Modified balloon vaginoplasty for high position vaginal atresia.

INTRODUCTION: The first-line approach for the management of distal vaginal atresia involves a pull-through vaginoplasty. If the proximal vagina is 3 cm or more from the introitus, the risk of stenosis is high, and an interposition graft may be necessary. We describe a safe, low-cost, and accessible approach for distal vaginal atresia ≥ 3 cm that we call the "modified balloon vaginoplasty" and validate the technical feasibility and anatomical outcomes. METHODS: Ten patients who underwent modified balloon vaginoplasty were retrospectively evaluated. Age, symptoms at presentation, length of atresia, operation time, and postoperative complications were analyzed. RESULTS: All the cases were successfully performed without any intraoperative morbidity. The postoperative complications included one case of stenosis ring in the distal vagina because not right used vagina model. All the girls had regular menstruation and were satisfied with the surgical outcome. CONCLUSION: Modified balloon vaginoplasty allows further distention of the distal vagina or thinning of the septum, which may decrease the risk of stenosis, is a beneficial choice for patients with distal vaginal atresia ≥ 3 cm.

A novel gene expression system for Ralstonia eutropha based on the T7 promoter.

BACKGROUND: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones. RESULTS: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. CONCLUSIONS: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

Engineering the Calvin-Benson-Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved autotrophic growth and polyhydroxybutyrate production.

BACKGROUND: CO2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO2 fixation efficiency of cells is an important subject of biological research with significant application potential. RESULTS: In this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD600 of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD600 to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%. CONCLUSIONS: To our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO2 assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.

Correction to: Development of an autotrophic fermentation technique for the production of fatty acids using an engineered Ralstonia eutropha cell factory.

The article Development of an autotrophic fermentation technique for the production of fatty acids using an engineered.

Development of an autotrophic fermentation technique for the production of fatty acids using an engineered Ralstonia eutropha cell factory.

Massive emission of CO2 into atmosphere from consumption of carbon deposit is causing climate change. Researchers have applied metabolic engineering and synthetic biology techniques for improving CO2 fixation efficiency in many species. One solution might be the utilization of autotrophic bacteria, which have great potential to be engineered into microbial cell factories for CO2 fixation and the production of chemicals, independent of fossil resources. In this work, several pathways of Ralstonia eutropha H16 were modulated by manipulation of heterologous and endogenous genes related to fatty acid synthesis. The resulting strain B2(pCT, pFP) was able to produce 124.48 mg/g (cell dry weight) free fatty acids with fructose as carbon source, a fourfold increase over the parent strain H16. To develop a truly autotrophic fermentation technique with H2, CO2 and O2 as substrates, we assembled a relatively safe, continuous, lab-scale gas fermentation system using micro-fermentation tanks, H2 supplied by a hydrogen generator, and keeping the H2 to O2 ratio at 7:1. The system was equipped with a H2 gas alarm, rid of heat sources and placed into a fume hood to further improve the safety. With this system, the best strain B2(pCT, pFP) produced 60.64 mg free fatty acids per g biomass within 48 h, growing in minimal medium supplemented with 9 × 103 mL/L/h hydrogen gas. Thus, an autotrophic fermentation technique to produce fatty acids was successfully established, which might inspire further research on autotrophic gas fermentation with a safe, lab-scale setup, and provides an alternative solution for environmental and energy problems.

Partial response to crizotinib + regorafenib + PD-1 inhibitor in a metastatic BRAF V600EMT colon cancer patient with acquired C-MET amplification and TPM4-ALK fusion: a case report.

