Uterine macrophages and NK cells exhibit population and gene-level changes after implantation but maintain pro-invasive properties.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.

Innate Immune-Enhancing Effect of Pinus densiflora Pollen Extract via NF-κB Pathway Activation.

Considering the emergence of various infectious diseases, including the coronavirus disease 2019 (COVID-19), people's attention has shifted towards immune health. Consequently, immune-enhancing functional foods have been increasingly consumed. Hence, developing new immune-enhancing functional food products is needed. Pinus densiflora pollen can be collected from the male red pine tree, which is commonly found in Korea. P. densiflora pollen extract (PDE), obtained by water extraction, contained polyphenols (216.29 ± 0.22 mg GAE/100 g) and flavonoids (35.14 ± 0.04 mg CE/100 g). PDE significantly increased the production of nitric oxide (NO) and reactive oxygen species (ROS) but, did not exhibit cytotoxicity in RAW 264.7 cells. Western blot results indicated that PDE induced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. PDE also significantly increased the mRNA and protein levels of cytokines and the phosphorylation of IKKα/ß and p65, as well as the activation and degradation of IκBα. Additionally, western blot analysis of cytosolic and nuclear fractions and immunofluorescence assay confirmed that the translocation of p65 to the nucleus after PDE treatment. These results confirmed that PDE increases the production of cytokines, NO, and ROS by activating NF-κB. Therefore, PDE is a promising nutraceutical candidate for immune-enhancing functional foods.

Harvest of Cell-Only Muscle Fibers Using Thermally Expandable Hydrogels with Adhesive Patterns.

Muscle tissue engineering has been the focus of extensive research due to its potential for numerous medical applications, including ex vivo actuator development and clinical treatments. In this study, we developed a method for harvesting muscle fiber in a floatable and translocatable manner utilizing thermally expandable hydrogels with a chemically patterned polydopamine (PD) layer generated by microcontact printing (µCP). The µCP of PD on the hydrogel facilitated the formation of stripe patterns with varying widths of printed/nonprinted area (50/50, 100/100, and 200/200 µm). The spatially controlled adhesion of C2C12 myoblasts on the PD patterns produced clearly distinguishable muscle fibers, and translocated muscle fibers exhibited preserved extracellular matrix and junction proteins. Furthermore, the development of anisotropic arrangements and mature myotubes within the fibers suggests the potential for functional control of engineered muscle tissues. Overall, the muscle fiber harvesting method developed herein is suitable for both translocation and floating and is a promising technique for muscle tissue engineering as it mimics the structure-function relationship of natural tissue.

Free radical-scavenging composite gelatin methacryloyl hydrogels for cell encapsulation.

Gelatin methacryloyl (GelMA) hydrogels have been widely used for cell encapsulation in tissue engineering due to their cell adhesiveness and biocompatibility. However, free radicals generated during gelation decrease the viability of the encapsulated cells by increasing intracellular oxidative stress, so appropriate strategies for scavenging free radicals need to be developed. To meet that need, we developed composite GelMA hydrogels incorporating nanofiber particles (EF) coated with epigallocatechin-gallate (EGCG). The GelMA composite hydrogels were successfully fabricated and had a storage modulus of about 5 kPa, which is similar to that of pristine GelMA hydrogel, and the drastic free radical scavenging activity of EGCG was highly preserved after gelation. In addition, human adipose-derived stem cells encapsulated within our composite hydrogels had better viability (about 1.5 times) and decreased intracellular oxidative stress (about 0.3 times) compared with cells within the pristine GelMA hydrogel. We obtained similar results with human dermal fibroblasts and human umbilical vein endothelial cells, indicating that our composite hydrogels are suitable for various cell types. Furthermore, we found that the ability of the encapsulated cells to spread and migrate increased by 5 times within the composite hydrogels. Collectively, our results demonstrate that incorporating EF into GelMA hydrogels is a promising way to enhance cell viability by reducing free-radical-derived cellular damage when fabricating 3D tissue ex vivo. STATEMENT OF SIGNIFICANCE: Gelatin methacryloyl (GelMA) hydrogels have been widely applied to various tissue engineering applications because of their biocompatibility and cell interactivity. However, free radicals generated during the GelMA hydrogel fabrication decrease the viability of encapsulated cells by elevating intracellular oxidative stress. Here, we demonstrate radical scavenging GelMA hydrogels incorporating epigallocatechin-gallate (EGCG)-coated nanofiber particles (EF). The composite GelMA hydrogels are successfully fabricated, maintaining their mechanical properties, and the viability of encapsulated human adipose-derived stem cells is greatly improved after the gelation, indicating that our composite GelMA hydrogel alleviates damages from free radicals. Collectively, the incorporation of EF within GelMA hydrogels may be a promising way to enhance the viability of encapsulated cells, which could be applied to 3D tissue fabrication.

