Patients were staged using the 8th edition of the AJCC [A retrospective review of institutional medical records between 2019 and 2021 was performed to identify cases where 3DMs and VSP were utilized during surgery for sinonasal or skull base malignancies with or without microvascular free flap reconstruction  and magnetic resonance imaging (MRI), with or without positron emission tomography (PET). All cases are discussed at a skull base multidisciplinary tumor board meeting prior to the initiation of treatment.® Maxillofacial, West Chester, PA, USA) or an alternative agent. A formal meeting between the operating surgeons  and a company representative is completed to review the VSP images and design surgical models and cutting guides pre-operatively. In select cases, alternative, reserve cutting guides are designed to permit multiple ablative and reconstructive options based on the intraoperative clinical findings. Reconstructive materials can be digitally superimposed on the model pre-operatively in order to plan precise reconstruction of the surgical defect. A final report detailing the planned cutting guides is presented to the team pre-operatively. In cases where cutting guides for osteotomy are not required, a 3DM may be produced to guide patient communication, education, or planning.The VSP imaging and 3DMs used for surgical planning are created using multiplanar  CT imaging using 0.5–1.25 mm slice thickness, which is converted into de-identified Digital Imaging and Communications in Medicine (DICOM) format with a 0.33 mm × 0.45 mm pixel size. Manufacture of the 3DM is typically completed by DePuy Synthes  involving the lacrimal duct received induction chemotherapy prior to open craniofacial resection incorporating nasal bones and medial maxilla. In illustrative Traditionally, to ensure safe and comprehensive tumor excision, anatomical landmarks based on radiological imaging are used to guide ablation intraoperatively. The surgeon will use two-dimensional (2D) images to understand the anatomic relationships between a tumor and its local anatomy and derive a mental 3D plan. This is combined with the use of intraoperative image guidance to maximize surgical accuracy. The use of VSP and a 3DM permits precise planning and demonstration of incision margins on a physical model based on patient imaging in advance of surgery, which may be particularly useful where anatomy is distorted or obliterated by the tumor. Partial response to induction chemotherapy, pre-operative radiation therapy, and a non-centripetal pattern of tumor response may make recognizing anatomical landmarks more challenging, further strengthening the case for the use of VSP as an aid during surgery. The authors highlight the use of 3DMs to provide precise cutting guides for bony cuts in the midface, which may serve to enhance the quality and accuracy of reconstruction. 3D-printed cutting guides may or may not be used based on tumor location and the ability to affix cutting guides to bone.Case 2 describes an 8-year-old male with a diagnosis of a cT2N0M0 nasopharyngeal embryonal rhabdomyosarcoma treated with chemotherapy and radiotherapy. The patient subsequently developed local recurrence high in the parapharyngeal space closely applied to both the internal carotid artery and external carotid artery branches. In this case, a 3DM and VSP allowed a greater appreciation of the relationship of the tumor and major arteries when communicating the need for preoperative embolization of the proximal internal carotid artery, the internal carotid at the foramen lacerum, and the maxillary branch of the external carotid artery to the patient’s family. This permitted a lip-split approach to en bloc removal of the tumor encompassing the ramus of the mandible, the masticator space, and the parapharyngeal space.Given the rarity and anatomical complexity of tumors in the sinonasal and skull base region, educating patients and their families regarding treatment plans, alternative options, and the associated risks is essential. Frequently, however, this presents a conceptual challenge for patients, who may be presented with 2D radiological imaging during discussion. In this type of case, a 3D rendered image A can be The use of a 3DM in this case assisted understanding of the need for a complex multi-stage treatment process. Improving communication and patient education is one way in which physicians can hope to improve patient satisfaction and outcomes using VSP and 3DMs.Case 3 describes a 69-year-old female who presented with nasal obstruction, facial pain, swelling, and nasal cavity mass. A complete workup confirmed a cT4aN0M0 ethmoid sinus adenoid cystic carcinoma with invasion of the maxillary sinus, sphenoid sinus, and anterior cranial fossa, with abutment of the internal carotid artery (d artery B,C. The Surgical residents and fellows involved in the workup, surgical management, and aftercare of patients with sinonasal and skull base tumors can benefit from the use of VSP and 3DMs A. TraineUsing the 3DM, residents and fellows were able to directly visualize the tumor extent and potential pitfalls of the endoscopic technique employed. This permitted them to assist in complete resection of the tumor with negative margins. Sound anatomical knowledge and competent surgical skills remain the most critical skills for trainees, with VSP and 3DM technology offering great potential to enhance resident and fellow education.Case 4, an 87-year-old female presented with a new diagnosis of a cT4aN0M0 left maxillary sinus SCC. The patient previously underwent multiple surgical resections for an inverted sinus papilloma at outside institutions and presented to our center following rapid expansion of the mass. The imaging highlighted an expansile, multicompartment maxillary sinus tumor measuring 5.5 cm × 3.0 cm, involving the infraorbital nerve and the floor of the orbit, with extraconal orbital spread and infiltration of all the walls of the maxillary sinus (In ry sinus B,C.The patient underwent total maxillectomy with reconstruction with a vertical rectus abdominis myocutaneous free flap and a titanium mesh to reconstruct the orbital floor. VSP and a 3DM were used in this case to demonstrate the anticipated osteotomies and the resulting defect to the reconstructive team in advance of surgery A. Using Use of VSP in this case was focused on assessing the best possible reconstructive options for this elderly patient and reducing the operative time while enhancing the patient’s functional and cosmetic outcomes. Further potential advantages of VSP and 3DMs are the ability to plan prosthodontic rehabilitation pre-operatively as well as applying cutting guides not only for the ablative osteotomies but also the reconstructive osteotomies, for example, in scapular or fibular free flap reconstruction. This patient underwent successful resection and reconstruction without complication.Case 5 describes a 54-year-old female presenting with a cT4aN0M0 spindle cell sclerosing rhabdomyosarcoma involving the anterior skull base, ethmoid sinus, frontal sinus, and orbit (nd orbit A,B. TreaAn open craniofacial resection with skull base reconstruction using a peri-cranial flap, calvarial bone graft, and radial forearm free tissue transfer was ultimately undertaken C. The 3DWe describe the role of VSP and 3DMs in sinonasal and skull base surgery with a thematic clinical case series demonstrating real-world applications in surgical ablation, reconstruction, resident and fellow education, and multidisciplinary teamwork. We particularly highlight the role of 3DMs and VSP in providing potentially more precise oncologic resection and more comprehensive planning for reconstruction. Additionally, the role of VSP and 3DMs in enhancing patient understanding of surgery and improving communication between team members has been discussed as additional benefits of these technologies in the setting of sinonasal and skull base surgery.The role of VSP and 3DMs in mapping tumor extent pre-operatively and to ensure adequate oncologic resection has been established in head and neck surgery, as well as other specialties, but is less completely explored in the skull base field ,8,9. As Precision cutting guides modelled on VSP have been validated in maxillofacial and craniofacial surgery . It is tWe highlight the role of 3DMs and VSP in bony and soft tissue microvascular free flap reconstruction of the skull base. Reconstruction of the skull base typically requires multiple forms of tissue to establish adequate functional reconstruction without CSF leak or other complications. We demonstrate in our series that estimating the size of the ablative defect more precisely using VSP in advance of surgery permits improved reconstruction, as seen in Case 4. We highlight improved bony contact allowing enhanced anatomical reconstruction where VSP/a 3DM is used to plan osteotomies, as well as the possibility to assess nasal anatomy and vasculature for nasoseptal flap reconstruction ,17. OutsThe use of VSP and 3DMs in place of cadaveric specimens has been described in both medical student and resident education . ObtainiNew technologies have also developed in tandem with VSP and 3DMs. Patient-specific instruments designed to conform to a patient’s particular pathology have been described. Manufacture of 3D-printed bone scaffolds which permit bony ingrowth has been reported . The addAs with all new technologies, potential disadvantages exist with VSP and 3DMs. Though some authors have demonstrated reduced costs, the cost of VSP and 3DM technology can be significant . AdditioVirtual surgical planning and 3D modeling offer a variety of benefits in sinonasal and skull base surgery to enhance both the quality of surgical ablation and guide reconstruction in these complex surgeries. In addition to their immediate clinical applications, VSP and 3DMs have potential for use as communication and education tools for both patients and trainees. We highlight 3DMs and VSP as useful tools in the future of the multidisciplinary management of sinonasal and skull base tumors.