Background: Colorectal cancer (CRC) with the Raf murine sarcoma viral oncogene homolog B (BRAF) V600E had a relatively poor prognosis. Anaplastic lymphoma kinase (ALK) fusion and the mesenchymal-to-epithelial transition factor (MET) amplification have been recognized as potentially important therapeutic targets in non-small cell lung cancer (NSCLC). However, both of them are of extremely lower frequencies (<2%) in metastatic CRC, and few studies have mentioned the real application of their inhibitors in CRC treatment. Case Description: A 49-year-old Chinese male was diagnosed with ascending colon adenocarcinoma (cT3N+?M1) with liver metastases. The patient performed next-generation sequencing (NGS) using tissue and circulating tumor DNA (ctDNA), and the results showed a BRAF V600E mutation. He received an initial combination treatment with cetuximab, dabrafenib, and trametinib with a partial response (PR) assessment. We changed the therapy regimen on this patient several times because of the patient's intolerance to the drugs or the inefficacy of the treatment. During this period, we detected the c-MET amplification and tropomyosin 4 (TPM4)-ALK fusion by NGS after triplet targeted therapy (tislelizumab, dabrafenib, and trametinib), thus he was finally treated with programmed cell death protein 1 (PD-1) inhibitor (tislelizumab), MET/ALK inhibitor (crizotinib) plus multikinase inhibitor (regorafenib). Imageological examinations showed that PR was achieved and ctDNA sequencing results indicated a significantly reduced BRAF mutation frequency, MET amplification and TPM4-ALK fusion were undetectable. NGS analysis of peripheral blood showed a recurrence of the MET acquired resistant amplification mutation over 2 months of ongoing treatment. but the patient was assessed as PR and still under treatment of crizotinib, tislelizumab and regorafenib within good physical condition. At the last follow-up on October 2021, the patient died of symptomatic treatment fail for obstructive jaundice. The patient finally achieved 11 months overall survival. Conclusions: This study reported a co-existence of a BRAF V600E mutation, c-MET amplification and TPM4-ALK fusion in a CRC patient. Administration of crizotinib combined with regorafenib and tislelizumab obtained an obvious response. Furthermore, continuous ctDNA detection appears to be a promising technique to monitor tumor burden, which may provide better clinical decision support during the disease course.

Drug-free in vitro activation combined with ADSCs-derived exosomes restores ovarian function of rats with premature ovarian insufficiency.

BACKGROUND: Drug-free in vitro activation (IVA) is a new protocol to activate residual dormant follicles for fertility restoration in patients with premature ovarian insufficiency (POI). However, several deficiencies have reduced its clinical efficacy rate. Our previous studies have confirmed that the combination of adipose-derived stem cells (ADSCs) and drug-free IVA can improve the effectiveness of drug-free IVA and restore ovarian function of rats with POI. Increasing evidence has demonstrated that mesenchymal stem cell-derived exosomes have similar therapeutic effects as their source cells. Here, we performed a preclinical study to evaluate the therapeutic effects of ADSCs-derived exosomes (ADSCs-Exos) combined with drug-free IVA in the POI rats and the mechanism in restoring ovarian function. RESULTS: In vivo, the effects of ADSCs-Exos were comparable to those of ADSCs, and the ADSCs-Exos combined with drug-free IVA was better than ADSCs-Exos alone therapy in promoting follicular development. Moreover, transplantation of ADSCs/ADSCs-Exos lead to up-regulation of BCL-2 expression and down-regulation of the expression of Bax and Cleaved Caspase-3, thus reducing the apoptosis of chemotherapy-induced follicle cells, and further promoting the development of the follicles and rescuing ovarian function in POI-damaged ovary. In vitro, ovarian fragmentation could activate follicular growth and development, and in combination with ADSCs-Exos could prevent the loss of follicles, promote follicular proliferation and inhibit apoptosis. CONCLUSIONS: ADSCs-Exos combined drug-free IVA had remarkable therapeutic effects in restoring ovarian function of POI rats, and markedly promoted follicular development and inhibited apoptosis of ovarian cells in vitro. Our study confirmed that the combination therapy might be a promising and effective treatment for POI.

A rare case report of Herlyn-Werner-Wunderlich syndrome: Unraveling unusual urinary anomalies and literature review.