Impact of imatinib administration on the mouse ovarian follicle count and levels of intra-ovarian proteins related to follicular quality.

OBJECTIVE: The impact of imatinib, a tyrosine kinase inhibitor, on ovarian follicles and several proteins related to follicular function and apoptosis was investigated in mice. METHODS: Saline, cyclophosphamide (Cp; 50 or 75 mg/kg), or imatinib (7.5 or 15 mg/kg) was injected once intraperitoneally into female B6D2F1 mice (18 mice in each group). In multiple ovarian sections, the number of various types of follicles and the proportion of good-quality (G1) follicles were counted. The levels of six proteins (anti-Müllerian hormone [AMH], BCL-xL, BAX, acid sphingomyelinase [A-SMase], caspase-3, and α-smooth muscle actin [α-SMA]) within the whole ovaries were quantified using Western blots. RESULTS: Compared to the saline group, a significant reduction of the primordial follicle count was observed in the group treated with imatinib 7.5 and 15 mg/kg, as well as in the group treated with Cp 75 mg/kg. Administration of Cp significantly decreased the proportion of G1 primordial follicles, but administration of imatinib did not. No differences in the AMH, anti-apoptotic BCLX-L, pro-apoptotic BAX, and A-SMase levels in the ovarian tissues were observed among the five groups. However, caspase-3 and α-SMA levels were significantly higher in the imatinib and Cp groups than in the saline group. CONCLUSION: The administration of imatinib to mice significantly reduced the primordial follicle count and increased the protein levels of caspase-3 and α-SMA. Our findings suggest that imatinib potentially exerts ovarian toxicity via apoptotic processes, similarly to Cp.

Spatially arranged encapsulation of stem cell spheroids within hydrogels for the regulation of spheroid fusion and cell migration.

Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. STATEMENT OF SIGNIFICANCE: Recently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.

Effects of mechanical properties of gelatin methacryloyl hydrogels on encapsulated stem cell spheroids for 3D tissue engineering.

Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (<1 kPa), has not yet been studied. In this study, we determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction.

Surface engineering of 3D-printed scaffolds with minerals and a pro-angiogenic factor for vascularized bone regeneration.

