An experimental model for ovarian cancer: propagation of ovarian cancer initiating cells and generation of ovarian cancer organoids.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological cancer due to the recurrence of drug-resistance. Cancer initiating cells (CICs) are proposed to be responsible for the aggressiveness of OC. The rarity and difficulty of in vitro long-term cultivation of CICs challenge the development of CIC-targeting therapeutics. Reprogramming cancer cells into induced cancer initiating cell (iCICs) could be an approach to solve these. Several inducible CICs have been acquired by activating the expression of stemness genes in different cancer cells. However, few reports have demonstrated the feasibility in OC. METHODS: Patients with primary OC receiving surgery were enrolled. Tumor tissue were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunohistochemistry (IHC) staining to investigate the association of stemness markers with overall survival (OS). An high-grade serous ovarian cancer (HGSOC) cell line, OVCAR-3 was reprogrammed by transducing Yamanaka four factors OCT4, SOX2, KLF4 and MYC (OSKM) to establish an iOCIC model, iOVCAR-3-OSKM. CIC characteristics of iOVCAR-3-OSKM were evaluated by RT-PCR, sphere formation assay and animal experiments. Drug-resistance and migration ability were accessed by dye-efflux activity assay, MTT assay and migration assay. Gene profile was presented through RNA-sequencing. Lineage differentiation ability and organoid culture were determined by in vitro differentiation assays. RESULTS: In OC patients, the co-expression of multiple stem-related transcription factors (OCT4, SOX2, and NANOG) was associated with worse OS. iOVCAR-3-OSKM cells generated by reprogramming successfully exhibited stemness characteristics with strong sphere-forming and tumorigenesis ability. iOVCAR-3-OSKM cells also showed malignant potential with higher drug resistance to chemodrug, Paclitaxel (PTX) and migration ability. iOVCAR-3-OSKM was maintainable and expandable on feeder-dependent culture condition, it also preserved ovarian lineage differentiation abilities, which could well differentiate into OC cells with CK-7 and CA125 expressions and develop into an organoid mimic poor prognostic OC histological feature. CONCLUSIONS: The establishment of iOVCAR-3-OSKM not only allows us to fill the gap in the information on induced CICs in OC but also provides a potential strategy to develop personalized CICs and organoid models for treating OC in the near future.

Lipofundin mediates major inhibition of intravenous propofol on phorbol myristate acetate and Escherichia coli-induced neutrophil extracellular traps.

BACKGROUND: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. METHODS: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. RESULTS: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. CONCLUSIONS: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.

Obesity alters ovarian folliculogenesis through disrupted angiogenesis from increased IL-10 production.

OBJECTIVE: Obesity has been reported to have a modulatory effect on the ovulatory functions of patients with polycystic ovary syndrome. The role of adipokines in this obesity-associated ovulatory disturbance has not been extensively explored. In this study, the relationships between obesity, adipokine production from visceral fat, and ovarian folliculogenesis were explored in a mice model of induced obesity. METHODS: Obesity was induced in female C57BL/6 mice fed ad libitum with high-fat feed and fructose water for 4 weeks. Follicular developments in the ovaries were assessed by histopathology in these diet-induced obese mice. Changes in adipokine expression in the peri-ovarian adipose tissues were screened with an adipokine array. The adipokine with the most significant increase over time was identified. The functions of the adipokine in angiogenic processes were evaluated in a cell model of endothelial proliferation. The in vivo effects of neutralizing this adipokine using specific antibodies were assessed in the same obesity model. RESULTS: A high-fat and fructose diet induced an accumulation of early ovarian follicles and a reduction in mature follicles and corpus lutea. The number of microvessels in the early follicles also decreased. The adipokine protein array of the peri-ovarian adipose tissues identified a progressive increase in IL-10 expression with the duration of the obesogenic diet. In vitro experiments in the endothelial cell model confirmed IL-10 as a disrupter of VEGF-induced angiogenesis. Administration of anti-IL-10 antibodies prevented the histopathological changes induced by the obesogenic diet and further highlighted the role of IL-10 in disrupting folliculogenesis. CONCLUSIONS: Obesity may disrupt normal folliculogenesis through increased production of IL-10 in visceral fats. This relationship may help clarify the reported association between obesity and ovulatory dysfunction, which has been found in patients with polycystic ovary syndrome. However, the duration of this study was short, which limited conclusions on the long-term reproductive outcomes.