Diabetes constitutes a world-wide pandemic that requires searching for new treatments to halt its progression. Cellular senescence of pancreatic beta cells has been described as a major contributor to development and worsening of diabetes. The concept of reversibility of cellular senescence is critical as is the timing to take actions against this “dormant” senescent state. The reversal of cellular senescence can be considered as rejuvenation of the specific cell if it returns to the original “healthy state” and doesn’t behave aberrantly as seen in some cancer cells. In rodents, treatment with senolytics and senomorphics blunted or prevented disease progression, however their use carry drawbacks. Modulators of cellular senescence is a new area of research that seeks to reverse the senescence. More research in each of these modalities should lead to new treatments to stop diabetes development and progression. The accumulation of senescent cells is one of the hallmarks of aging, which also include telomere shortening, mitochondrial dysfunction, genomic instability, cell exhaustion, inflammation, abnormal cell to cell communication, fibrosis and microbiome dysregulation –4. CelluThe phenotype of a senescent cell has been considered as a static and permanent cellular state, in which a cell can no longer proliferate but remains metabolically active, secreting the cytokines, chemokines and other molecules that constitute the senescence-associated secretory phenotype (SASP). Characteristics of a senescent cell include flattened and enlarged shape, heterochromatin foci, lipofuscin granules, DNA scars, and dysfunction in protein processing and gene expression. In addition, senescent cells present increased lysosomal activity  and macrAs it has been described, cellular senescence is a double-edge sword. Beneficial effects of treatments for cancers, such as therapy-induced senescence (TIS), come from drugs that target specific proteins of the cell cycle machinery to stop proliferation and halt tumor’s growth and metastasis –13. HoweIn this review, we will focus on the recent findings that support the notion that a major contribution to development and worsening of diabetes is the accumulation of senescent cells in metabolically related tissues and specially in the pancreas , 19, 20 Cellular senescence was described for the first time by Hayflick and Moorhead in 1961 , when thCellular senescence is the response to cellular stress and is promoted by different stressors such as DNA damage, telomere dysfunction, oncogene activation and organelle stress. It has been shown that accumulation of senescent cells in tissues promotes organismal aging and dysfunction.WAF1/CIP1 and the p16INK4a/pRB pathways. Both axes of control of cellular senescence involve upstream and downstream regulators that could serve as targets for treatments to suppress senescent cell accumulation in tissues or for tumor treatments , which is regulated by miR-29 and miR-124 miRNAs (p53 responsive miRNAs), triggered cellular senescence while its overexpression delayed it and improved cell viability  triggers reprogramming, but also leads to accumulated DNA damage resulting in some cells entering senescence. They demonstrated that tissues lacking p16INK4a/ARF do not undergo senescence but they also lack reprograming ability. By pharmacological manipulation of cellular senescence, using palbociclib (mimics p16INK4a) to increase senescence or navitoclax (proapoptotic drug) to reduce it, reprograming was increased or decreased respectively in vivo, showing the direct correlation between reprograming and cellular senescence. In fact, they identified one of the SASP factors, interleukin-6 (IL-6), as a key factor secreted by senescent cells promoting reprogramming. It was suggested that the same process occurs in conditions of injury or aging as well as in physiological conditions to promote tissue repair by inducing cell dedifferentiation. Other studies found SASP factors contribute to the expression of stem cells signature in mouse primary keratinocytes and in vivo in the liver, but that prolonged exposure to the SASP promoted cell cycle arrest (Other examples related to “escape/reverse cellular senescence” are found in the reprograming of induced pluripotent stem cells (IPSCs). The use of Yamanaka factors  in combie arrest .What is perhaps surprising is that there is variation in the senescence characteristics, the timing of its development, and its SASP factors depending on the inducer and the cell type studied . Such vaIn conclusion, it seems that under certain circumstances of senescence-cell states, cells can escape from induced cellular senescence before it becomes chronic and irreversible Figure 2Diabetes is a global pandemic that increases every year . BesidesIn the last decade, several studies identified accumulation of senescent cells in important metabolic tissues and correlated their increase with development of the disease or its worsening . The numInk4a expressing cells after administration of a B/B homodimerizer that promotes activation of caspase 8 only in p16Ink4a positive cells)  +quercetin (a flavonoid with antioxidant and anti-inflammatory effects that induces apoptosis of senescent cells by inhibiting the anti-apoptotic Bcl-xL protein) (D+Q), significantly reduced liver fat accumulation. In addition, they demonstrated a correlation between hepatocyte senescence and the severity of NAFLD . They concluded that hepatocyte senescence is a major driver of fat accumulation in liver, potentially by mitochondrial dysfunction and impaired lipid metabolism. Such findings in different hepatic diseases suggest potential new effective treatments focused on removal of senescent cells and restoration of the functional hepatocytes.The liver is another metabolic organ in which senescence was found to participate in the development of its pathologies or in worsening them. Primary hepatocytes from patients with cirrhosis had significantly shortened telomeres compared to non-cirrhotic human samples. Telomere shortening correlated with expression of the cellular senescence marker β-galactosidase and was restricted to hepatocytes and was not observed in other cells in the liver, such as lymphocytes or stellate cells in areas of fibrosis . Anothere cells)  reduced Interestingly, these authors also reported that obesity induced by high fat diet or leptin deficiency increased anxiety and impaired neurogenesis in mice and that