Herlyn-Werner-Wunderlich syndrome (HWWS) is a rare congenital genitourinary abnormality defined by uterine didelphys, obstructed hemivagina, and ipsilateral urological anomalies. Accurate diagnosis and prompt commencement of therapy can be difficult owing to heterogeneous genitourinary malformation among different patients. This is a case report of a patient with rare HWWS with uterine didelphys, obstructed hemivagina, vagina-ureteral remnant fistula (Gartner's duct cyst), and ipsilateral kidney dysgenesis who complained of intermittent abdominal pain during menstruation. The right ureteral remnant of the patient was distinctive, with three portions. The upper section was connected to the right dysplastic kidney, the lower section formed the fistulous tract with the vagina and bladder, while the middle section communicated with Gartner's Duct Cyst, which merged with the vagina and opened to the posterior cavity of hemivagina. The lower section of the right ureter was excised and ligated during laparoscopic surgery, while the upper section was excised. The patient recovered after surgery. We presented this rare case and conducted a literature review to provide a more comprehensive understanding of HWWS. This could help gynecologists effectively reduce misdiagnosis and missed diagnosis, especially when combined with complicated urinary malformation.

Clinical features and mutation analysis of class 1/2/3 BRAF mutation colorectal cancer.

BACKGROUND: BRAF (B-Raf proto-oncogene, serine/threonine kinase)-mutated colorectal cancer (CRC) still has poor prognostic. The efficacy of BRAF inhibitor is unpredictable just that intrinsic genetic complexity, immune microenvironment and partially unknown reason. Understanding the co-mutation mechanism can help improve treatment and follow-up strategies. METHODS: We retrospectively analyzed 35 (BRAF-mutated/BRAF wild-type) Chinese CRC and 125 Western CRC who underwent next-generation sequencing (NGS). Co-occurrence mutation analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was enabled in this study. RESULTS: Thirty-five (10.32%) patients were BRAF-mutated, with 17 patients were BRAF V600E in Beijing Hospital. Patients with BRAF mutation had significant association with high tumor mutational burden (TMB-H) (P=0.0004) and high microsatellite instability (MSI-H) (P=0.0003) than those with BRAF wild-type. In 125 BRAF-mutated Western CRC patients, the frequency of age at diagnosis, gender, sample type, Tumor-Node-Metastasis (TNM), MSI, TMB, and BRAF mutation type was consistent with Chinese data. However, the primary tumor location showed significant statistical differences (P<0.0001). Class 1 were more likely to occur in elder and female. Western cohort was consistent with above in Chinese cohort. Other clinicopathological features were not significantly associated with mutation type. However, Western cohort showed class 1 exhibited primary sample type predominance in both class 1 vs. others (P<0.05) and class 1 vs. class 3 (P<0.05). Meanwhile, the data showed TMB-H (57.69% vs. 11.76%, P<0.001) and MSI-H (28.21% vs. 0%, P<0.05) of the class 1 BRAF mutation proportion were significantly higher, compared with class 3 BRAF mutation. In concurrent oncogenic mutations, compared with non-class 1 BRAF mutation, class 1 are more likely to co-occur with passenger mutation. Data from Western populations showed similar results. We also found that the class 1 mutation was mutually exclusive with co-KRAS (Kirsten rat sarcoma viral oncogene homologue) mutation in CRC, and co-APC (APC regulator of WNT signaling pathway) mutation appeared more frequently in non-class 1 BRAF mutation. KEGG pathway showed that fewer proto-cancer signaling pathways were enriched in the class 1, which further confirmed that this type had stronger tumorigenicity. GO enrichment also proved that class 1 had stronger tumorigenicity. Finally, prognostic analysis showed median overall survival (mOS) of 19.43 months in class 1 vs. 47.57 months in non-class 1 (P=0.0002). Further study showed that the mOS of class 1, class 2, class 3 and class NA (unknown) was 19.43, 28.50, 47.57 months and not reached (P=0.0001), respectively. CONCLUSIONS: This study showed class 1/non-class 1 BRAF mutation in CRC had significantly differences in co-mutation features, genomic markers and prognostic. Understanding BRAF mutation types and co-mutation mechanism will contribute to accurately grasping treatment and follow-up strategies and promoting the development of precision therapy for CRC in the future.