Scaffolds functionalized with biomolecules have been developed for bone regeneration but inducing the regeneration of complex structured bone with neovessels remains a challenge. For this study, we developed three-dimensional printed scaffolds with bioactive surfaces coated with minerals and platelet-derived growth factor. The minerals were homogeneously deposited on the surface of the scaffold using 0.01 M NaHCO3 with epigallocatechin gallate in simulated body fluid solution (M2). The M2 scaffold demonstrated enhanced mineral coating amount per scaffold with a greater compressive modulus than the others which used different concentration of NaHCO3. Then, we immobilized PDGF on the mineralized scaffold (M2/P), which enhanced the osteogenic differentiation of human adipose derived stem cells in vitro and promoted the secretion of pro-angiogenic factors. Cells cultured in M2/P showed remarkable ratio of osteocalcin- and osteopontin-positive nuclei, and M2/P-derived medium induced endothelial cells to form tubule structures. Finally, the implanted M2/P scaffolds onto mouse calvarial defects had regenerated bone in 80.8 ± 9.8% of the defect area with the arterioles were formed, after 8 weeks. In summary, our scaffold, which composed of minerals and pro-angiogenic growth factor, could be used therapeutically to improve the regeneration of bone with a highly vascularized structure. STATEMENT OF SIGNIFICANCE: Surface engineered scaffolds have been developed for bone regeneration but inducing the volumetric regeneration of bone with neovessels remains a challenge. In here, we developed 3D printed scaffolds with bioactive surfaces coated with bio-minerals and platelet-derived growth factors. We proved that the 0.01 M NaHCO3 with polyphenol in simulated body fluid solution enhanced the deposition of bio-minerals and even distribution on the surface of scaffold. The in vitro studies demonstrated that the attached cells on the bioactive surface showed the enhanced osteogenic differentiation and secretion of pro-angiogenic factors. Finally, the scaffold with bioactive surface not only improved the regenerated volume of bone tissues but also increased neovessel formation after in vivo implantation onto mouse calvarial defect.

Evaluation of the anti-oxidative and ROS scavenging properties of biomaterials coated with epigallocatechin gallate for tissue engineering.

In tissue engineering, excessively generated reactive oxygen species (ROS) during biomaterial implantation or cell transplantation is a one of major causes of diminishing therapeutic effects. In this study, we prepared biomaterial surfaces coated with antioxidant epigallocatechin gallate (EGCG) and metal ions, and evaluated their anti-oxidative and ROS scavenging properties. We revealed that EGCG-coating on polycaprolactone (PCL) film surface increased hydrophilicity and anti-oxidative properties as a function of total phenol content (TPC) potentially due to the increase in phenolic -OH and π-electrons from structural maintenance and directly removed the hydrogen peroxide (H2O2) by resonance-stabilization. Furthermore, EGCG-coated PCL film increased attachment, spreading area, and viability of human adipose-derived stem cells (hADSCs) against H2O2 treatment while stimulated the cellular signaling to reduce apoptotic gene and enhance anti-oxidative enzyme expression. Further, we applied EGCG coating on the surface of poly-L-lactic acid (PLLA) fibers. Spheroids incorporating EGCG-coated PLLA fibers were able to maintain their shape and showed improved viability and anti-oxidative activities in response to H2O2-induced oxidative stress than control spheroids. Therefore, metal-phenolic network (MPN) coating of EGCG is a suitable method to impart the anti-oxidative properties to biomaterials by evaluating the structural properties and biological effects.

One-step harvest and delivery of micropatterned cell sheets mimicking the multi-cellular microenvironment of vascularized tissue.

