Superfertility and subfertility in patients with recurrent pregnancy loss: A comparative analysis of clinical characteristics and etiology based on differences in fertile ability.

This study aimed to elucidate the etiologies of and risk factors for recurrent pregnancy loss (RPL) according to fertile ability, focusing on the differences between superfertile and subfertile patients. This retrospective observational study included 828 women with RPL between July 2017 and February 2020. Patients were divided into three groups based on time to pregnancy (TTP): superfertile (SUP) (TTP ≤3 months for all previous pregnancies), subfertile (SUB) (previous TTP ≥12 months and use of assisted reproductive technology [ART]), and Normal (N) (TTP >3 or <12 months without ART). All patients were assessed for uterine anatomy, antiphospholipid antibodies (APAs), thyroid function, and thrombophilia. Of the 828 patients, 22%, 44%, and 34% were assigned to the SUP, SUB, and N groups, respectively. The mean ages were 33.9, 38.2, and 35.9 years in the SUP, SUB, and N groups, respectively, revealing a significant difference (P < 0.001). The anti-CL ß2GPI antibody positivity rate was significantly higher in the SUP group (4.6%) than in the N group (0.8%; P = 0.016). The prevalence of APA positivity was lowest in the N group. Overall, the clinical characteristics and etiologies of RPL associated with superfertility and subfertility were strikingly similar, with comparable positivity rates after adjusting for maternal age. Further investigation including chromosomal analysis of products of conception is needed to elucidate the clinical impact of differences in fertility on patients with RPL.

Calculation of Human Melatonin Partial Metabolic Clearance in Healthy Adult Volunteers for Investigation of a Novel in Vivo CYP1A2 Phenotyping Method: A Pilot Study.

The method of administering caffeine as a probe to evaluate the phenotypic activity of the CYP1A2, has not yet been applied clinically. In contrast, if endogenous melatonin (MEL) metabolism can be used to assess CYP1A2 activity, it could be a simple method that does not require substance administration. The study aim was to calculate the MEL partial metabolic clearance (CLm(MEL)) from plasma MEL and its urinary metabolites and to test the potential of this approach as a novel CYP1A2 phenotyping method. Nine subjects were included in the study; 3 had 6 blood and 4 urine samples collected between 10:00 and 18:00 (collectively, the intraday sample). Nine subjects had 3 blood samples and 2-h urine samples collected between 10:00 and 12:00 once a week for 3 weeks (interday sample). The CLm(MEL) was calculated from the plasma area under the curve (AUC) of MEL (AUCMEL) and urinary MEL metabolites excretion (X6MEL). Among the intraday samples, the AUCMEL ranged from 6.45-13.17 pmol·h/L and X6MEL ranged from 0.204-0.899 nmol/2 h, showing a decrease in concentration over time. In contrast, the CLm(MEL) ranged from 30.52-69.57 L/h (within-individual percent relative standard deviation: 9.2-20.1%), showing no time-dependent variation. Large interindividual variability was observed in AUCMEL and X6MEL in the interday sample, but CLm(MEL) showed small interindividual variabilities. The CLm(MEL) was 1.8-fold higher for smokers than for nonsmokers. The results obtained in this study may be valuable in future studies of evaluating novel CYP1A2 phenotyping method.

Development and Validation of an LC-MS/MS-Based Method for Quantifying Urinary Endogenous 6-Hydroxymelatonin.

Evaluation of endogenous melatonin (MEL) secretion using its urinary metabolites is useful for the treatment of circadian rhythm sleep disorders. The primary melatonin metabolites excreted in the urine are 6-hydroxymelatonin (6-O-MEL) sulfate (S-O-MEL) and 6-O-MEL glucuronate, which result from sequential MEL metabolism by phases I and II drug metabolizing enzymes. To determine the accurate MEL secretion level, these urinary metabolites should be enzymatically deconjugated and converted into MEL. Furthermore, the use of LC-tandem mass spectrometry (LC-MS/MS) is preferable for the precision of this determination. Therefore, as part of our ongoing efforts to ultimately determine the level of MEL secretion, we herein aimed to develop an LC-MS/MS-based quantification method for 6-O-MEL and optimize deconjugation conditions. We determined the LC-MS/MS conditions of 6-O-MEL measurement and optimized the conditions of enzymatic reactions. The most efficient S-O-MEL deconjugation (102.1%) was achieved with Roche Glucuronidase/Arylsulfatase (from Helix pomatia) at 37 °C, pH-4.0 reaction buffer, and 60 min of reaction time. For human urine samples, the minimum amount of the enzyme required was 5944 units. Under these conditions, the accuracy and precision values of the 6-O-MEL determination (relative errors and standard deviation) were -3.60--0.47% and <6.80%, respectively. Finally, we analyzed the total amount of MEL metabolites excreted in 24-h urine samples; it was 6.70-11.28 µg in three subjects, which is comparable with the values reported till date. Thus, we have established a new method of measuring the total 6-O-MEL in human urine samples using an LC-MS/MS coupled with the prerequisite deconjugation reaction.

