Detection of Interleukin-1 ß (IL-1ß) in Single Human Blastocyst-Conditioned Medium Using Ultrasensitive Bead-Based Digital Microfluidic Chip and Its Relationship with Embryonic Implantation Potential.

The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of cytokines produced by a single embryo in successful implantation is largely unknown. This study aimed to investigate the role of IL-1ß expressed in a single-embryo-conditioned medium (ECM) in embryo implantation. Seventy samples of single ECM were analyzed by a specially designed magnetic-beads-based microfluidic chip from 15 women. We discovered that IL-1ß level increased as the embryo developed, and the difference was significant. In addition, receiver operator characteristic (ROC) curves analysis showed a higher chance of pregnancy when the IL-1ß level on day 5 ECM was below 79.37 pg/mL and the difference between day 5 and day 3 was below 24.90 pg/mL. Our study discovered a possible association between embryonic proteomic expression and successful implantation, which might facilitate single-embryo transfer in the future by helping clinicians identify the embryo with the greatest implantation potential.

Gut Microbiome-Estrobolome Profile in Reproductive-Age Women with Endometriosis.

Microbiota is associated with our bodily functions and microenvironment. A healthy, balanced gut microbiome not only helps maintain mucosal integrity, prevents translocation of bacterial content, and contributes to immune status, but also associates with estrogen metabolism. Gut dysbiosis and estrobolome dysfunction have hence been linked to certain estrogen-dependent diseases, including endometriosis. While prior studies on microbiomes and endometriosis have shown conflicting results, most of the observed microbial differences are seen in the genital tract. This case-control study of reproductive-age women utilizes their fecal and urine samples for enzymatic, microbial, and metabolic studies to explore if patients with endometriosis have distinguishable gut microbiota or altered estrogen metabolism. While gut ß-glucuronidase activities, microbial diversity, and abundance did not vary significantly between patients with or without endometriosis, fecal samples of patients with endometriosis were more enriched by the Erysipelotrichia class and had higher folds of four estrogen/estrogen metabolites. Further studies are needed to elucidate what these results imply and whether there indeed is an association or causation between gut microbiota and endometriosis.

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Anti-angiogenic drug aggravates the degree of anti-resorptive drug-based medication-related osteonecrosis of the jaw by impairing the proliferation and migration function of gingival fibroblasts.

BACKGROUND: Long-term use of anti-resorptive or anti-angiogenic drugs in cancer patients with odontogenic infections may lead to medication-related osteonecrosis of the jaw (MRONJ). This study investigated whether anti-angiogenic agents aggravate MRONJ occurrence in anti-resorptive-treated patients. METHODS: The clinical stage and jawbone exposure of MRONJ patients caused by different drug regimens were analyzed to ascertain the aggravation effect of anti-angiogenic drugs on anti-resorptive drug-based MRONJ. Next, a periodontitis mice model was established, and tooth extraction was performed after administering anti-resorptive and/or anti-angiogenic drugs; the imaging and histological change of the extraction socket were observed. Moreover, the cell function of gingival fibroblasts was analyzed after the treatment with anti-resorptive and/or anti-angiogenic drugs in order to evaluate their effect on the gingival tissue healing of the extraction socket. RESULTS: Patients treated with anti-angiogenic and anti-resorptive drugs had an advanced clinical stage and a bigger proportion of necrotic jawbone exposure compared to patients treated with anti-resorptive drugs alone. In vivo study further indicated a greater loss of mucosa tissue coverage above the tooth extraction in mice treated with sunitinib (Suti) + zoledronate (Zole) group (7/10) vs. Zole group (3/10) and Suti group (1/10). Micro-computed tomography (CT) and histological data showed that the new bone formation in the extraction socket was lower in Suti + Zole and Zole groups vs. Suti and control groups. In vitro data showed that the anti-angiogenic drugs had a stronger inhibitory ability on the proliferation and migration function of gingival fibroblasts than anti-resorptive drugs, and the inhibitory effect was obviously enhanced after combining zoledronate and sunitinib. CONCLUSION: Our findings provided support for a synergistic contribution of anti-angiogenic drugs to anti-resorptive drugs-based MRONJ. Importantly, the present study revealed that anti-angiogenic drugs alone do not induce severe MRONJ but aggravate the degree of MRONJ via the enhanced inhibitory function of gingival fibroblasts based on anti-resorptive drugs.