Techniques for harvest and delivery of cell sheets have been improving for decades. However, cell sheets with complicated patterns closely related to natural tissue architecture were hardly achieved. Here, we developed an efficient method to culture and harvest cell sheets with complex shape (noted as microtissues) using temperature-responsive hydrogel consisting of expandable polyethylene oxide polymer at low temperature. Firstly, a temperature-responsive hydrogel surface with honeycomb patterns (50 and 100 µm in width) were developed through microcontact printing of polydopamine (PD). The human dermal fibroblasts (HDFBs) and human umbilical vein endothelial cells (HUVECs) spontaneously formed honeycomb-shaped microtissues on the patterned hydrogel surface. The microtissues on the hydrogel were able to be harvested and directly delivered to the desired target through thermal expansion of the hydrogel at 4 °C with an efficiency close to 80% within 10 min which is faster than conventional method based on poly(N-isopropylacrylamide). The microtissues maintained their original honeycomb network and intact structures. Honeycomb-patterned cell sheets also were fabricated through serial seeding of various cell lines, including HDFBs, HUVECs, and human adipose-derived stem cells, in which cells were attached along the honeycomb pattern. The underlying honeycomb patterns in the cell sheets were successfully maintained for 3 days, even after delivery. In addition, patterned cell sheets were successfully delivered in vivo while maintaining an intact structure for 7 days. Together, our findings demonstrate that micropatterned temperature-responsive hydrogel is an efficient method of one-step culturing and delivery of complex microtissues and should prove useful in various tissue engineering applications. STATEMENT OF SIGNIFICANCE: Scaffold-free cell delivery techniques, including cell sheet engineering, have been developed for decades. However, there is limited research regarding culture and delivery of microtissues with complex architecture mimicking natural tissue. Herein, we developed a micro-patterned hydrogel platform for the culture and delivery of honeycomb-shaped microtissues. Honeycomb patterns were chemically engineered on the temperature-responsive hydrogel through microcontact printing of polydopamine to selectively allow for human dermal fibroblast or human umbilical vein endothelial cell adhesion. They spontaneously formed honeycomb-shaped microtissues within 24 hr upon cell seeding and directly delivered to various target area including in vivo via thermal expansion of the hydrogel at 4 °C, suggesting that the micro-patterned hydrogel can be an efficient tool for culture and delivery of complex microtissue.

Impact of imatinib or dasatinib coadministration on in vitro preantral follicle development and oocyte acquisition in cyclophosphamide-treated mice.

OBJECTIVE: We investigated the impact of tyrosine kinase inhibitor (imatinib or dasatinib) coadministration with cyclophosphamide (Cp) on preantral follicle development in an in vitro mouse model. METHODS: Seventy-three female BDF1 mice were allocated into four experimental groups: group A, saline; group B, Cp (25 mg/kg); group C, Cp (25 mg/kg) and imatinib (7.5 mg/kg); and group D, Cp (25 mg/kg) and dasatinib (7.5 mg/kg). Preantral follicles were isolated and cultured in vitro up to 12 days. Final oocyte acquisition and spindle integrity of metaphase II (MII) oocytes were assessed. Levels of 17ß-estradiol and anti-Müllerian hormone (AMH) in the final spent media were measured by enzyme-linked immunosorbent assays, and the mRNA levels of Star, Sod1, Mapk3, and Casp3 in the final follicular cells were quantified by real-time polymerase chain reaction. RESULTS: The percentage of MII oocytes per initiated follicle, the proportion of MII oocytes with normal spindles, and the 17ß-estradiol level were similar in all four groups. The median AMH level in group B (7.74 ng/mL) was significantly lower than that in group A (10.84 ng/mL). However, the median AMH levels in group C (9.96 ng/mL) and group D (9.71 ng/mL) were similar to that in group A. The mRNA expression levels of Star, Sod1, Mapk3, and Casp3 were similar in all four groups. CONCLUSION: Coadministration of imatinib or dasatinib with Cp could preserve AMH production capacity in this in vitro mice preantral follicle culture model, and it did not affect MII oocyte acquisition.

Effect of a dual trigger on oocyte maturation in young women with decreased ovarian reserve for the purpose of elective oocyte cryopreservation.

OBJECTIVE: The aim of this study was to determine whether co-administration of a gonadotropin-releasing hormone (GnRH) agonist and human chorionic gonadotropin (hCG) for final oocyte maturation improved mature oocyte cryopreservation outcomes in young women with decreased ovarian reserve (DOR) compared with hCG alone. METHODS: Between January 2016 and August 2019, controlled ovarian stimulation (COS) cycles in women (aged ≤35 years, anti-Müllerian hormone [AMH] <1.2 ng/mL) who underwent elective oocyte cryopreservation for fertility preservation were retrospectively analyzed. RESULTS: A total of 76 COS cycles were triggered with a GnRH agonist and hCG (the dual group) or hCG alone (the hCG group). The mean age and serum AMH levels were comparable between the two groups. The duration of stimulation, total dose of follicle-stimulating hormone used, and total number of oocytes retrieved were similar. However, the number of mature oocytes retrieved and the oocyte maturation rate were significantly higher in the dual group than in the hCG group (p=0.010 and p<0.001). After controlling for confounders, the dual-trigger method remained a significant factor related to the number of mature oocytes retrieved (p=0.016). CONCLUSION: We showed improved mature oocyte collection and maturation rate with the dual triggering of oocyte maturation in young women with DOR. A dual trigger appears to be more beneficial than hCG alone in terms of mature oocyte cryopreservation for young women with DOR.