clearance of senescent cells from areas of brain implicated in regulation of food-control reduced the anxiety and restored the impaired neurogenesis . They foWAF1/CIP1 promoted atrophy in muscle from older subjects . SASP in MIN6 cells correlated with Cdkn1a expression, corresponding to early senescence. To test if the senescence seen in the beta cells in response to acute insulin resistance were also early stage and reversible, they compared single cell RNA seq data from murine islets after two weeks of treatment with insulin receptor antagonist S961 to accelerate beta cell senescence, islets from mice that then had two weeks of recovery  and control  mice. While analysis of the beta cells showed most of the S961-treated beta cells had changed gene expression with loss of identity genes, those from the recovery animals had mostly returned to control expression levels. The gene expression significantly correlated with Cdkn1a expression (p21), therefore suggesting that early senescence could be reversed when the inducer of cellular senescence was removed. One might speculate that in humans, reversal of the earlier stages of senescence may be the basis of the remission from type 2 diabetes seen with intensive treatment with insulin or gastric bypass . The effectiveness of senolytics, as well as senomorphics, has been studied Ink4a in islets from treated animals  has been used to target the HIF1α pathway, which is significantly upregulated in β-galactosidase positive beta cells, and ABT263 for targeting the Bcl2 pathway. Combination of D+Q effectively removed β−Gal positive beta cells from mice treated with S961 and improved blood glucose levels. Treatment with ABT263 improved glucose metabolism, beta cell identity gene expression and selectively reduced p16 animals . In typeWhile there may be benefits by deletion of the senescent cells, senolytics would reduce the mass of beta cells which is already reduced in early Type 1 or Type 2 diabetes Figure 3Additionally, investigators are searching for other treatments against cellular senescence. One approach is to target senescent cells using a sophisticated method of galacto-oligosaccharide-coated nanoparticles, based in the increased lysosomal mass and β-galactosidase activity of senescent cells . AnotherAnother approach is searching for molecules that can modulate cellular senescence existence in tissues. Modulators of cellular senescence (MCS) can act at different levels. Some MCS target pathways that evade cell cycle arrest, such as the inhibitor of mTOR pathway rapamycin, shown to reverse cellular senescence in embryonic fibroblasts . RapamycTranscription factors can also act as MCS. E2F1 plays an essential role in regulation of the cell cycle progression and is a main target for the tumor suppressor pRB. In human cancer cells the knockdown of E2F1 transcription factor induced replicative senescence and stable cell cycle arrest whereas its overexpression made the cells resistant to induced senescence . The dowThe anti-apoptotic protein Bcl-xl that serves as target for senolytic drugs  is also considered a modulator of senescence. During earlier stages of senescence induction, Bcl-xl helps mitochondria cope with the high metabolic demand to secrete the SASP needed to recruit the immune system for clearance of the senescent cells. However, as senescence become more established, senescent cells acquire a dysfunctional mitochondrial phenotype; Bcl-xl expression prevents the accumulation of these impaired mitochondria and their detrimental effect on tissue homeostasis. In addition, Bcl-xl contributes to preserve immunosurvillance of senescent cells , 99.WAF1/CIP1  mice compared to young (2-month-old) revealed the upregulation of several p53-responsive miRNAs, including miR124, miR34a/b/c and miR-29a/b/c , 100–102AF1/CIP1  or p16IN1 . Murine studies administering PUFAs, showed attenuation of several markers of age-related immunosenescence (described as the lower precision and activity of the adaptative immune system), inflammaging (indicating an increased pro-inflammatory activity of the innate immune system) and Th1/Th2 cytokine imbalance , 132. PUin vitro models for metabolic stress in MIN6 cells and in human islets, we showed that PAHSAs directly prevented, as well as reversed, expression of genes and proteins related to cellular senescence in pancreatic beta cells. The mechanism by which PAHSAs exert their effects on beta cells is by acting on p53’s negative regulator Mdm2 and other factors that control the p53/p21 pathway, as well as in the above mentioned E2F1 factors. PAHSAs exerted a modulatory effect preventing activation of p53 and the cascade of downstream effectors that induce senescence. PAHSAs treatment in NOD mice resulted in significantly decreased senescent proteins  in beta cells as well as in alpha cells. In addition, a novel feature of treatment with these lipids was the enhanced expression of DNA-repair and protective (glutathione metabolism) genes, which in cellular senescence are down-regulated. Therefore, we believe that these lipids (PAHSAs) constitute a great target to generate potential therapies against cellular senescence in pancreatic beta cells.Our own studies recently reported Palmitic acid hydroxy stearic acids (PAHSAs) as novel endogenous modulators of cellular senescence, with the potential to prevent and reverse cellular senescence in metabolically stressed, pancreatic beta cells . PAHSAs Therefore, in the context of the detrimental effects of cellular senescence observed in pancreatic beta cells, the use of such modulators of cellular senescence, with the potential to reverse senescence markers and restore the dysfunctional beta cells would preserve the functional beta cell mass in contrast to senolytics.Since cellular senescence has been shown as the target to ameliorate or halt the progression of diabetes, efforts have focused on the effectiveness of senolytics and senomorphics. However, the co-lateral damages of senolytics push the need of finding new therapies that erase cellular senescence from pancreatic beta cells as well as the other involved metabolic tissues. More research on possible therapies that “rejuvenate” or “reverse” is needed, including compounds that target genes and proteins identified to be involved in development of cellular senescence.MFRC and SB-W generated the ideas and wrote the manuscript. All authors contributed to the article and approved the submitted version.