Desktop-Stereolithography 3D Printing of a Decellularized Extracellular Matrix/Mesenchymal Stem Cell Exosome Bioink for Vaginal Reconstruction.

BACKGROUND: 3D-printing is widely used in regenerative medicine and is expected to achieve vaginal morphological restoration and true functional reconstruction. Mesenchymal stem cells-derived exosomes (MSCs-Exos) were applyed in the regeneration of various tissues. The current study aimed to explore the effctive of MSCs-Exos in vaginal reconstruction. METHODS: In this work, hydrogel was designed using decellularized extracellular matrix (dECM) and gelatin methacrylate (GelMA) and silk fibroin (SF). The biological scaffolds were constructed using desktop-stereolithography. The physicochemical properties of the hydrogels were evaluated; Some experiments have been conducted to evaluate exosomes' effect of promotion vaginal reconstruction and to explore the mechanism in this process. RESULTS: It was observed that the sustained release property of exosomes in the hydrogel both in vitro and in vitro.The results revealed that 3D scaffold encapsulating exosomes expressed significant effects on the vascularization and musule regeneration of the regenerative vagina tissue. Also, MSCs-Exos strongly promoted vascularization in the vaginal reconstruction of rats, which may through the PI3K/AKT signaling pathway. CONCLUSION: The use of exosome-hydrogel composites improved the epithelial regeneration of vaginal tissue, increased angiogenesis, and promoted smooth muscle tissue regeneration. 3D-printed, lumenal scaffold encapsulating exosomes might be used as a cell-free alternative treatment strategy for vaginal reconstruction.

Alternative Biological Material for Tissue Engineering of the Vagina: Porcine-Derived Acellular Vaginal Matrix.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a severe congenital disorder characterized by vaginal hypoplasia caused by dysplasia of the Müllerian duct. Patients with MRKH syndrome often require nonsurgical or surgical treatment to achieve satisfactory vaginal length and sexual outcomes. The extracellular matrix has been successfully used for vaginal reconstruction. METHODS: In this study, we developed a new biological material derived from porcine vagina (acellular vaginal matrix, AVM) to reconstruct the vagina in Bama miniature pigs. The histological characteristics and efficacy of acellularization of AVM were evaluated, and AVM was subsequently transplanted into Bama miniature pigs to reconstruct the vaginas. RESULTS: Macroscopic analysis showed that the neovaginas functioned well in all Bama miniature pigs with AVM implants. Histological analysis and electrophysiological evidence indicated that morphological and functional recovery was restored in normal vaginal tissues. Scanning electron microscopy showed that the neovaginas had mucosal folds characteristics of normal vagina. No significant differences were observed in the expression of CK14, HSP47, and α-actin between the neovaginas and normal vaginal tissues. However, the expression of estrogen receptor (ER) was significantly lower in the neovaginas than in normal vaginal tissues. In addition, AVM promoted the expression of ß-catenin, c-Myc, and cyclin D1. These results suggest that AVM might promotes vaginal regeneration by activating the ß-catenin/c-Myc/cyclin D1 pathway. CONCLUSION: This study reveals that porcine-derived AVM has potential application for vaginal regeneration.

Interactions between miRNAs and the Wnt/ß-catenin signaling pathway in endometriosis.