Engineering Multi-Cellular Spheroids for Tissue Engineering and Regenerative Medicine.

Multi-cellular spheroids are formed as a 3D structure with dense cell-cell/cell-extracellular matrix interactions, and thus, have been widely utilized as implantable therapeutics and various ex vivo tissue models in tissue engineering. In principle, spheroid culture methods maximize cell-cell cohesion and induce spontaneous cellular assembly while minimizing cellular interactions with substrates by using physical forces such as gravitational or centrifugal forces, protein-repellant biomaterials, and micro-structured surfaces. In addition, biofunctional materials including magnetic nanoparticles, polymer microspheres, and nanofiber particles are combined with cells to harvest composite spheroids, to accelerate spheroid formation, to increase the mechanical properties and viability of spheroids, and to direct differentiation of stem cells into desirable cell types. Biocompatible hydrogels are developed to produce microgels for the fabrication of size-controlled spheroids with high efficiency. Recently, spheroids have been further engineered to fabricate structurally and functionally reliable in vitro artificial 3D tissues of the desired shape with enhanced specific biological functions. This paper reviews the overall characteristics of spheroids and general/advanced spheroid culture techniques. Significant roles of functional biomaterials in advanced spheroid engineering with emphasis on the use of spheroids in the reconstruction of artificial 3D tissue for tissue engineering are also thoroughly discussed.

Cumulative live birth rate after up to three consecutive embryo transfer cycles in women with poor ovarian response.

OBJECTIVE: In the present study, we aimed to retrospectively evaluate the cumulative live birth rate (LBR) after up to three consecutive embryo transfer (ET) cycles, either fresh or frozen, in women with expected poor ovarian response (ePOR). METHODS: We selected 115 women who entered the first in vitro fertilization (IVF) cycle between August 2013 and July 2016. The women were divided into an ePOR group (37 women) and a non-ePOR group (78 women). All women in the ePOR group were ≥40 years old or had serum anti-Müllerian hormone levels of less than 1.1 ng/mL at the time of the first IVF cycle. Live birth outcomes were monitored until December 2017. The cumulative LBR (with both conservative and optimistic estimates) was calculated according to the serial number of ET cycles. RESULTS: After up to three ET cycles, the overall cumulative LBR was significantly lower in the ePOR group than in the non-ePOR group (conservative estimate, 10.8% vs. 44.9%, respectively; optimistic estimate, 14.7% vs. 56.1%, respectively; log-rank test, p=0.003). CONCLUSION: Women with ePOR exhibited a lower cumulative LBR than women in the non-ePOR group, and this information should be provided to ePOR women during counseling before starting IVF.

Oocyte cryopreservation for fertility preservation in women with ovarian endometriosis.