Human pluripotent stem cell-derived DDX4 and KRT-8 positive cells participate in ovarian follicle-like structure formation.

Understanding the mechanisms of human pluripotent stem cells (hPSCs) specification, development and differentiation to gametes are useful for elucidating the causes of infertility and potential treatment. This study aims to examine whether hPSCs can be induced to DDX4 extracellularly expressing primordial germ cell-like cells (DDX4ec PGCLCs) and further into ovarian follicle stage in a combined in vitro and in vivo model. The transcriptional signatures show that these DDX4ec PGCLCs are characteristic of PGCs and express ovarian folliculogenesis markers. We also verify that keratin (KRT)-8 is highly expressed in the DDX4ec PGCLCs and plays a crucial role in germ cell migration. By co-culturing DDX4ec PGCLCs with human granulosa cells (GCs), these cells are further induced into ovarian follicle-like structures in a xenograft mice model. This approach can in the future design practical strategies for treating germ cell-associated issues of infertility.

Four-Dimensional Introital Ultrasound in Assessing Perioperative Pelvic Floor Muscle Functions of Women with Cystoceles.

PURPOSE: Research on the use of ultrasound to explore the pelvic floor in women is rarely done with introital ultrasound. This study aimed to investigate the performance of four-dimensional (4D) introital ultrasound in the perioperative assessment of pelvic floor muscle (PFM) function in women with cystocele. MATERIALS AND METHODS: The reliability and agreement of ultrasound measurements were determined by intraclass correlation coefficients (ICC) with 95 % confidence interval and Bland-Altman analysis in 20 women. The validity of ultrasound parameters was assessed by correlating squeezing ultrasound measurements with the modified Oxford scale (MOS) in 317 women. 4D introital ultrasound data of 241 women with (n = 29) and without (n = 212) postoperative cystocele at the 12-month postoperative assessment were retrospectively analyzed. Levator avulsion was diagnosed using tomographic ultrasound imaging. Involuntary and voluntary PFM functions were explored by dynamic changes in the bladder neck and genital hiatus, respectively, upon coughing and squeezing on 4D introital ultrasound. RESULTS: The ICC for the reliability of all tested ultrasound parameters was good to very good. The changes and change ratios of most ultrasound measurements from resting to squeezing were fairly correlated with MOS. Women with postoperative cystocele demonstrated more rates of complete levator avulsion [41.3 % vs. 4.7 %, P < 0.001, odds ratio (OR) 14.26, 95 % confidence interval (CI) 4.88-42.42] and fewer rates of capable voluntary PFM contraction (65.5 % vs. 92.5 %, P < 0.001, OR 0.16, 95 % CI 0.06-0.43) than those without postoperative cystocele postoperatively. CONCLUSION: 4D introital ultrasound is feasible to assess perioperative PFM function in women with cystocele.

Copy number variant hotspots in Han Taiwanese population induced pluripotent stem cell lines - lessons from establishing the Taiwan human disease iPSC Consortium Bank.

BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.

Five-year clinical and imaging outcomes of primary transobturator midurethral sling procedures for uncomplicated urodynamic stress incontinence.

OBJECTIVES: To report 5-year clinical and imaging outcomes of primary transoburator midurethral sling (TOT) procedures for uncomplicated urodynamic stress incontinence (USI). MATERIALS AND METHODS: We retrospectively investigated the data of 136 women who underwent primary TOT procedures for uncomplicated USI. All women received preoperative as well as 1-year and 5-year postoperative assessments comprising clinical interview, pelvic examination, and introital four-dimensional (4D) ultrasound. The primary outcome was stress urinary incontinence (SUI), defined as the report of SUI in patient interview, a positive response to item 3 of the short form of the Urogenital Distress Inventory (UDI-6), or a positive cough stress test and negative dysuria or urinalysis. Secondary outcomes included SUI severity, SUI bother, scores on the short forms of the UDI-6 and Incontinence Impact Questionnaire (IIQ-7), rates of de novo overactive bladder (OAB) symptoms, de novo voiding dysfunction, groin/thigh pain, and sling exposure, as well as ultrasound manifestations of bladder neck, midurethra, and sling. RESULTS: At 1 and 5 years, rates for SUI (7.4% vs 8.8%, P = 0.824), de novo OAB symptoms (4.4% vs 5.1%, P = 1.000), de novo voiding dysfunction (11.2% vs 10.3%, P = 1.000), groin/thigh pain (3.7% vs 0.7%, P = 0.216), and sling exposure (2.2% vs 0.0%, P = 0.246) were similar. Scores on the UDI-6 and IIQ-7 were significantly decreased postoperatively. Sling location and a more cranioventral midurethral location were sustained during follow-up. CONCLUSIONS: For uncomplicated USI, TOT has good and sustained clinical and imaging outcomes, though a notable rate of de novo voiding dysfunction.