The human gut microbiota environment is constantly changing and some specific changes influence the host’s metabolic, immune, and neuroendocrine functions. Emerging evidence of the gut microbiota’s role in the development of cardiovascular disease (CVD) including hypertension is remarkable. There is evidence showing that alterations in the gut microbiota and especially the gut-dependant metabolite trimethylamine N-oxide is associated with hypertension. However, there is a scarcity of literature addressing the role of trimethylamine N-oxide in hypertension pathogenesis. In this review, we discuss the impact of the gut microbiota and gut microbiota dependant trimethylamine N-oxide in the pathogenesis of hypertension. We present evidence from both human and animal studies and further discuss new insights relating to potential therapies for managing hypertension by altering the gut microbiota. The gut microbiota is a community of micro-organisms consisting of approximately 100 trillion micro-organisms mainly bacteria, but also viruses, fungi, and protozoa that exist in the human gastrointestinal tract, interact with one another and with the host and greatly impact human health and physiology . The gutRecently, high levels of TMAO have been found to be associated with an increased risk of cardiovascular disease (CVD) including heart failure, and chronic kidney disease . Emerginvia the TMAO metabolite and summarize evidence of its importance in the pathogenesis of CVD and hypertension.Given the high prevalence of hypertension and its impact on cardiovascular health, a deeper understanding of the mechanisms involved in the aetiology and potential therapeutic targets is crucial. By exploring the complex relationship between TMAO, gut microbiota, and hypertension, we can gain new insights into the pathogenesis of hypertension and develop novel strategies to prevent or treat this condition. In this review, we provide insights into the current understanding of the host-gut microbiota interaction Bacteroides or Prevotella) and the Firmicutes (predominantly gram positive Clostridium and Lactobacillus) phyla, respectively  gene and are significant producers of trimethylamine, a precursor of TMAO  deposition in peripheral tissues , a proceKng1, Cxcl1, Cxcl2, Cxcl6, and Il6 in bone-marrow-derived macrophage  include:via apoptotic inflammatory responses and ROS generation leading to increased risk for hypertension  . In the d stress . UPR is d stress . Apoptosd stress , Figure TMAO selectively activates the PERK pathway of the UPR and the PERK in turn induces FoxO1. This response is attenuated by terminating FMO3 function, an enzyme that activates TMAO, as well as by manipulating gut microbiota that may have enzymatic pathways leading to TMAO production . This invia promotion of atherosclerotic processes such as formation of foam cells by upregulation of macrophage scavenger receptors  resulting in increased deposition of cholesterol in arterial blood vessel and activation of scavenger receptors and atherosclerotic processes . TMAO reAs discussed above, TMAO induces activation of the NF-kB resulting in the generation of proinflammatory cytokines that contribute to atherogenesis and vascular injury and remodelling . TMAO alTMAO has been implicated in contributing to development of renal insufficiency and mortality risk in chronic kidney disease patients by directly inducing progressive renal fibrosis and dysfunction . TMAO isvia formation of advanced glycation end-products (AGEs) and superoxide-stimulated oxidative stress, which in consent increases intrinsic wall stiffness and can occur with the aging process  . In othe–HF axis  and func–HF axis . HoweverHypertension can also be driven by high dietary salt (sodium) intake, hence the term salt-sensitive hypertension . Salt seTMAO has been demonstrated to be affected by high dietary salt intake . A studyClostridium and Bacilli species) are trimethylamine producing gut bacteria, a precursor to TMAO , the major modulators of the immune response leading to a proinflammatory state  and its activating enzyme CutD gene responsible for TMAO production  mainly of plant-based foods, such as whole grains, vegetables, fruits, seeds, legumes, nuts, herbs, spices and extra virgin olive oil . The conry fibre , extra vry fibre , some hery fibre  and garlry fibre .Although the current relationship between the Mediterranean diet and specific gut microbiota associated with hypertension from literature is not yet established to be a cause-to-effect, this diet is reported to increase biodiversity or what is termed as alpha diversity of the gut microbiota associated with reduced CVD risk . Reducedvia the action of hepatic FMO3. Accumulation of TMAO is linked to atherogenesis, hyperactivation of platelets resulting in adverse CVD events such as stroke, heart attack and mortality , harboured esterase activity and degraded the ester ACEIs; quinapril and ramipril in vitro. In addition, C. comes reduced the antihypertensive effect of quinapril in spontaneously hypertensive rats (SHR). On the other hand, The reports that increasing TMAO has potential to increase blood pressure suggests that lowering blood TMAO concentrations can reduce BP levels and several studies have demonstrated this using probiotics, minocycline and enalapril . AntihypMounting evidence indicates that faecal microbiota transplantation also treats metabolic syndrome, diabetes, and hypertension , and a pvia increased NO production and reduction of NADPH oxidase-driven ROS production (CutC) and its activating enzyme CutD gene responsible for TMAO production  and most importantly the sodium-hydrogen exchanger 3 (NHE3)  . NHE3 is3 (NHE3) . Gut NHE3 (NHE3) .−/−) mice have shown a shift in the bacteria phyla with contraction of Firmicutes and expansion of Bacteroidetes along the gastrointestinal tract  in NHE3−/− compared to wild type (−/− may play a role in reducing the Firmicutes/Bacteroidetes ratio, hence TMA and consequently TMAO production (−/− mice (−/− resulted in a significant promotion of the pressure natriuretic response and reduction in blood pressure compared to wild-type. Therefore, blood pressure lowering NHE3 inhibition could therefore be considered as a therapeutic target for hypertension, although more animal and human studies are needed to confirm that NHE3 inhibition is a treatment option, as some reports suggest that NHE3 deficiency can cause irritable bowel disease-like symptoms, gut dysbiosis, and an inflammatory immune system (Studies evaluating the gut microbiota in NHE3-knock-out (NH3ild type . This suoduction . Both anoduction . On the oduction . These r−/− mice . Li et ae system .While the role of gut microbiota in human health has been a topic of interest for decades , the intSeveral studies have demonstrated the potential for TMAO to be used in clinical settings. For example, TMAO can be used as a prognostic biomarker in patients with pulmonary arterial hypertension where increased TMAO levels are associated with poor prognosis . TMAO miIf the associations around TMAO prove to be causal, then TMAO could be targeted to potentially reduce the risk associated with these conditions including the risk for hypertension and hypertension related adverse events.TMAO and gut microbiota play a critical role in propagating and sustaining cardiovascular risks such as hypertension through various mechanisms that are still not well understood. Future studies should concentrate efforts in considering these aspects to further drive the clinical management of CVDs and hypertension.• The gut microbiota contributes to CVD risk• TMAO is associated with CVD risk and hypertension• There are several mechanisms by which plasma TMAO levels might influence the pathogenesis and progression of hypertension. Vascular injury and inflammation is the hallmark underlying this pathogenesis.• Antihypertensive drugs can modulate blood pressure through interactions with the gut microbiota and TMAO• Faecal microbiota transplantation is a promising intervention for future research• High TMAO concentrations correlates with high BP levels• Plasma levels of TMAO can be used as a predictive and prognostic biomarker for CVD, hypertension and metabolic diseases.