RESEARCH-QUESTION: What is the clinical usefulness of oocyte cryopreservation for fertility preservation in women with ovarian endometriosis? DESIGN: Clinical characteristics were retrospectively analysed in 34 women with endometrioma before a planned ovarian cystectomy. Ovarian stimulation outcomes were compared according to laterality. A one-to-one propensity score-matched analysis was conducted to compare ovarian stimulation outcomes of the first cycle in patients with endometrioma undergoing fertility preservation with those in infertile patients without endometrioma who underwent IVF treatment. The number of oocytes cryopreserved in repeated ovarian stimulation cycles was analysed. RESULTS: The mean endometrioma size at diagnosis was 6.0 ± 2.5 cm. The mean age, serum anti-Mullerian hormone levels and number of oocytes cryopreserved were 30.7 ± 5.9 years, 1.85 ± 1.14 ng/ml, and 4.8 ± 3.2, respectively. The number of oocytes cryopreserved in bilateral endometrioma compared with unilateral endometrioma patients was 4.1 ± 2.9 versus 5.7 ± 3.4 (P = 0.600). In the propensity score-matched cohort (n = 22 per group), the number of oocytes retrieved was significantly lower in the patients with endometrioma undergoing fertility preservation compared with that in infertile patients without endometrioma (5.4 ± 3.8 versus 8.1 ± 4.8; P = 0.045). A total of 13 (38.2%) patients with endometrioma underwent repeated stimulation. The median (interquartile range) number of cryopreserved oocytes at the first and the second cycle were 3.0 (2.5-6.0) and 5.0 (2.5-7.5), respectively. CONCLUSIONS: Women with endometrioma should be counselled about oocyte cryopreservation for fertility preservation before surgery. The number of cryopreserved oocytes can be increased by repeated oocyte retrieval.

Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells.

Despite the diverse roles of cell-secreted proteases in the extracellular matrix (ECM), classical methods to analyze protease activity have not been explored at the cell culture site. Here, we report a stable, matrix-sticky, and protease-sensitive extracellular reporter that comprises a collagen-binding protein and a Förster resonance energy transfer (FRET) coupler of an enhanced green fluorescent protein and a small dye molecule. The extracellular FRET reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by matrix metalloproteinase-2 (MMP2) activity during living cell culture without impeding cell viability. When a proMMP2 mutant (Y581A) with altered protease secretion and activity was transfected into cancer cells, the reporter revealed a dramatic reduction in MMP2 activity in both two- and three-dimensional culture systems, compared with cells transfected with wild-type proMMP2. Our reporter is immediately amenable to monitor protease activity in diverse ECM-resident cells as well as to study protease-related extracellular signaling and tissue remodeling.

Pathologic discrepancies between colposcopy-directed biopsy and loop electrosurgical excision procedure of the uterine cervix in women with cytologic high-grade squamous intraepithelial lesions.

OBJECTIVE: To investigate pathologic discrepancies between colposcopy-directed biopsy (CDB) of the cervix and loop electrosurgical excision procedure (LEEP) in women with cytologic high-grade squamous intraepithelial lesions (HSILs). METHODS: We retrospectively identified 297 patients who underwent both CDB and LEEP for HSILs in cervical cytology between 2015 and 2018, and compared their pathologic results. Considering the LEEP to be the gold standard, we evaluated the diagnostic performance of CDB for identifying cervical intraepithelial neoplasia (CIN) grades 2 and 3, adenocarcinoma in situ, and cancer (HSIL+). We also performed age subgroup analyses. RESULTS: Among the study population, 90.9% (270/297) had pathologic HSIL+ using the LEEP. The diagnostic performance of CDB for identifying HSIL+ was as follows: sensitivity, 87.8%; specificity, 59.3%; balanced accuracy, 73.6%; positive predictive value, 95.6%; and negative predictive value, 32.7%. Thirty-three false negative cases of CDB included CIN2,3 (n=29) and cervical cancer (n=4). The pathologic HSIL+ rate in patients with HSIL- by CDB was 67.3% (33/49). CDB exhibited a significant difference in the diagnosis of HSIL+ compared to LEEP in all patients (p<0.001). In age subgroup analyses, age groups <35 years and 35-50 years showed good agreement with the entire data set (p=0.496 and p=0.406, respectively), while age group ≥50 years did not (p=0.036). CONCLUSION: A significant pathologic discrepancy was observed between CDB and LEEP results in women with cytologic HSILs. The diagnostic inaccuracy of CDB increased in those ≥50 years of age.

The effects of letrozole on women with endometriosis undergoing ovarian stimulation for in vitro fertilization.