Endometriosis is a disease characterized by the ectopic growth of endometrial tissue (glands and stroma) outside the confines of the uterus and often involves vital organs such as the intestines and urinary system. Endometriosis is considered a refractory disease owing to its enigmatic etiology, propensity for recurrence following conservative or surgical interventions, and the absence of radical treatment and long-term management. In recent years, the incidence of endometriosis has gradually increased, rendering it a pressing concern among women of childbearing age. A more profound understanding of its pathogenesis can significantly improve prognosis. Recent research endeavors have spotlighted the molecular mechanisms by which microRNAs (miRNAs) regulate the occurrence and progression of endometriosis. Many miRNAs have been reported to be aberrantly expressed in the affected tissues of both patients and animal models. These miRNAs actively participate in the regulation of inflammatory reactions, cellular proliferation, angiogenesis, and tissue remodeling. Their capacity to modulate crucial signaling pathways, such as the Wnt/ß-catenin signaling pathway, reinforces their potential utility as diagnostic markers or therapeutic agents for endometriosis. In this review, we provide the latest insights into the role of miRNAs that interact with the Wnt/ß-catenin pathway to regulate the biological behaviors of endometriosis cells and disease-related symptoms, such as pain and infertility. We hope that this review will provide novel insights and promising targets for innovative therapies addressing endometriosis.

Unveiling the novel immune and molecular signatures of ovarian cancer: insights and innovations from single-cell sequencing.

Ovarian cancer is a highly heterogeneous and lethal malignancy with limited treatment options. Over the past decade, single-cell sequencing has emerged as an advanced biological technology capable of decoding the landscape of ovarian cancer at the single-cell resolution. It operates at the level of genes, transcriptomes, proteins, epigenomes, and metabolisms, providing detailed information that is distinct from bulk sequencing methods, which only offer average data for specific lesions. Single-cell sequencing technology provides detailed insights into the immune and molecular mechanisms underlying tumor occurrence, development, drug resistance, and immune escape. These insights can guide the development of innovative diagnostic markers, therapeutic strategies, and prognostic indicators. Overall, this review provides a comprehensive summary of the diverse applications of single-cell sequencing in ovarian cancer. It encompasses the identification and characterization of novel cell subpopulations, the elucidation of tumor heterogeneity, the investigation of the tumor microenvironment, the analysis of mechanisms underlying metastasis, and the integration of innovative approaches such as organoid models and multi-omics analysis.

Role of non­coding RNA intertwined with the Wnt/ß­catenin signaling pathway in endometrial cancer (Review).

Endometrial cancer (EC) ranks as the sixth most common malignancy in women around the world. Although low­grade and early­stage EC commonly have an excellent prognosis, ~20% of EC patients experience an unfavorable prognosis. Identifying the pathogenesis and novel therapeutic targets may help address this group of patients. Non­coding (nc)RNAs, such as long non­coding RNAs (lncRNAs), microRNAs and circular RNAs (circRNAs), have been associated with EC occurrence and development. In addition, the aberrant activation of the Wnt/ß­catenin signaling pathway can promote the proliferation, invasion, migration and epithelial­to­mesenchymal transition (EMT) of EC cells. The network of ncRNAs has also been demonstrated to inhibit or activate the Wnt/ß­catenin signaling pathway. In the present review, ncRNAs, the Wnt/ß­catenin signaling pathway, and their crosstalk in EC were summarized and highlighted. This information is expected to provide novel insights into improving the management of EC using RNA as therapeutics.

Human umbilical cord mesenchymal stem cell­derived exosomes improve ovarian function in natural aging by inhibiting apoptosis.