Livestock farming provides a major source of animal protein and occupation opportunities for a large proportion of the world’s population, and its profitability could be effectively increased by improvement of either feed efficiency  or reproThis Research Topic aimed to collect new research in the field of “Genetics of reproduction in livestock species”. After announcing the Research Topic, a total of 20 original articles were received. However, after intensive review processes, only five articles were accepted and published in this Research Topic. A summary of the results of the articles in this Research Topic is as follows.Yang et al.).1. Identification of genes related to sexual differentiation and sterility in embryonic gonads of mule ducks by transcriptome analysis 2. Genetic parameters estimation and genome molecular marker identification for gestation length in pigs  as the molecular breeding markers 3. Genome-wide analysis for the melatonin trait associated genes and SNPs in dairy goat  4. Comparative gonad transcriptome analysis in cobia 5. The alternative transcription and expression characterization of Cobia is known as a fast-growing fish with a high commercial value, and many fish producers favor a monosex culture because of the higher body size of female fish. However, the effects of genetic factors associated with the sexual development and sexual phenotype in cobia are largely unknown. This study is possibly the first attempt to detect sex-biased differentially expressed genes (DEGs) in cobia fish gonads, using comparative gonad transcriptome sequencing analysis. A total of 76 candidate genes, including 42 known testis-biased and 34 known ovary-biased DEGs were identified as the genes involved in sex determination cascades or sex differentiation pathways. A total of 11 pathways, including Wnt signaling, oocyte meiosis, TGF-β signaling, and MAPK signaling pathways, were identified as functionally related to sex determination and differentiation. The results of this study provide a molecular basis for further studies on cobia’s sex determination mechanism and facilitation of monosex culturing.Dmc1 gene (DNA meiotic recombinase 1) in autotetraploid goldfish. This study showed different expressions of Dmc1 in diploid and tetraploid goldfish, and specific expression of the duplicated Dmc1 in the autotetraploid individuals is probably involved in meiosis progression of diploid-like chromosome pairing.The different fertilities and meiosis stabilities of established and neo-autotetraploids are known . This stIn addition to the subjects published in this Research Topic, there are several other interesting areas, including high-throughput methods, that should be subjected to future studies on the genetics of reproduction.Genome-wide association studies (GWAS) are widely used for detection of genomic regions associated with productive and reproductive traits. Previous GWASs resulted in detection of many quantitative trait loci and candidate genes associated with reproductive traits in different livestock species, such as cattle , sheep , goats , PoultryBMPR1B (BMP15 knockout in goats (GDF9 (OCT4) gene in cattle  in a single generation by using the CRISPR/Cas9 approach. This technology has been used for the identification of three well-known major fecundity genes in farm animals, including the Booroola mutation in ovine BMPR1B , the BMPin goats , and thets (GDF9 , as welln cattle . HoweverGene drive comprises the introduction of synthetic fragments involving a driving element, the CRISPR/Cas9 system, into the target genome, which can increase the spreading chance of specific alleles through the population compared to the standard Mendelian inheritance . A main Understanding major genes/mutations associated with the reproductive performance of farm animals could have a great impact on the economic efficiency of animal production enterprises. The articles published in this Research Topic improved our understanding of the genetics of reproduction in livestock species. However, there are huge numbers of questions about genetic factors associated with reproduction performance in farm animals, which should be considered in future studies.


Mammalian eggs (oocytes) are formed during fetal life and establish associations with somatic cells to form primordial follicles that create a store of germ cells . The size of this pool is influenced by key events during the formation of germ cells and by factors that influence the subsequent activation of follicle growth. These regulatory pathways must ensure that the reserve of oocytes within primordial follicles in humans lasts for up to 50 years, yet only approximately 0.1% will ever be ovulated with the rest undergoing degeneration. This review outlines the mechanisms and regulatory pathways that govern the processes of oocyte and follicle formation and later growth, within the ovarian stroma, through to ovulation with particular reference to human oocytes/follicles. In addition, the effects of aging on female reproductive capacity through changes in oocyte number and quality are emphasized, with both the cellular mechanisms and clinical implications discussed. Finally, the details of current developments in culture systems that support all stages of follicle growth to generate mature oocytes in vitro and emerging prospects for making new oocytes from stem cells are outlined. CLINICAL HIGHLIGHTSFollicle formation and the onset of oocyte meiosis occur before birth; thus a female fetus can be directly influenced by exposure to environmental chemicals and maternal factors such as smoking. These can result in transgenerational effects on health and reproductive potential.The regulation of follicle activation and growth remains incompletely understood but is a potential therapeutic target, either to slow it as an approach for protection against damage  or to increase it and thus the number of follicles that may be available for stimulation during assisted reproduction.At present, only the very last stages of oocyte maturation can be supported in vitro for clinical use, and even this is infrequently used. Further development of in vitro maturation and progress toward support of follicle and oocyte growth from the earliest stages may lead to dramatic changes in assisted conception in the future.Increasing maternal age at childbirth has major implications for both societies and individuals. Current clinical applications are based on the identification of abnormalities, but a growing understanding of the molecular mechanisms underpinning oocyte meiosis and early embryo development may lead to new therapeutic approaches.The mammalian oocyte is the largest cell in the body and develops within the ovarian follicle. The concept of “omne vivum ex ovo”—“all living things come from eggs”—was strongly advocated by William Harvey in 1651 in his classic publication “on the generation of animals” . A greatThe mammalian ovary has dual functions of producing female germ cells (oocytes) and synthesizing hormones that will regulate the processes of ovulation, fertilization, early embryonic development, and implantation. However, the origin of mammalian germ cells was the subject of fierce scientific debate from the time the mammalian oocyte was correctly identified . One sidweek 6 of gestation (WNT4) and forkhead box transcription factor 2 (FOXL2) in the somatic cells induces ovarian development,  are the progenitors of the germline in all mammals, with the capacity to become either oocytes or spermatogonia depending on the gonadal environment , 13. Durestation , and it estation . The absla ovary . It has la ovary , 28, 29:la ovary . The tesla ovary  as femalhe males .Tex14 homozygous mutant fetal ovaries, fewer cysts are formed and germ cells are connected via syncytia or fragmented cell membranes leading to the production of fewer oocytes and many morphologically abnormal oocytes thus demonstrating a direct link between cyst formation, intercellular bridges, and oocyte development  a transcriptional activator that enhances the expression of several cell cycle and meiotic prophase 1 genes leading to inhibition of mitotic activity  plays a key role in triggering the onset of meiosis , 42. RA activity , 43; in regation . Stra8 h meiosis . STRA8 