An overview of the current and emerging platforms for preimplantation genetic testing for aneuploidies (PGT-A) in in vitro fertilization programs.

Preimplantation genetic testing for aneuploidies (PGT-A) and PGT for monogenic disorders (PGT-M) have currently been used widely, aiming to improve IVF outcomes. Although with many years of unsatisfactory results, PGT-A has been revived because new technologies have been adopted, such as platforms to examine all 24 types of chromosomes in blastocysts. This report compiles current knowledge regarding the available PGT platforms, including quantitative PCR, array CGH, and next-generation sequencing. The diagnostic capabilities of are compared and respective advantages/disadvantages outlined. We also address the limitations of current technologies, such as assignment of embryos with balanced translocation. We also discuss the emerging novel PGT technologies that likely will change our future practice, such as non-invasive PGT examining spent culture medium. Current literature suggest that most platforms can effectively reach concordant results regarding whole-chromosome ploidy status of all 24 types of chromosomes. However, different platforms have different resolutions and experimental complexities; leading to different turnaround time, throughput and differential capabilities of detecting mosaicism, segmental mutations, unbalanced translocations, concurrent PGT-A and PGT-M etc. Based on these information, IVF staff can more appropriately interpret PGT data and counsel patients, and select suitable platforms to meet personalized needs. The present report also concisely discusses some crucial clinical outcomes by PGT, which can clarify the role of applying PGT in daily IVF programs. Finally the up-to-date information about the novel use of current technologies and the newly emerging technologies will also help identify the focus areas for the design of new platforms for PGT in the future.

The significance of pelvic floor support on the pelvic floor muscle functions of women with lower urinary tract symptoms.

OBJECTIVE: To explore the significance of pelvic organ prolapse (POP) on pelvic floor muscle (PFM) function among women with lower urinary tract symptoms (LUTS). MATERIALS AND METHODS: Four-dimensional ultrasound data of 577 women with LUTS were retrospectively analyzed. The bladder neck and genital hiatus were assessed during resting, coughing, and squeezing. The bladder neck location, genitohiatal size, and genitohiatal location were evaluated with bladder neck distance (BNd) and bladder neck angle (BNa), genitohiatal dimension (GHd) and genitohiatal area (GHAR), and genitohiatal angle (GHa), respectively. RESULTS: Compared with women without POP (n = 306), women with POP (n = 271) exhibited higher rates of levator complete avulsion (6.5% vs. 40.2%, P < 0.001), shorter BNd (2.84 ± 1.56 cm vs. 2.45 ± 0.45 cm, P = 0.018), larger BNa (92 ± 15° vs. 101 ± 21°, P < 0.001), longer GHd (5.25 ± 0.72 cm vs. 5.60 ± 0.87 cm, P < 0.001), larger GHa (141 ± 10° vs. 145 ± 9°, P = 0.004), and larger GHAR (20.0 ± 4.7 cm2 vs. 24.2 ± 5.6 cm2, P < 0.001) during resting. Fewer women with POP were able to maintain stable bladder neck location (79.5% vs. 65.5%, P < 0.001), genitohiatal size (60.7% vs. 51.9%, P = 0.042), and genitohiatal location (61.6% vs. 52.8%, P = 0.044) following coughing. Fewer women with POP were capable of squeezing (77.8% vs. 58.3%, P < 0.001). CONCLUSION: Among women with LUTS, the presence of POP is associated with weaker resting, involuntary, and voluntary PFM functions.

Medium-term outcomes for primary native tissue reconstructive surgeries with and without transobturator vaginal meshes in stress continent women with stage 3 or higher pelvic organ prolapse.