Prolonging the reproductive lifespan is beneficial for preserving the physical and psychological health of women. The transplantation of mesenchymal stem cell (MSC)­derived exosomes (MSC­Exos) has been reported to be a promising regenerative therapeutic strategy for restoring the function of aging ovaries. The present study thus evaluated the therapeutic efficacy of exosomes derived from human umbilical cord­MSCs (hUCMSC­Exos) in a mouse model of natural ovarian aging (NOA), and further investigated the role of exosomal microRNAs (miRNAs/miRs) in the mechanisms of this creative therapy. Specifically, following the administration of hUCMSC­Exos in mice with NOA, ovarian function was found to improve, as indicated by the restoration of follicle numbers and hormone levels. These exosomes were found to exhibit the ability to inhibit PTEN expression and suppress apoptosis both in vivo and in vitro. Subsequently, miRNA sequencing of the exosomes was performed, following which bioinformatics analysis was used to identify the highly expressed miRNAs that are capable of targeting PTEN expression. Through high­throughput sequencing and molecular analyses, miR­21­5p was found to be the highest in ranking in terms of expression, suggesting that hUCMSC­Exos can preserve ovarian function by suppressing PTEN expression to inhibit apoptosis by delivering miR­21­5p. On the whole, the results of the present study suggest that the application of exosomes can be used to restore ovarian function in mice with NOA. These positive findings also suggest that the transplantation of exosomes derived from MSCs holds promise as an agent against ovarian aging.

Vaginal reconstruction with a double-sided biomembrane-a preclinical experimental study on large animals.

Tissue engineering techniques bring the promise of vaginal reconstruction with low invasiveness and fewer complications. However, existing biomaterial scaffolds remain limited in efficient vaginal recovery, focusing only on regenerating an epithelial layer, but muscle layers are missing or abnormal. The lack of a multi-tissue hierarchical structure in the reconstructed vagina leads to shrinking, stenosis, and fibrosis. Here, an acellular matrix named a double-sided biomembrane (DBM) is demonstrated for vaginal recovery. The regeneration of epithelial and muscle layers is achieved simultaneously since the smooth side of the DBM is helpful for guiding epithelial cell growth, while its loose and porous side guides muscle cell growth. In addition, the DBM demonstrates excellent mechanical properties similar to vaginal tissue, and hydrophilicity. Therefore, neovaginas were observed in the fourth and twelfth weeks after DBMs were transplanted to repair full-thickness vaginal defects (4 cm) that we established in large animals. The DBMs can effectively promote rapid epithelialization, the formation of large muscle bundles, higher rates of angiogenesis, and the restoration of physiological function in a neovagina. That is, the injured vagina achieves nearly complete recovery in anatomy and function, similar to a normal vagina. These preclinical results indicate that the DBM has prospects for vaginal injury repair.

Gallbladder neuroendocrine carcinoma: A report of two cases and literature review.

Gallbladder neuroendocrine carcinoma (GB-NEC) is a rare, aggressive neuroendocrine carcinoma that arises from the gallbladder. Patients with GB-NEC usually have a poor prognosis. The present study described two cases diagnosed with GB-NEC and reviewed the literature to improve knowledge of GB-NEC. The present study reported on two cases of GB-NEC in male patients aged 65 and 66 years, respectively. Both patients underwent surgical resection. Postoperative pathology confirmed that one case had mixed adeno-neuroendocrine carcinoma and the other had large cell neuroendocrine carcinoma. In addition, both patients had uneventful recoveries following surgery and received cisplatin-etoposide combination chemotherapy. The present study summarized the two cases and reviewed the literature to improve understanding of GB-NEC. The results revealed that radiological findings of GB-NEC are non-specific. The present study demonstrated that surgical resection was still the most effective therapy and that postoperative adjuvant chemotherapy could markedly improve the prognosis of patients with GB-NEC.

Ovarian pregnancy after bilateral salpingectomy in a patient with in vitro fertilization: a case report.

Ovarian pregnancy is rare but may occur with in vitro fertilization-embryo transfer in women who have undergone bilateral salpingectomy. We report a case of an approximately 30-year-old woman who had in vitro fertilization and a history of bilateral salpingectomy, and was diagnosed with an ovarian pregnancy. Laparoscopic enucleation of the gestational product in the ovary and ovarian remnant reconstruction were performed. The patient recovered well after surgery and was discharged home 5 days postoperatively. ß-human chorionic gonadotropin was undetectable 3 weeks after the surgery. Awareness of the possibility of ovarian pregnancy after in vitro fertilization-embryo transfer is the most important step in an early diagnosis and treatment. Salpingectomy should be carefully performed to eliminate the risk of heterotopic pregnancy, especially in cases where a subsequent gestation is desired.