This study aimed to analyze the effects of a new protocol with letrozole on the outcomes of in vitro fertilization (IVF) cycles in women with endometriosis. This retrospective cohort study was conducted for women diagnosed with endometriosis undergoing IVF from an infertility clinic. A new protocol, combination therapy with letrozole and gonadotropin, was used from August 2016 to January 2018 ('protocol 1', n = 38). From March 2014 to July 2016, conventional IVF with gonadotropin was administered ('protocol 2', n = 26). Age and ovarian reserve were comparable between the two groups. The patients who received protocol 1 resulted in a significantly lower peak estradiol level in IVF compared with those received protocol 2 (722 ± 1076 pg/mL versus 2168 ± 1521 pg/mL, p < .001). The length of stimulation, the total dose of gonadotropin, number of oocytes retrieved, fertilization rates, and number of embryos obtained were similar between the two groups. The mean percentage of mature oocytes was lower (69.9 ± 23.7% versus 80.2 ± 21.0%, p = .029) in patients with protocol 1. While maintaining low estrogen levels, the combination therapy with letrozole and gonadotropin produce similar oocyte and embryo yield to the conventional IVF protocol in women with endometriosis.

Effect of sperm selection using hyaluronan on fertilization and quality of cleavage-stage embryos in intracytoplasmic sperm injection (ICSI) cycles of couples with severe teratozoospermia.

This study aimed to evaluate the effect of hyaluronan-selected/physiological intracytoplasmic sperm injection (PICSI) on fertilization and quality of cleavage-stage embryos in infertile couples with ≤1% of spermatozoa with normal strict morphology (severe teratozoospermia). Seventy-seven couples underwent PICSI between October 2017 and December 2018 (PICSI group), while 75 couples underwent conventional intracytoplasmic sperm injection (ICSI) between January 2016 and September 2017 (ICSI group). Good quality embryos (GQEs) were evaluated based on morphology. Patient and cycle characteristics were comparable between the PICSI and ICSI groups, except for age and anti-Müllerian hormone (AMH) level (38.4 ± 3.9 years vs. 36.3 ± 4.3 years, p = .002 and 2.06 ± 1.99 ng/mL vs. 2.97 ± 3.25 ng/mL, p = .040). The fertilization rate per oocyte inseminated and GQE rate were significantly higher in the PICSI group than in the ICSI group (82.7% vs. 71.7%, p ˂ .001 and 52.8% vs. 34.0%, p ˂ .001). Furthermore, the absence of GQEs was found to be lower in the PICSI group (13.0% vs. 30.7%, p = .008). Multivariate analysis adjusted for age and AMH level identified PICSI as an unfavorable and independent factor for the absence of GQEs (adjusted odds ratio, 0.333; 95% confidence interval, 0.125-0.890). PICSI seems to be superior to ICSI in terms of fertilization and embryo quality in couples with severe teratozoospermia.

Current progress in application of polymeric nanofibers to tissue engineering.

Tissue engineering uses a combination of cell biology, chemistry, and biomaterials to fabricate three dimensional (3D) tissues that mimic the architecture of extracellular matrix (ECM) comprising diverse interwoven nanofibrous structure. Among several methods for producing nanofibrous scaffolds, electrospinning has gained intense interest because it can make nanofibers with a porous structure and high specific surface area. The processing and solution parameters of electrospinning can considerably affect the assembly and structural morphology of the fabricated nanofibers. Electrospun nanofibers can be made from natural or synthetic polymers and blending them is a straightforward way to tune the functionality of the nanofibers. Furthermore, the electrospun nanofibers can be functionalized with various surface modification strategies. In this review, we highlight the latest achievements in fabricating electrospun nanofibers and describe various ways to modify the surface and structure of scaffolds to promote their functionality. We also summarize the application of advanced polymeric nanofibrous scaffolds in the regeneration of human bone, cartilage, vascular tissues, and tendons/ligaments.