AIMS: To compare the surgical outcomes of conventional surgeries with or without concomitant transobturator vaginal mesh (TVM) for ≥Stage 3 pelvic organ prolapse (POP). METHODS: We retrospectively investigated 166 women who received conventional surgery including vaginal total hysterectomy, modified McCall culdoplasty, and AP-repair (conventional group) and 98 women with concomitant TVM (mesh group). Follow-up at 3, 12, and 24 months comprised symptom interview, pelvic examination, and ultrasound assessments. The primary outcome was anatomical success defined as ≤Stage 1 POP. Secondary outcomes were subjective symptoms, ultrasound manifestations, and complications. RESULTS: Both groups showed improvements in functional and anatomical outcomes after operations. Compared with the conventional group, the mesh group had higher rates of de novo stress urinary incontinence (SUI) at 3-month (3.6% vs 19.4%; P < .001), 12-month (3.7% vs 26.4%; P < .001), and 24-month (2.4% vs 21.4%; P = .001) follow-up, a higher POP-C point (-7.3 ± 0.7 cm vs -7.6 ± 0.6 cm; P < .001) at 3-month follow-up, a smaller straining bladder neck angle indicating a more cranioventral straining bladder neck position (117 ± 25° vs 102 ± 20°; P < .001) at 3-month follow-up, and a less bladder neck mobility at 3-month (19 ± 24° vs 8 ± 14°; P = .002) and 12-month (26 ± 18° vs 12 ± 15°; P = .003) follow-up. CONCLUSIONS: Concomitant TVM is associated with a higher rate of de novo SUI, more cranioventral straining bladder neck position, and less bladder neck mobility.

Preimplantation Genetic Diagnosis of Neurodegenerative Diseases: Review of Methodologies and Report of Our Experience as a Regional Reference Laboratory.

Preimplantation genetic diagnosis (PGD) has become a crucial approach in helping carriers of inherited disorders to give birth to healthy offspring. In this study, we review PGD methodologies and explore the use of amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) and/or linkage analysis for PGD in neurodegenerative diseases that are clinically relevant with typical features, such as late onset, and which are severely debilitating. A total of 13 oocyte retrieval cycles were conducted in 10 cases with various neurodegenerative diseases. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. Of the 12 embryo transfer cycles, three resulted in pregnancy, and all pregnancies were delivered. The implantation rate and livebirth rate were 23.1% (3/13) per oocyte retrieval cycle and 25.0% (3/12) per embryo transfer cycle. Allele dropout (ADO) was noted in two embryos that were classified as unaffected by ARMS-qPCR but were evidenced as affected after prenatal diagnosis, rendering the false negative rate as 6.3% (2/32). Four among the 13 cycles underwent PGD by ARMS-qPCR coupled with linkage analysis, and all were correctly diagnosed. We conclude that PGD by ARMS-qPCR and/or linkage analysis is a feasible strategy, whereas ADO is a concern when ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases.

Generation of induced pluripotent stem cell line-NTUHi001-A from a premature ovarian failure patient with Turner's syndrome mosaicism.

Turner's syndrome (TS) is one of the main causes of premature ovarian failure (POF). However, the mechanisms underlying POF are difficult to study due to the lack of suitable disease models. Herein, we have generated a human induced pluripotent stem cell (hiPSC) line derived from the peripheral blood mononuclear cells of a female patient with Turner's syndrome mosaicism via integration-free Sendai-virus system. The hiPSCs were confirmed with a 45, X karyotype and the acquisition of pluripotency. It's likely that hiPSCs can serve as a feasible cellular model for further pathophysiological studies of POF cases, especially for those originating in TS.

WW Domain-Containing Proteins YAP and TAZ in the Hippo Pathway as Key Regulators in Stemness Maintenance, Tissue Homeostasis, and Tumorigenesis.

The Hippo pathway is a conserved signaling pathway originally defined in Drosophila melanogaster two decades ago. Deregulation of the Hippo pathway leads to significant overgrowth in phenotypes and ultimately initiation of tumorigenesis in various tissues. The major WW domain proteins in the Hippo pathway are YAP and TAZ, which regulate embryonic development, organ growth, tissue regeneration, stem cell pluripotency, and tumorigenesis. Recent reports reveal the novel roles of YAP/TAZ in establishing the precise balance of stem cell niches, promoting the production of induced pluripotent stem cells (iPSCs), and provoking signals for regeneration and cancer initiation. Activation of YAP/TAZ, for example, results in the expansion of progenitor cells, which promotes regeneration after tissue damage. YAP is highly expressed in self-renewing pluripotent stem cells. Overexpression of YAP halts stem cell differentiation and yet maintains the inherent stem cell properties. A success in reprograming iPSCs by the transfection of cells with Oct3/4, Sox2, and Yap expression constructs has recently been shown. In this review, we update the current knowledge and the latest progress in the WW domain proteins of the Hippo pathway in relevance to stem cell biology, and provide a thorough understanding in the tissue homeostasis and identification of potential targets to block tumor development. We also provide the regulatory role of tumor suppressor WWOX in the upstream of TGF-ß, Hyal-2, and Wnt signaling that cross talks with the Hippo pathway.