i9 and 11 . Culture9 and 11 , 48 and 9 and 11 .RA was identified as a prosurvival and proliferation factor for culturing germ cells from fetal mouse ovaries , and at During these early stages of meiosis the synaptonemal complex (SC), a protein structure that facilitates pairing of homologous chromosomes, is formed. The SC enables the exchange of genetic material by crossing over and accurate segregation of homologs . The proDAZ is a Y chromosome gene so it is male specific, but DAZL and BOLL are autosomal. Mice where Dazl has been knocked out are infertile and germ cells do not progress beyond leptotene of meiotic prophase I  and its homologues DAZ (deleted in azoospermia) and BOLL (Bol-like)  , is alsorequency .GDF9), whose expression also increases immediately before follicle formation in the human ovary  of pre-mRNAs with follicle formation. Specifically, serine/arginine-rich splicing factor 1 (SRSF1) is a key posttranscriptional regulator of gene expression in several processes . Conditiimpaired . These fThe human ovary can contain ∼500,000 to 1,000,000 oocytes at the time of birth . The actThere are several points during an oocyte’s developmental pathway where tightly coordinated cell death pathways ultimately determine the size of the ovarian reserve and eventually the number of ovulated oocytes  as early as the last mitotic division . FollowiCasp3-deficient female mice show no differences in numbers of healthy primordial, primary, or small preantral follicles compared to wild-type littermates on day 4 postpartum (TAF4b) null mice exhibit a significant increase in activated Casp3 immunostaining on day 1 postpartum compared to control ovaries  polymerase 1 (PARP1) and DNA fragmentation being apparent at this time in both mice and humans , 114. Instpartum , suggest ovaries , concomiof CASP2 . Further oocytes . Interesollicles . It is iBAX and PUMA (proapoptotic) and BCL2 (antiapoptotic)  , 120–122optotic) . BAX is optotic) . Similaroptotic) .Bax expression can regulate the size of the ovarian reserve and even extend the reproductive lifespan  12; however, these differences were no longer apparent by PND 30–60 . This su ovaries . CollectCASP3 and CASP9 activation , which are by-products of mitochondrial activity . Recent nt phase . Dormantnt phase .Primordial follicles are maintained in a state of growth arrest, characterized by a low transcriptional and translational activity, essential for preserving chromosomal stability and prolonged reproductive life. Primordial follicle activation (PFA) is the process by which primordial follicles are selected into the growing follicle pool and is characterized by the differentiation of the flattened granulosa cells into mitotic cuboidal granulosa cells before oocyte growth –181.The balance between primordial follicle dormancy and activation is complex, involving numerous molecular, cellular, and biochemical events, whose interactions continue to be characterized  FIGURE . Given t2) into phosphatidylinositol-3,4,5-triphosphate (PIP3). This reaction is reversed by the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which converts PIP3 into PIP2. PIP3 recruits phosphoinositide dependent-kinase 1 (PDK1) and Akt to the membrane, where PDK1 phosphorylates AKT. Further phosphorylation of AKT by the mammalian target of rapamycin complex 2 (mTORC2) leads to the full activation of the protein, which mediates the regulation of protein synthesis, cell survival, and cell cycle entry through targeting forkhead box O3 (FOXO3), Bad, tuberous sclerosis complexes 1/2 (TSC1/2), and Cdk inhibitor p27 (1 arrest (The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and mammalian target of rapamycin (mTOR) signaling pathways are crucial for fundamental cellular processes, from cell growth and proliferation, survival, and migration to metabolism . In mammitor p27 . Notablyitor p27 . AKT-ind1 arrest  , and the cell cycle regulator protein c-Myc  has emerged as a new therapy that initially relied on the combination of Hippo disruption and PI3K/Akt upregulation to activate residual primordial/small follicles in women who had a low ovarian reserve. The procedure involved the surgical removal of cortical strips from the ovary, their fragmentation, and short-term culture with Akt stimulators and then their transplantation back into the patient , 239. ThIn addition to the pathways described above, additional pathways have been described as potential regulators of PFA C–E. A reAnti-Müllerian hormone (AMH) is produced by granulosa cells, and its synthesis, initiated in primary follicles, reaches its highest levels in preantral and small antral follicles and then sharply declines in large follicles , 271. ThDensity-dependent interfollicular regulation of PFA via local diffusing inhibitory factors has been postulated , and folAs well as the mechanisms outlined, it should be highlighted that an additional level of regulation exists via noncoding micro-RNAs (miRNA) that likely affect each stage of follicle development, including PFA. These small molecules regulate gene expression at the posttranscriptional level and are conserved across animal species . There iPFA is a complex process with more than 1,000 genes being differentially expressed in the oocyte and granulosa cells during the human primordial-to-primary follicle transition , 269. SiA reduction in primordial follicle numbers occurs throughout life with attrition being greatest during prepubertal ages. Activation of follicles begins as soon as the follicle population is formed and follicle growth increases prepubertally , 309. AMStages of human follicle development are classified as primordial, secondary, antral, preantral, and preovulatory follicles according to size and number of cell layers  FIGURE . The ear1) nurturing the oocyte to achieve developmental competence to be fertilized and support embryo development , and 2) producing the sex steroids needed to support the menstrual cycle, endometrial function, and the establishment of pregnancy.Follicle development has the dual role of 1) undergo substantial growth with an increase in diameter from ∼20 µm to 110 µm (human); 2) acquire competence to resume and complete meiosis; and 3) acquisition of developmental competence, i.e., capacity for fertilization and formation of embryos. All of these processes are critically dependent on intercellular communication between the growing oocyte and the developing granulosa cells and therefore support and maintenance of these connections are essential as follicles progress through each developmental stage  [aromatase and 17β-hydroxysteroid dehydrogenase (17β-HSD)] are expressed by the granulosa cells regulated by FSH. The combination of these two cell types and two gonadotropins leads to the production of estradiol  and the theca externa defining the outer layer of the follicle where blood vessels will form. Formation of new blood vessels (angiogenesis) plays an important role in the ovary, and investment of blood vessels within the theca externa differs in individual follicles making angiogenesis a critical regulator in determining the fate of follicles . The vasIt is clear that the fate of each follicle is influenced by a myriad of factors and that the majority are destined to degenerate. Once follicles reach the antral stage of development, they are acutely dependent on endocrine factors and in particular gonadotropins. The majority of follicles that reach this stage will undergo degeneration and this loss is termed atresia. While primordial follicle death appears to be triggered by the oocyte itself  , 398, lo1) continuous recruitment, where antral follicles between 4 and 6 mm in diameter are recruited to grow throughout the menstrual cycle independent of the gonadotropins FSH and LH and the dominant follicle is selected on the basis of it being in the right place at the right time (2) cyclic recruitment, a cyclical increase in antral follicles between 2 and 5 mm recruited from a continuous supply of growing preantral follicles, which is referred to as secondary recruitment to distinguish it from PFA (3) follicular waves, the idea that synchronous growth of a cohort of antral follicles (2–5 mm) occurs at regular intervals has been described as waves. Waves of follicle development have been