A Lifelong Impact on Endometriosis: Pathophysiology and Pharmacological Treatment.

Endometriosis is a chronic inflammatory disease associated with bothersome symptoms in premenopausal women and is complicated with long-term systemic impacts in the post-menopausal stage. It is generally defined by the presence of endometrial-like tissue outside the uterine cavity, which causes menstrual disorders, chronic pelvic pain, and infertility. Endometriotic lesions can also spread and grow in extra-pelvic sites; the chronic inflammatory status can cause systemic effects, including metabolic disorder, immune dysregulation, and cardiovascular diseases. The uncertain etiologies of endometriosis and their diverse presentations limit the treatment efficacy. High recurrence risk and intolerable side effects result in poor compliance. Current studies for endometriosis have paid attention to the advances in hormonal, neurological, and immunological approaches to the pathophysiology and their potential pharmacological intervention. Here we provide an overview of the lifelong impacts of endometriosis and summarize the updated consensus on therapeutic strategies.

Altered Expression of Interleukin-18 System mRNA at the Level of Endometrial Myometrial Interface in Women with Adenomyosis.

Adenomyosis is a uterine pathology characterized by a deep invasion of endometrial glands and stroma, disrupting the endometrial−myometrial interface (EMI). Interleukin-18 (IL-18) system is a dominant cytokine involved in the menstrual cycle of human endometrium. IL-18 may play a defensive role against maternal immune response in the uterine cavity. The present study was designed to determine IL-18-mediated immune response at the level of EMI. We uncovered that mRNA of IL-18 system, including IL-18, IL-18 receptor (IL-18R), and its antagonist, IL-18 binding protein (IL-18BP), expressed in eutopic, ectopic endometrium, and corresponding myometrium in patients with adenomyosis. IL-18 system was demonstrated in paired tissue samples by immunochemistry and immunofluorescence study. According to RT-PCR with CT value quantification and 2−∆∆Ct method, a significant down-regulation of IL-18BP in corresponding myometrium in comparison to eutopic endometrium (p < 0.05) indicates that the IL-18 system acts as a local immune modulator at the level of EMI and regulating cytokine networks in the pathogenesis of adenomyosis. Furthermore, an increased IL-18 antagonist to agonist ratio was noted in ectopic endometrium compared with corresponding myometrium. We suggest that altered IL-18 system expression contributes to immunological dysfunction and junctional zone disturbance in women with adenomyosis.

Extracellular Vesicle-Associated MicroRNA-138-5p Regulates Embryo Implantation and Early Pregnancy by Adjusting GPR124.

Functional embryo-maternal interactions occur during the embryo implantation and placentation. Extracellular vesicles with microRNA (miR) between cells have been considered of critical importance for embryo implantation and the programming of human pregnancy. MiR-138-5p functions as the transcriptional regulator of G protein-coupled receptor 124 (GPR124). However, the signaling pathway of miR138-5p- and GPR124-adjusted NLRP3 inflammasome activation remains unclear. In this study, we examine the roles of the miR138-5p and GPR124-regulated inflammasome in embryo implantation and early pregnancy. Human decidual stromal cells were isolated from the abortus tissue and collected by curettage from missed abortion patients and normal pregnant women at 6- to 12-week gestation, after informed consent. Isolated extracellular vesicles from decidua and decidual stromal cells were confirmed by transmission electron microscopy (TEM). Next-Generation Sequencing (NGS) and microarray were performed for miR analysis. The predicated target genes of the differentially expressed miR were analyzed to identify the target genes and their pathway. We demonstrated the down-regulation of miR-138-5p and the overexpression of GPR124 in spontaneous miscarriage compared to normal pregnancy. We also showed the excessive activation of the NLRP3 inflammasome in spontaneous miscarriage compared to normal pregnancy. Here, we newly demonstrate that the miR-138-5p and GPR124-adjusted NLRP3 inflammasome were expressed in extracellular vesicles derived from decidua and decidual stromal cells, indicating that the miR-138-5p, GPR124 and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome have a potential modulatory role on the decidual programming and placentation of human pregnancy. Our findings represent a new concept regarding the role of extracellular vesicles, miR-138-5p, GPR124, and the NLRP3 inflammasome in normal early pregnancy and spontaneous miscarriage.

Single-type reporter multiplexing with A single droplet through bead-based digital microfluidics.