Polybenzyl Glutamate Biocompatible Scaffold Promotes the Efficiency of Retinal Differentiation toward Retinal Ganglion Cell Lineage from Human-Induced Pluripotent Stem Cells.

Optic neuropathy is one of the leading causes of irreversible blindness caused by retinal ganglion cell (RGC) degeneration. The development of induced pluripotent stem cell (iPSC)-based therapy opens a therapeutic window for RGC degeneration, and tissue engineering may further promote the efficiency of differentiation process of iPSCs. The present study was designed to evaluate the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors. The iPSC-derived neural spheres cultured on PBG scaffold increased the differentiated retinal neurons and promoted the neurite outgrowth in the RGC progenitor layer. Additionally, iPSCs cultured on PBG scaffold formed the organoid-like structures compared to that of iPSCs cultured on cover glass within the same culture period. With RNA-seq, we found that cells of the PBG group were differentiated toward retinal lineage and may be related to the glutamate signaling pathway. Further ontological analysis and the gene network analysis showed that the differentially expressed genes between cells of the PBG group and the control group were mainly associated with neuronal differentiation, neuronal maturation, and more specifically, retinal differentiation and maturation. The novel electrospinning PBG scaffold is beneficial for culturing iPSC-derived RGC progenitors as well as retinal organoids. Cells cultured on PBG scaffold differentiate effectively and shorten the process of RGC differentiation compared to that of cells cultured on coverslip. The new culture system may be helpful in future disease modeling, pharmacological screening, autologous transplantation, as well as narrowing the gap to clinical application.

Induced pluripotent stem cell-derived endothelial progenitor cells attenuate ischemic acute kidney injury and cardiac dysfunction.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI), which is associated with high morbidity and mortality. AKI is a serious and costly medical condition. Effective therapy for AKI is an unmet clinical need, and molecular mechanisms underlying the interactions between an injured kidney and distant organs remain unclear. Therefore, novel therapeutic strategies should be developed. METHODS: We directed the differentiation of human induced pluripotent stem (iPS) cells into endothelial progenitor cells (iEPCs), which were then applied for treating mouse AKI. The mouse model of AKI was induced by I/R injury. RESULTS: We discovered that intravenously infused iEPCs were recruited to the injured kidney, expressed the mature endothelial cell marker CD31, and replaced injured endothelial cells. Moreover, infused iEPCs produced abundant proangiogenic proteins, which entered into circulation. In AKI mice, blood urea nitrogen and plasma creatinine levels increased 2 days after I/R injury and reduced after the infusion of iEPCs. Tubular injury, cell apoptosis, and peritubular capillary rarefaction in injured kidneys were attenuated accordingly. In the AKI mice, iEPC therapy also ameliorated apoptosis of cardiomyocytes and cardiac dysfunction, as indicated by echocardiography. The therapy also ameliorated an increase in serum brain natriuretic peptide. Regarding the relevant mechanisms, indoxyl sulfate and interleukin-1ß synergistically induced apoptosis of cardiomyocytes. Systemic iEPC therapy downregulated the proapoptotic protein caspase-3 and upregulated the anti-apoptotic protein Bcl-2 in the hearts of the AKI mice, possibly through the reduction of indoxyl sulfate and interleukin-1ß. CONCLUSIONS: Therapy using human iPS cell-derived iEPCs provided a protective effect against ischemic AKI and remote cardiac dysfunction through the repair of endothelial cells and the attenuation of cardiomyocyte apoptosis.

Feasibility of corifollitropin alfa/GnRH antagonist protocol combined with GnRH agonist triggering and freeze-all strategy in polycystic ovary syndrome patients.