identified using transvaginal ultrasonography in conjunction with endocrine profiling, and 2–3 waves of 4–14 follicles were detected in healthy women, although not in all  cyclfrom PFA ; and 3) t in all . This cot in all  and the t in all , and modt in all . A singlt in all , 411. Set in all   signal transduction pathway. Synthesis of cAMP is from ATP via adenylyl cyclase (AC) and is degraded by phosphodiesterases (PDEs). In rodent oocytes, a constitutively active G-protein coupled receptor 3 (GPR3) has been located on the oocyte membrane (oolema) and has also been identified in human oocytes  system. C-type natriuretic peptide (CNP) is produced in mural granulosa cells, and its receptor NPR2 (a member of the guanyl cyclase receptor family) is expressed within cumulus granulosa cells. The production of cGMP in the granulosa cells inhibits the degradation of cAMP by inhibiting PDE3 activity in the oocyte . The regThe LH surge triggers ovulation and stimulates a sequence of events in the ovulatory follicle leading to the resumption of meiosis in the oocyte, mucification, and expansion of the cumulus, and rupture of the follicle resulting in the release of the cumulus-oocyte complex (COC) into the fallopian tube. Once the COC is released, the granulosa and thecal cells become luteinized and form the corpus luteum.The resumption of meiosis to metaphase II is initiated by the surge of pituitary LH that occurs midcycle and triggers ovulation. LH stimulates oocyte maturation via its action on the theca and granulosa cells  and indiLH binds to the LH receptor (LHR) a G protein-coupled receptor (GPCR) expressed on the membrane of mural granulosa cells and theca cells , 441. HoLH signaling reduces the cAMP level within the oocyte through downregulating the NPPC/NPR2 system  and closThe first morphological sign that meiosis has resumed is that the nuclear envelope [germinal vesicle (GV)] breaks down (GVBD). This marks the start of metaphase I with compaction of chromosomes and the homologs orient on the metaphase I plate and segregating at anaphase I leading to the extrusion of one set of chromosomes in a small bleb of cytoplasm, i.e., the first polar body (PB1) . This fiCytoplasmic maturation indicates the molecular and cytoskeletal changes as well as organelle reorganization and other processes that prepare the oocyte for fertilization and preimplantation development. Just as competence to undergo nuclear maturation is acquired in a stepwise manner during follicle development so is cytoplasmic maturation. Defining the process of cytoplasmic maturation remains vague despite the search for markers of cytoplasmic and developmental competence .As oocytes grow through the stages of follicle development, they are transcriptionally active, producing the mRNAs required to support oocyte maturation and early embryonic development. Before ovulation oocytes are transcriptionally repressed. The coordinated changes that take place in organelle and cytoskeletal structure necessary for maturation are regulated through the translation of prestored RNA in the oocyte and RNA binding proteins. Key to driving these processes is the maternally derived mitochondria that provide energy to support nuclear and cytoplasmic maturation .With each ovarian and menstrual cycle, normally one oocyte will be ovulated and either fertilized or degenerated. With time, there is a progressive diminishing of the pool of follicles/oocytes and the aging process impacts on oocyte quality. As well as changes in oocyte number and quality, several other changes occur in the aging ovary.The changes in the ovary that accompany aging are of increasing importance both in society and clinically. There has been a consistent rise in age at motherhood across many developed societies resulting in many women not starting their families until they are in their thirties, which translates clinically into age having an ever-growing importance in the prognosis for many patients attending for medical assistance in conceiving. Women tend to have slightly older male partners, and while the impact of age on male fertility is much less than on women, it can also have an impact . While fThis in turn reflects one of the two key aspects of ovarian aging, which is the incessant decline in the number of primordial follicles, the ovarian reserve, across life. Despite the clinical difficulties in counting nongrowing follicles in a large structure such as a whole human ovary and the inevitable scarcity of such samples, a number of models have been developed over the years to examine this decline. Initial analyses by Faddy and Gosden , 457 andWhile recognizing the substantial variation between individuals, analysis identifies that the number of nongrowing follicles in the ovary halves approximately every 5 years from the midtwenties . VariatiThese changes in ovarian function with age are reflected in endocrine changes. A monotropic rise in FSH concentrations has long been recognized as a hallmark of ovarian aging before menopause and has been shown to be due to reduced feedback at the pituitary from lower inhibin B concentration from the reduced number of antral follicles , 465. ThWhile the original endowment of the primordial follicle pool formed in utero  is likelIn addition to research on extrinsic influences on remaining follicle number and by implication the function and quality of the growing follicles and oocytes, large-scale genetic studies have also sought to identify key genetic associations with reproductive lifespan. Given the relationship between reproductive lifespan and fertility, such studies can also be seen as pointers toward the genetic regulation of oocyte quality. In addition to important though rare single gene defects , recent In addition to the major changes in follicle/oocyte number with age, there are also changes in oocyte “quality” that have a major impact on female fertility and the success of assisted reproduction. The importance of oocyte quality, i.e., the potential to develop into a normal pregnancy and ultimately a heathy baby, has long been recognized. Classic studies have documented the falling success rate of IVF with age and how that did not occur when the oocytes used were donated from young women , and theThe key basis for this decline in oocyte quality with age is the increased risk of chromosomal aneuploidy. Meiosis is initiated in fetal life, with oocytes progressing through the early stages of meiosis I before arresting in the diplotene stage of prophase. This includes the process of chromosomal crossover (recombination) after pairing of homologous chromosomes, through which genetic material is exchanged between the parental chromosomes. This is then maintained until meiosis resumes at the time of ovulation, triggered by a cascade of biochemical changes initiated by the LH surge . In womeWhile several mechanisms for oocyte aneuploidy have been identified, including recombination/crossover formation defects, cohesin loss, spindle deformation, spindle assembly checkpoint malfunction, microtubule-kinetochore attachment failure, kinetochore misorientation, and mitochondria dysfunction with increases in reactive oxygen species [detailed in recent reviews –488] witsirt1 show reduced fertility and oocyte quality without an effect on follicle number  to older mice improved meiotic spindle assembly and increased both oocyte yield and the number of pups born, with improved mitochondrial function and reductions in oocyte reactive oxidative species  on a blastomere biopsied from a cleavage stage embryo to next-generation DNA sequencing of several cells from a trophectoderm biopsy, generally 5 days after fertilization . PreimplAn additional question of clinical importance is whether the oocytes of women with a reduced ovarian reserve for their age are also of poorer quality, or whether in that situation, quality and quantity are not related. An analysis of 9,489 cycles among 8,214 patients found no association between live birth and ovarian reserve among pregnant IVF patients under the age of 35 years , and in There is a growing interest in the contribution of the ovarian stroma to ovarian aging and thus to its important role in supporting normal follicle development throughout life. In the human ovary, the stroma