With the limited sample volume, the droplet-based microfluidic becomes attractive in biomedical diagnosis, especially for measuring multiple analytes. Usually, for multiplexing by parallel processing, a larger sample volume is required. In our previous study, simultaneously detecting two analytes from a single droplet was first achieved by measuring different fluorescence wavelengths for different analytes. However, the number of detectable analytes could be limited by the spectral resolution of fluorescence. Here a different approach is proposed for multiplexing by sharing a single droplet in multiple sub-assays. Therefore, only a single-type reporter, i.e., the fluorescence with the same wavelength, is needed for detection of different analytes from a single sample droplet, called single-type reporter multiplexing (STRM). The standard curves of two analytes, human IL-1ß and human TNF-α, are demonstrated. The required sample volume for one measurement is only 520 nL; the total duration of the on-chip process is less than 50 min. The limits of detection (LOD) of human IL-1ß and human TNF-α are about 1.14 and 0.97 pg/mL, respectively. It is shown that the proposed bead-based digital microfluidic immunoassay can achieve multiple analytes detection with low LOD from a single sample droplet using the single-type reporter, which has never been achieved before.

Bead Number Effect in a Magnetic-Beads-Based Digital Microfluidic Immunoassay.

In a biomedical diagnosis with a limited sample volume and low concentration, droplet-based microfluidics, also called digital microfluidics, becomes a very attractive approach. Previously, our group developed a magnetic-beads-based digital microfluidic immunoassay with a bead number of around 100, requiring less than 1 µL of sample volume to achieve a pg/mL level limit of detection (LOD). However, the bead number in each measurement was not the same, causing an unstable coefficient of variation (CV) in the calibration curve. Here, we investigated whether a fixed number of beads in this bead-based digital microfluidic immunoassay could provide more stable results. First, the bead screening chips were developed to extract exactly 100, 49, and 25 magnetic beads with diameters of less than 6 µm. Then, four calibration curves were established. One calibration curve was constructed by using varying bead numbers (50-160) in the process. The other three calibration curves used a fixed number of beads, (100, 49, and 25). The results indicated that the CVs for a fixed number of beads were evidently smaller than the CVs for varying bead numbers, especially in the range of 1 pg/mL to 100 pg/mL, where the CVs for 100 beads were less than 10%. Furthermore, the calculated LOD, based on the composite calibration curves, could be reduced by three orders, from 3.0 pg/mL (for the unfixed bead number) to 0.0287 pg/mL (for 100 beads). However, when the bead numbers were too high (more than 500) or too low (25 or fewer), the bead manipulation for aggregation became more difficult in the magnetic-beads-based digital microfluidic immunoassay chip.

DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform.

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 µL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 µL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept.

Impact of growth hormone-releasing hormone antagonist on decidual stromal cell growth and apoptosis in vitro†.

Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.

Supplementation with human menopausal gonadotropin in the gonadotropin-releasing hormone antagonist cycles of women with high AMH: Pregnancy outcomes and serial hormone levels.

OBJECTIVE: To evaluate the value of using both HMG and recombinant FSH (r-FSH) in the GnRH antagonist protocol for women with high AMH. MATERIALS AND METHODS: This retrospective, single-center cohort study was conducted from January 2013 to December 2018. Of 277 GnRH antagonist IVF/ICSI cycles in women with anti-Mullerian hormone (AMH) ≥5 µg/L, 170 cycles receiving the combination of r-FSH and HMG (77 with HMG added at the beginning of the GnRH antagonist cycle and 93 with HMG added after GnRH antagonist administration) and 107 cycles receiving r-FSH alone were analyzed. The dynamic hormone profiles and embryonic and clinical outcomes of the patients were evaluated. RESULTS: We observed significantly lower serum LH levels in the r-FSH + HMG groups during ovarian stimulation. The serum estradiol and progesterone levels were lower in the r-FSH + HMG groups on the trigger day. Nevertheless, there were no significant differences with respect to the number of oocytes retrieved, maturation, fertilization, blastocyst formation rate or ovarian hyperstimulation syndrome (OHSS). The implantation and live birth rates were increased in the r-FSH + HMG groups compared with the r-FSH alone group, with no statistical significance. CONCLUSIONS: HMG for LH supplementation in the GnRH antagonist protocol for patients with high AMH is not significantly superior to r-FSH alone in terms of ovarian response and pregnancy outcome. Nevertheless, HMG supplementation might be appropriate for women with an initially inadequate response to r-FSH or intracycle LH deficiency.