BACKGROUND/PURPOSE: The long-acting corifollitropin alfa is comparable to FSH in terms of pregnancy outcomes in normal responders and poor responders. Corifollitropin alfa has never been studied in polycystic ovary syndrome (PCOS) patients because of concerns of excessive ovarian stimulation and ovarian hyperstimulation syndrome (OHSS). The purpose of the study was to evaluate if corifollitropin alfa can be used in PCOS patients. METHODS: Forty PCOS patients who were going to undergo in vitro fertilization were enrolled in this study. A single injection of corifollitropin alfa was administered on cycle day 2 or day 3. From stimulation day 8 onwards, daily FSH was administered until the day of final oocyte maturation. Cetrorelix was administered from stimulation day 5 to prevent premature LH surge. Final oocyte maturation was triggered by: acetate. All embryos were cryopreserved and replaced in subsequent cycles. RESULTS: All 40 patients were subjected to oocyte retrieval, and none developed moderate or severe ovarian hyperstimulation syndrome (0%, 95% CI 0-0.088). For each patient, an average of 23.4 (±7.4; 95% CI 21.0-25.7) oocytes were retrieved and a mean of 11.7 (±6.4; 95% CI 9.6-13.8) embryos were frozen. Mean serum estradiol level on the day of GnRHa triggering was 7829.9 pg/ml (±3297; 95% CI 6775-8885). The cumulated ongoing pregnancy rate after 3 frozen-thawed embryo transfers was 75.0% (95% CI 61.6%-88.4%). CONCLUSION: The results suggest that corifollitropin alfa/GnRH antagonist protocol can be used in PCOS patients, in combination with GnRHa triggering and embryo cryopreservation.

Preimplantation genetic diagnosis and screening: Current status and future challenges.

Preimplantation genetic diagnosis (PGD) is a clinically feasible technology to prevent the transmission of monogenic inherited disorders in families afflicted the diseases to the future offsprings. The major technical hurdle is it does not have a general formula for all mutations, thus different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scarce, whereas timely result is sometimes requested if fresh embryo transfer is desired. On the other hand, preimplantation genetic screening (PGS) screens embryo with aneuploidy and was also known as PGD-A (A denotes aneuploidy) in order to enhance the implantation rates as well as livebirth rates. In contrasts to PGD, PGS is still under ferocious debate, especially recent reports found that euploid babies were born after transferring the aneuploid embryos diagnosed by PGS back to the womb and only very few randomized trials of PGS are available in the literature. We have been doing PGD and/or PGS for more than 10 years as one of the core PGD/PGS laboratories in Taiwan. Here we provide a concise review of PGD/PGS regarding its current status, both domestically and globally, as well as its future challenges.

Noninvasive prenatal diagnosis of fetal aneuploidy by circulating fetal nucleated red blood cells and extravillous trophoblasts using silicon-based nanostructured microfluidics.

BACKGROUND: Noninvasive prenatal testing (NIPT) based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community since 2011 but limitations, such as maternal malignancy and fetoplacental mosaicism, preclude its full replacement of invasive prenatal diagnosis. We present a novel silicon-based nanostructured microfluidics platform named as "Cell Reveal™" to demonstrate the feasibility of capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cell-based noninvasive prenatal diagnosis (cbNIPD). METHODS: The "Cell Reveal™" system is a silicon-based, nanostructured microfluidics using immunoaffinity to capture the trophoblasts and the nucleated RBC (nRBC) with specific antibodies. The automated computer analysis software was used to identify the targeted cells through additional immunostaining of the corresponding antigens. The identified cells were retrieved for whole genome amplification for subsequent investigations by micromanipulation in one microchip, and left in situ for subsequent fluorescence in situ hybridization (FISH) in another microchip. When validation, bloods from pregnant women (n = 24) at gestational age 11-13+6 weeks were enrolled. When verification, bloods from pregnant women (n = 5) receiving chorionic villus sampling or amniocentesis at gestation age 11+4-21 weeks with an aneuploid or euploid fetus were enrolled, followed by genetic analyses using FISH, short tandem repeat (STR) analyses, array comparative genomic hybridization, and next generation sequencing, in which the laboratory is blind to the fetal genetic complement. RESULTS: The numbers of captured targeted cells were 1-44 nRBC/2 ml and 1-32 EVT/2 ml in the validation group. The genetic investigations performed in the verification group confirmed the captured cells to be fetal origin. In every 8 ml of the maternal blood being blindly tested, both fnRBC and EVT were always captured. The numbers of captured fetal cells were 14-22 fnRBC/4 ml and 1-44 EVT/4 ml of maternal blood. CONCLUSIONS: This report is one of the first few to verify the capture of fnRBC in addition to EVT. The scalability of our automated system made us one step closer toward the goal of in vitro diagnostics.

PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1-KLF4 complex in telomerase expression in cancer and stem cells.

Comparative global immune-related gene profiling of somatic cells, human pluripotent stem cells and their derivatives: implication for human lymphocyte proliferation.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.