is densely packed with spindle-shaped fibroblast-like cells, with a key change during aging being increased fibrosis. The increased collagen content changes the physical properties of the ovary, with increasing stiffness . A detai+ T cells, B cells, and macrophages as well as increased expression of proinflammatory cytokines and the inflammasome genes ASC and NLRP3 (517). Knockout mouse models of these two inflammasome genes also show some evidence of reduced loss of primordial follicles with age, associated with reduced macrophage numbers within the ovary and reduced expression of inflammatory cytokines  with clinically available drugs used to treat pulmonary fibrosis resulted in a reduction in ovarian cortical collagen content and, in most but not all animals, restoration of the ovarian response to superovulation . RemarkaThe consequences of age-related increased fibrosis have not been greatly explored, but it is possible that it also plays a role following the recovery of ovarian function following chemotherapy treatment. Such treatment has been demonstrated to increase collagen deposition in the human ovary  and thusThe poor vascularization of the superficial ovarian cortex has related to ongoing quiescence of the primordial follicle pool  and the It is possible that these changes in the extracellular matrix are in part endocrine related. In mice, LH has been associated with ovarian fibrosis and this was reduced by treatment with a GnRH antagonist . These sAs detailed in earlier sections, our understanding of the processes and regulation of oocyte/follicle development has been founded on a large body of experimental studies utilizing a range of models that have relied heavily on rodents with extrapolation to humans. While rodent transgenic and knockout models have been essential in elucidating regulatory pathways and mechanisms that may apply across species, there is a need for tractable models to study human-specific processes relating to germline development , 530. BaAs far back as 1934 Pincus and Enzman  posed thImproved knowledge of factors regulating oocyte development led to advances in culture systems being made . It was Supporting this complex multilayered process in vitro is technically challenging; however, complete human oocyte development from primordial/unilaminar follicles to meiotic maturation  has been achieved , 544. CuAs discussed in detail earlier, PFA is a defining step for oocyte development. During this process, primordial follicles respond to a combination of inhibitory and stimulatory factors, within the context of complex intrafollicular networks that integrate signals from various signaling pathways  FIGURE . Early aA spontaneous shift from quiescent primordial to growing follicles occurs within the cultured ovarian cortex over 6–8 days in vitro , 543 FIB. ExtensTissue preparation and ovarian cortex remodeling have been linked to a disruption of the Hippo signaling pathway. Fragmentation of the ovarian cortex leads to an imbalance in the G-actin/F-actin ratio of the Hippo cascade, which alters the balance of negative regulators of growth leading to the translocation of Hippo’s main effector YAP into the nucleus of granulosa cells. Nuclear YAP allows the transcription of growth factors and apoptosis inhibitors, resulting in follicle awakening , 239.This microcortex that has been fragmented and mechanically loosened supports activation of human primordial follicles that develop to multilaminar (secondary) stages within 6 days , 543 FIB AND C. Multilaminar follicles can be isolated from ovarian stroma using enzymes such as collagenase and DNAse , 560 or Culture systems have been developed to support isolated multilaminar  growing follicles isolated from the human ovarian cortex –568 or dNovel scaffolds to support human follicle growth in vitro have been developed; these include decellularized ovarian tissue , 575 andstep 3 of the multistep system  of the human multistep culture system is informed by this mouse system as culturing human preantral follicles to in vivo preovulatory sizes (20 mm or more in diameter in vivo) is technically challenging. Thus, the aim of step 3 is to promote oocyte growth as oocyte size is an indicator of meiotic and developmental potential  , and the1) ESCs (2) epiblast stem cells (EpiSCs)  or induced Pluripotent stem cells (iPSCs). Two types of stem cell lines have been derived from the inner cell mass of the developing blastocyst that forms the pluripotent epiblast cells: 1) ESCs , and 2) (EpiSCs) . These c(EpiSCs) . ESCs ha(EpiSCs)  and are (EpiSCs) .While research with mouse ESCs (mESCs) gives us insight into cell lineage development and proof of principle, the use of human ESCs clinically is fraught with practical difficulties and ethical concerns. There are ethical concerns surrounding the use of human embryos and stem-based research in general . ConcernMethodology to dedifferentiate and induce pluripotency in adult cells was developed by Takahashi and Yamanaka , 605 wheA, gdf9, zp1, zp2, and zp3) when cocultured with granulosa cells (Differentiating germ cells have been derived from mouse embryonic stem cells (mESCs) . Epiblassa cells , with sisa cells . Howeversa cells , confirmFollicle-like structures containing oocyte-like cells (OLCs) have been formed by combining PGCLCs with embryonic ovarian somatic cells following transplantation to the ovarian bursa of immune-deficient recipient mice. The OLCs were capable of being matured and fertilized in vitro and embryos were produced, albeit with lower efficiency than with normal oocytes, resulting in healthy offspring with normal imprinting patterns . These sThe ability to generate all cell types including germ cells from mouse iPSCs has been demonstrated to be similar to ESCs , 613. MoThe ability to derive oocytes from stem cells entirely in vitro is a huge step, but this protocol  relied oGerm cell differentiation from human ESCs has been investigated, and PGCLCs have been derived from hESCs with gene expression patterns similar to PGCs . The difThis review has highlighted the complexity of human oocyte development within the context of the ovarian follicle and the dynamic ovarian environment and its alterations through aging. The control of these processes is multilayered involving paracrine, autocrine, and endocrine regulators, as well as new layers of complexity such as through small noncoding miRNAs that modify gene expression. New factors and pathways continue to be identified through single-cell sequencing and bioinformatics. Our understanding of these processes has been informed by mouse models and using large mammals, enhanced by human in vitro models, and these have facilitated the identification of targets that may have utility for therapeutic purposes. We are moving closer to developing therapies that will protect the follicle reserve from damaging chemotherapeutic agents and environmental insults to alleviate or prevent POI and even potentially reduce the adverse effects of aging on oocyte quality. Indeed, remarkable data have been very recently obtained using drugs that are already in clinical practice, making treatments for a range of ovarian conditions within immediate reach. As well as facilitating a greater understanding of the mechanisms regulating normal human oocyte development, the processes of IVD and IVG could ultimately contribute to new approaches to the treatment of infertility and, with further developments in IVM, become an integral part of fertility preservation for young girls and women. Before these methodologies can progress toward clinical application they need to be shown to be reliable and safe, but it is clear that such developments are approaching.The authors’ work in this field is supported by grants from the Wellcome Trust (215625/Z/19/Z to E.E.T. and R.A.A.) the Medical Research Council , Wellbeing of Women (PRF005 to R.R.), and Biotechnology and Biological Sciences Research Council (BB/R015635/1 to R.A.A.).No conflicts of interest, financial or otherwise, are declared by the authors.E.E.T., J.G., Y.L.O., and R.R. prepared figures; E.E.T. and R.A.A. drafted manuscript; E.E.T., J.G., Y.L.O., R.R., and R.A.A. edited and revised manuscript; E.E.T., J.G., Y.L.O., R.R., and R.A.A. approved final version of manuscript.