Basic study on improvement of plastic drape adhesion for surgery: effect of adhesive layer thickness on drape detachment from wound edge.

BACKGROUND: Plastic drapes are used in surgery for a wide range of purposes, but currently marketed drapes often become detached from the wound edge during surgery. The purpose of this study was to determine the appropriate adhesive layer thickness for optimal peel and shear strength and the smallest peeled area to improve surgical drape wound adhesion. METHODS: Thirty-two rats were randomly assigned to four groups of different adhesive layer thickness (50, 100, 300 and 800-1000 µm). The rats were anaesthetized, and drapes were applied to the dorsal chest. After incision, the peeled area was visualized by dropping ink in the wound site to measure the peeled area over time. RESULTS: All drapes peeled off from the wound edge, and the peeled area increased over time. The peeled area decreased in the order of 50 µm > 100 µm > 800-1000 µm > 300 µm. CONCLUSIONS: It is possible to control the peeling of plastic drapes during surgery by limiting the peeled area at the time of cutting. Three-hundred micrometres is the suitable adhesive layer thickness to minimize the peeled area at cutting.

Intrathoracic retention of insoluble hyaluronic acid and its absorption process in rats.

BACKGROUND: Post-thoracotomy adhesions are frequent postoperative complications. It has been reported that insoluble hyaluronic acid may prevent adhesions. MATERIALS AND METHODS: This study had two objectives: first, to determine the in vivo degradation and absorption process, as well as the intrathoracic retention, of solid insoluble hyaluronic acid membrane; and second, to elucidate the association between postoperative intrathoracic retention and the morphological changes of insoluble hyaluronic acid in 12 Wistar rats. Insoluble hyaluronic acid membranes were cut into 2.0 cm × 1.0 cm rectangles in a dry state. After weighing, the test membranes were soaked and washed with saline to be implanted after pericardiotomy via thoracotomy. At Days 4, 7, 10, 14, and 28 after implantation, the rats were euthanized, the chest was opened, and the condition and implantation site of the inserted test membrane were examined. RESULTS: Although approximately 10 days were required for the test membrane to decrease to half in the thoracic cavity, the intrathoracic remnant decreased to a mean of ~2% just 4 days later. CONCLUSION: This study clarified the time-dependent degradation process and remnants of insoluble hyaluronic acid in the thoracic cavity. A close relationship between the intrathoracic remnant of insoluble hyaluronic acid and its morphological change associated with degradation was demonstrated.

Elucidation of the time-dependent degradation process in insoluble hyaluronic acid formulations with a controlled degradation rate.

Degradation rate of hyaluronic acid to prolong its stability in vivo would be beneficial. We investigated a potential solution for prolonging the stability of hyaluronic acid within the body. We focused on decreasing the swelling ratio to slow the degradation rate of hyaluronic acid by insolubilizing sodium hyaluronate without using potentially harmful substances such as crosslinkers or modifiers. Hyaluronic acid formulations were created with three different swelling ratios and time-dependent morphological changes in hyaluronic acid formulations and were scored based on each swelling ratio. In vivo degradation was modeled in simulated body fluid and the extent of decay of test membranes were monitored over time. Results showed that, by adjusting the swelling ratio, the degradation rate of hyaluronic acid formulation could be controlled. Our research could lead to improvements in many products, not only preventive materials for postoperative adhesions, but also pharmaceutical products such as osteoarthritis treatments and cosmetic medicines.

Assessment of the subcutaneous degradation process of insoluble hyaluronic acid in rats.