A Review on Microfluidics: An Aid to Assisted Reproductive Technology.

Infertility is a state of the male or female reproductive system that is defined as the failure to achieve pregnancy even after 12 or more months of regular unprotected sexual intercourse. Assisted reproductive technology (ART) plays a crucial role in addressing infertility. Various ART are now available for infertile couples. Fertilization in vitro (IVF), intracytoplasmic sperm injection (ICSI) and intrauterine insemination (IUI) are the most common techniques in this regard. Various microfluidic technologies can incorporate various ART procedures such as embryo and gamete (sperm and oocyte) analysis, sorting, manipulation, culture and monitoring. Hence, this review intends to summarize the current knowledge about the application of this approach towards cell biology to enhance ART.

A randomised controlled trial to clinically validate follitropin delta in its individualised dosing regimen for ovarian stimulation in Asian IVF/ICSI patients.

STUDY QUESTION: Is ovarian stimulation with follitropin delta in its individualised fixed-dose regimen at least as efficacious as follitropin alfa in a conventional dosing regimen in Asian population? SUMMARY ANSWER: Ovarian stimulation with individualised follitropin delta dosing resulted in a non-inferior ongoing pregnancy rate, a significantly higher live birth rate and a significantly lower incidence of early ovarian hyperstimulation syndrome (OHSS) and/or preventive interventions compared to conventional follitropin alfa dosing. WHAT IS KNOWN ALREADY: Previous randomised controlled trials conducted in Japan as well as in Europe, North- and South America have demonstrated that ovarian stimulation with the individualised follitropin delta dosing regimen based on serum anti-Müllerian hormone (AMH) level and body weight modulated the ovarian response and reduced the risk of OHSS without compromising pregnancy and live birth rates. STUDY DESIGN, SIZE, DURATION: Randomised, controlled, multi-centre, assessor-blind trial conducted in 1009 Asian patients from mainland China, South Korea, Vietnam and Taiwan, undergoing their first IVF/ICSI cycle. Randomisation was stratified by age (<35, 35-37, 38-40 years). The primary endpoint was ongoing pregnancy rate assessed 10-11 weeks after embryo transfer in the fresh cycle (non-inferiority limit -10.0%; analysis adjusted for age stratum). PARTICIPANTS/MATERIALS, SETTING, METHODS: The follitropin delta treatment consisted of a fixed daily dose individualised according to each patient's initial AMH level and body weight (AMH <15 pmol/l: 12 µg; AMH ≥15 pmol/l: 0.19 to 0.10 µg/kg; min-max 6-12 µg). The follitropin alfa dose was 150 IU/day for the first 5 days with subsequent potential dose adjustments according to individual response. A GnRH antagonist protocol was applied. OHSS was classified based on Golan's system. Women with an ongoing pregnancy were followed until live birth and 4 weeks after. MAIN RESULTS AND THE ROLE OF CHANCE: The number of oocytes retrieved was significantly (P < 0.001) lower with individualised follitropin delta versus conventional follitropin alfa (10.0 ± 6.1 versus 12.4 ± 7.3). Nevertheless, compared to the conventional dosing approach, the individualised follitropin delta dosing regimen resulted in on average 2 more oocytes (9.6 ± 5.3 versus 7.6 ± 3.5) in potential low responders as indicated by AMH <15 pmol/l, and on average 3 fewer oocytes (10.1 ± 6.3 versus 13.8 ± 7.5) in potential high responders as indicated by AMH ≥15 pmol/l. Among women with AMH ≥15 pmol/l, excessive response occurred less frequently with individualised follitropin delta than with follitropin alfa (≥15 oocytes: 20.2% versus 39.1%; ≥20 oocytes: 6.7% versus 18.5%; both P < 0.001). The incidence of early OHSS and/or preventive interventions for early OHSS was significantly (P = 0.004) reduced from 9.6% with follitropin alfa to 5.0% with individualised follitropin delta. The total gonadotropin use was significantly (P < 0.001) reduced from an average of 109.9 ± 32.9 µg (1498 ± 448 IU) follitropin alfa to 77.5 ± 24.4 µg follitropin delta. Non-inferiority of follitropin delta in its individualised dosing regimen to conventional follitropin alfa was established with respect to the primary endpoint of ongoing pregnancy rate which was 31.3% with follitropin delta compared to 25.7% with follitropin alfa (estimated mean difference 5.4% [95% CI: -0.2%; 11.0%]). The live birth rate was significantly higher at 31.3% with individualised follitropin delta compared to 24.7% with follitropin alfa (estimated mean difference 6.4% [95% CI: 0.9%; 11.9%]; P = 0.023). The live birth rate for each stratum were as follows for follitropin delta and follitropin alfa, respectively; <35 years: 31.0% versus 25.0%, 35-37 years: 35.3% versus 26.7%, 38-40 years: 20.0% versus 14.3%. LIMITATIONS, REASONS FOR CAUTION: The trial only covered the clinical outcome of one treatment cycle with fresh cleavage-stage embryo transfers. WIDER IMPLICATIONS OF THE FINDINGS: The present trial shows that in addition to reducing the early OHSS risk, follitropin delta in its individualised fixed-dose regimen has the potential to improve the success rate in fresh cycles across all ages and with a lower gonadotropin consumption compared to conventional follitropin alfa dosing. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Ferring Pharmaceuticals. J.Q., Y.Z., X.L., T.H., H.-Y.H. and S.-H.K. have received institutional (not personal) clinical trial fees from Ferring Pharmaceuticals. M.G., B.M. and J.-C.A. are employees of Ferring Pharmaceuticals. J.-C.A. has pending and issued patent applications in the WO 2013/020996 and WO 2019/043143 patent families that comprise allowed and granted patent rights related to follitropin delta. TRIAL REGISTRATION NUMBER: NCT03296527 (clinicaltrials.gov). TRIAL REGISTRATION DATE: 28 September 2017. DATE OF FIRST PATIENT'S ENROLMENT: 1 December 2017.

Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro.

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 Vpp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).

Extraction of Cell-free Dna from An Embryo-culture Medium Using Micro-scale Bio-reagents on Ewod.

As scientific and technical knowledge advances, research on biomedical micro-electromechanical systems (bio-MEMS) is also developing towards lab-on-a-chip (LOC) devices. A digital microfluidic (DMF) system specialized for an electrowetting- on-dielectric (EWOD) mechanism is a promising technique for such point-of-care systems. EWOD microfluidic biochemical analytical systems provide applications over a broad range in the lab-on-a-chip field. In this report, we treated extraction of cell-free DNA (cf-DNA) at a small concentration from a mouse embryo culture medium (2.5 days & 3.5 days) with electro-wetting on a dielectric (EWOD) platform using bio-reagents of micro-scale quantity. For such extraction, we modified a conventional method of genomic-DNA (g-DNA) extraction using magnetic beads (MB). To prove that extraction of cf-DNA with EWOD was accomplished, as trials we extracted designed-DNA (obtained from Chang Gung Memorial Hospital (CGMH), Taiwan which shows properties similar to that of cf-DNA). Using that designed DNA, extraction with both conventional and EWOD methods has been performed; the mean percentage of extraction with both methods was calculated for a comparison. From the cycle threshold (Ct) results with a quantitative polymerase chain reaction (q-PCR), the mean extraction percentages were obtained as 14.8 percent according to the conventional method and 23 percent with EWOD. These results show that DNA extraction with EWOD appears promising. The EWOD extraction involved voltage 100 V and frequency 2 kHz. From this analysis, we generated a protocol for an improved extraction percentage on a EWOD chip and performed cf-DNA extraction from an embryo-culture medium (KSOM medium) at 3.5 and 2.5 days. The mean weight obtained for EWOD-extracted cf-DNA is 0.33 fg from the 3.5-day sample and 31.95 fg from the 2.5-day sample. All these results will pave a new path towards a renowned lab-on-a-chip concept.

[Differences in Patient Safety Reporting Attitudes and Knowledge Among Different Hospital Levels].