Ovary-derived Decellularized Extracellular Matrix-based Bioink for Fabricating 3D Primary Ovarian Cells-laden Structures for Mouse Ovarian Failure Correction.

Fertility preservation is becoming a clinical duty in practice. Three-dimensional (3D) bioprinting technology is potentially realize ovarian morphological repair and reproductive endocrine function rebuild. There is no published work on 3D bioprinting ovary using a decellularized extracellular matrix (dECM)-based bioink, though dECM is the preferred matrix choice for an artificial ovary. The study aimed to explore swine ovarian dECM-based bioink to fabricate 3D primary ovarian cells (POCs)-laden structures for mouse ovarian failure correction. In this study, the ovarian dECM was converted to dECM-based bioink by dECM solution mixed with a seaweed gelatin blend solution of bioink that was characterized using scanning electron microscopy, circular dichroism, rheology, hematoxylin and eosin staining, and immunohistochemistry. The 3D scaffolds were, then, printed with or without POCs by the extrusion 3D bioprinter. The laden POCs viability was detected with the live/dead assay kit. A female castrated mouse model was established, and the mice were treated with five different methods. The results revealed that the 3D scaffold encapsulating POCs group had more positive signals in neoangiogenesis, cell proliferation and survival than the 3D scaffold group, and ensured sex hormone secretion. Meanwhile, the expression of germ cells in the 3D scaffold encapsulating POCs group was more intensely than the non-printed hydrogel encapsulating POCs group. The work shows that the 3D bioprinting ovary employing ovarian dECM-based bioink is a promising approach for ovarian failure correction.

Drug-free in vitro activation combined with 3D-bioprinted adipose-derived stem cells restores ovarian function of rats with premature ovarian insufficiency.

BACKGROUND: Emerging drug-free in vitro activation (IVA) technique enables patients with premature ovarian insufficiency (POI) to restore ovarian function and conceive their own genetic offspring. However, various issues have greatly restricted its clinical application. Transplantation of adipose-derived stem cells (ADSCs) has promising roles in restoring ovarian function of rats with POI, but insufficient retention has greatly hampered their efficiency. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and ADSCs, which may prolong the retention of ADSCs and construct an early vascular microenvironment, thus compensating for the disadvantages of drug-free IVA to some extent and ameliorating impaired ovarian function in the POI rats. METHODS: After intraperitoneal injection of cyclophosphamide, the POI model rats were randomized into 5 groups: (1) POI group; (2) ovarian fragments group; (3) 3D scaffold combined with ovarian fragments group; (4) ovarian fragments combined with ADSCs group; (5) 3D scaffold with ADSCs combined with ovarian fragments as 3D-bioprinted engineering ovary group. Normal rats were identified as the control group. The localization of CM-Dil-labeled ADSCs and co-localization with CD31 were observed to examine the distribution and underlying mechanism of differentiation. Histomorphological and immunohistochemical analyses were performed to calculate follicle number and assess proliferation and apoptosis of granulosa cells (GCs). Immunofluorescence staining was used to evaluate angiogenesis. Hormone levels were measured to evaluate the restoration of endocrine axis. Western blot analysis and RT-PCR were conducted to explore the potential mechanism. RESULTS: CM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Several regions of the co-staining for CM-Dil and CD31 were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation, neoangiogenesis, and hormone levels were significantly improved in the 3D-bioprinted engineering ovary group, as compared with other groups. Furthermore, the ovarian function was ameliorated and angiogenesis was promoted through regulating the PI3K/AKT pathway. CONCLUSION: Our results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of rats with POI, which could compensate for the disadvantages of drug-free IVA to some extent.