BACKGROUND: Establishing a positive reporting culture, which helps medical and healthcare workers learn from errors and reduce the risks of future adverse events, is essential to fostering a culture of patient safety. PURPOSE: The objectives of this study were to investigate the differences among the three levels of hospitals in terms of the knowledge and attitudes of hospital staff regarding the patient safety reporting system and to identify the potential factors affecting these differences. METHODS: This cross-sectional study was carried out in six hospitals, including two academic medical centers, two regional hospitals, and two district hospitals. The subjects were physicians, nurses, medical technicians, and administrative staffs. Data were collected using a patient safety reporting questionnaire. RESULTS: Three hundred and forty-eight participants were recruited, with 348 valid questionnaires returned (response rate: 100%). The average score for knowledge of patient safety reporting was 12.76 (total possible score: 14). Age, work position, and work experience were significantly associated with knowledge of patient safety reporting (p < .01). The patient safety reporting attitudes questionnaire comprised 21 items, each of which was scored using a five-point Likert scale. The mean score for each item was 3.92 ± 0.50. Gender, age, work position, work experience, and job discipline were significantly associated with attitude toward reporting (p < .01). The level of hospital was found to significantly impact attitudes toward patient safety reporting (p = .01), with participants working at medical centers scoring the highest. In addition, participants who were older and in more-senior positions scored higher and more positively for both knowledge and attitudes. CONCLUSIONS: The key factors to successfully fostering a strong patient safety reporting culture are staff security, a reliable reporting system, and a user-friendly interface. Improving attitudes toward reporting requires more resources and time than improving knowledge of reporting, which may be improved using education and promotion. Regional hospitals may invest more resources to enhance positive attitudes toward reporting and increase the willingness of staff to report.

Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip.

The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1ß and human TNF-α, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520 nL; the total duration of the on-chip process is less than 40 min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-α change little from day 3 to day 5-6, but the concentrations of IL-1ß for some embryos might double from day 3 to day 5-6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1ß, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.

Unveiling the Potential of Droplet Generation, Sorting, Expansion, and Restoration in Microfluidic Biochips.

Microfluidic biochip techniques are prominently replacing conventional biochemical analyzers by the integration of all functions necessary for biochemical analysis using microfluidics. The microfluidics of droplets offer exquisite control over the size of microliter samples to satisfy the requirements of embryo culture, which might involve a size ranging from picoliter to nanoliter. Polydimethylsiloxane (PDMS) is the mainstream material for the fabrication of microfluidic devices due to its excellent biocompatibility and simplicity of fabrication. Herein, we developed a microfluidic biomedical chip on a PDMS substrate that integrated four key functions-generation of a droplet of an emulsion, sorting, expansion and restoration, which were employed in a mouse embryo system to assess reproductive medicine. The main channel of the designed chip had width of 1200 µm and height of 500 µm. The designed microfluidic chips possessed six sections-cleaved into three inlets and three outlets-to study the key functions with five-day embryo culture. The control part of the experiment was conducted with polystyrene (PS) beads (100 µm), the same size as the murine embryos, for the purpose of testing. The outcomes of our work illustrate that the rate of success of the static droplet culture group (87.5%) is only slightly less than that of a conventional group (95%). It clearly demonstrates that a droplet-based microfluidic system can produce a droplet in a volume range from picoliter to nanoliter.

Obstetric and perinatal outcomes of pregnancy in patients with repeated implantation failure.

OBJECTIVE: Despite the great advance of assisted reproductive technology (ART) in recent decades, many IVF patients failed to achieve a pregnancy even after multiple IVF-ET attempts. These patients are considered to have repeated implantation failure (RIF). While exhausting efforts have been devoted to the improvement of pregnancy rate in RIF patients, it is not clear whether RIF patients have aberrant obstetric or perinatal outcomes after they eventually achieved a pregnancy. MATERIALS AND METHODS: Taking advantage of a relatively large database of IVF-ET cycles at the Chang Gung Memorial Hospital, we compared obstetric and perinatal outcomes of RIF patients who have a successful pregnancy after IVF-ET treatment(s) to those of control IVF-ET patients. RESULTS: Because multiple pregnancies are associated with a high risk of obstetric complications, we restricted the analysis to patients who had singleton pregnancies. Analysis of a total of 596 control and 46 RIF cases showed the rates of almost all obstetric and perinatal outcomes investigated are not different between the two groups. However, the rate of placental abruption in the RIF group (4.35%) appeared to be significantly higher than that of controls (0.50%; OR = 8.99). This difference is still statistically significant after adjustment with the age (adjusted OR = 8.2). CONCLUSION: While the rates of a spectrum of obstetric and perinatal outcomes are normal in RIF patients, these patients could have an enhanced risk of placental abruption. However, investigations with a large sample size are needed to substantiate this inference.