Insoluble hyaluronic acid (IHA) may prevent adhesions by forming a physical barrier during the period when postoperative adhesions form. This study was performed to verify the changes that a solid IHA membrane undergoes as it is degraded in vivo, and to ascertain the swelling rate of IHA required for it to function as a physical barrier during the postoperative adhesion formation period. Nine female WI rats weighing 300-400 g were used. Discs 8 mm in diameter were cut out of dry IHA membranes made of IHA with a swelling rate (wet weight/dry weight) of either 2.47 (high-swelling IHA) or 1.94 (low-swelling IHA). They were placed in saline to swell and then washed with saline before subcutaneous implantation in four pockets in each rat. The high-swelling IHA started to degrade more rapidly than the low-swelling IHA. There was no evidence of degradation of the low-swelling IHA until day 7, but once it had started, the speed of degradation tended to be similar to that of the high-swelling IHA. The present results showed that, when IHA is implanted subcutaneously in rats, it is degraded over time in a phased process. The swelling rate required for the use of IHA as a postoperative adhesion barrier was also suggested.

Screening for long-lived genes identifies Oga1, a guanine-quadruplex associated protein that affects the chronological lifespan of the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe and Saccharomyces cerevisiae are excellent model organisms to study lifespan. We conducted screening to identify novel genes that, when overexpressed, extended the chronological lifespan of fission yeast. We identified seven genes, among which we focused on SPBC16A3.08c. The gene product showed similarity to Ylr150w of S. cerevisiae, which has affinity for guanine-quadruplex nucleic acids (G4). The SPBC16A3.08c product associated with G4 in vitro and complemented the phenotype of an S. cerevisiae Ylr150w deletion mutant. From these results, we proposed that SPBC16A3.08c encoded for a functional homolog of Ylr150w, which we designated ortholog of G4-associated protein (oga1 (+)). oga1 (+) overexpression extended the chronological lifespan and also decreased mating efficiency and caused both high and low temperature-sensitive growth. Deleting oga1 (+) resulted in caffeine-sensitive and canavanine-resistant phenotypes. Based on these results, we discuss the function of Oga1 on the chronological lifespan of fission yeast.

Influence of systemic administration of atelocollagen on mouse livers: an ideal biomaterial for systemic drug delivery.

Atelocollagen (AC), a biomaterial with low antigenicity and high bioaffinity, has been widely used in implantable materials in clinical practice. Preclinical studies have demonstrated that AC is a potential drug carrier for local and systemic delivery of cytokines, growth factors, plasmid DNA, small interfering RNA, and microRNA. AC is also believed to have low systemic toxicity on the basis of the safety of implant usage; however, this is not enough determined. Therefore, we performed whole genome expression profiling in mouse liver after systemic administration of AC or the cationic liposome carrier DOTAP/cholesterol (LP) and compared the changes of gene expressions associated with hepatotoxicity. Microarray analysis revealed that systemic LP administration significantly increased expression of toxicity-related genes, i.e., those for lipocalin-2, cyclin-dependent kinase inhibitor 1A, serum amyloid A isoforms, chemokine ligands, and granzyme B. Alternatively, AC administration did not alter the expression of any of these genes. Further gene ontology (GO) enrichment analysis highlighted the characteristic annotations extracted from genes upregulated after LP administration, and most of them were related to toxicity annotations such as immune response, inflammatory response, and apoptosis induction. In contrast, GO enrichment analysis of genes induced after AC administration revealed that only three annotations, all of which were unrelated to toxicity. These findings indicate that AC is potentially far less hepatotoxic than LP after systemic administration, suggesting that AC may be an excellent biomaterial for nontoxic drug delivery system carriers.

Insight into unusual impurity absorbability of GeO(2) in GeO(2)∕Ge stacks.

Adsorbed species and its diffusion behaviors in GeO(2)∕Ge stacks, which are future alternative metal-oxide-semiconductor (MOS) materials, have been investigated using various physical analyses. We clarified that GeO(2) rapidly absorbs moisture in air just after its exposure. After the absorbed moisture in GeO(2) reaches a certain limit, the GeO(2) starts to absorb some organic molecules, which is accompanied by a structural change in GeO(2) to form a partial carbonate or hydroxide. We also found that the hydrogen distribution in GeO(2) shows intrinsic characteristics, indicative of different diffusion behaviors at the surface and at the GeO(2)∕Ge interface. Because the impurity absorbability of GeO(2) has a great influence on the electrical properties in Ge-MOS devices, these results provide valuable information in realizing high quality GeO(2)∕Ge stacks for the actual use of Ge-MOS technologies.

Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.

In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons. Two transgenic lines, L- and H-, were generated that carried cDNAs encoding the L- and H-chains of a mouse IgG mAb, respectively, under the control of the enhancer-linked sericin-1 promoter. Cocoon protein analysis indicated that the IgG L- or H-chain was secreted into the cocoons of each line. We also produced a transgenic line designated L/H, which carried both cDNAs, by crossing the L- and H-lines. This line efficiently produced the recombinant mAb as a fully assembled H(2)L(2) tetramer in its cocoons, with negligible L- or H-chain monomer and H-chain dimer production. Thus, the H(2)L(2) tetramer was synthesized in, and secreted from, the middle silk gland cells. Crossing of the L/H-line with a transgenic line expressing a baculovirus-derived trans-activator produced a 2.4-fold increase in mAb expression. The recombinant mAb was extracted from the cocoons with a buffer containing 3 m urea and purified by protein G affinity column chromatography. The antigen-binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma. Analysis of the structure of the N-glycans attached to the recombinant mAb revealed that the mAb contained high mannose-, hybrid- and complex-type N-glycans. By contrast, insect-specific paucimannose-type glycans were not detected. Fucose residues alpha-1,3- and alpha-1,6-linked to the core N-acetylglucosamine residue, both of which are found in insect N-glycans, were not observed in the N-glycans of the mAb.

Structure and function of 5'-flanking regions of Bombyx mori fibroin heavy chain gene: identification of a novel transcription enhancing element with a homeodomain protein-binding motif.

We studied the promoter activity of a 5'-flanking region from -5000 to +24 (-5000/+24) in Bombyx mori fibroin heavy chain gene (fibH), fibH(-5000/+24). A luciferase reporter vector carrying fibH(-5000/+24) was bombarded to isolated posterior silk glands (PSGs). The PSGs showed a high luciferase activity when transplanted to larvae, indicating its potent promoter activity. Deletion experiments showed the requirement of fibH(-5000/-3844) and fibH(-2211/-542) for the promoter activity. These two regions and fibH(-541/+24) that contained the basal promoter were tandem fused to yield fibH(-5000/-3844:-2211/-542:-541/+24), which was found to retain 88% of the activity of fibH(-5000/+24). Germline transgenic silkworms bearing fibH(-5000/-3844:-2211/-542:-541/+24) as a promoter and enhanced green fluorescent protein (EGFP) gene as a reporter efficiently secreted EGFP in cocoons. The promoter activity of fibH(-2211/-542) was further investigated, because this contained a DNase I-hypersensitive site. The transient expression assay demonstrated that the activity of fibH(-2211/-542) required fibH(-1659/-1590), which contained the homeodomain protein-binding motif. Mutation experiments suggested a critical role of the motif for the promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that a nuclear protein of PSGs bound to the motif. We propose fibH(-1659/-1590) as a novel transcription enhancer that plays a key role for the expression by recruiting a homeodomain protein.

A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.

A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system. The results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.

Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.

In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.

Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues.

Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated.

Production of L-lactic acid from fresh cassava roots slurried with tofu liquid waste by Streptococcus bovis.

To reduce the production cost of biodegradable plastics, the fermentation performance of L-lactic acid for a new fermentation medium, fresh cassava roots (FCRs) as a substrate slurried with tofu liquid waste (TLW) as basal medium, was investigated by batch fermentation of Streptococcus bovis. The fermentation properties of the three substrates, namely, FCR, tapioca (cassava starch) and glucose, which were independently mixed with TLW, were compared with those independently mixed with the standard basal medium, trypto-soya broth (TSB). Experiments were conducted at various sugar concentrations of the substrates with CaCO(3) as a neutralizer. The maximum L-lactic acid concentrations (C(La)) obtained using the three substrates in TLW were about 75% of those obtained using TSB caused by less nutrients in the TLW. The L-lactic acid productivities (P(La)) and the specific growth rates of S. bovis (mu) in TLW were about 1/4 to 1/3 and 1/5 to 1/4 of those in TSB, respectively. The maximum C(La), P(La) and mu were obtained at 10% w/w sugar concentration. Total yields (eta) were nearly constant up to 10% w/w sugar concentration for TSB and TLW, that is, 80% to 85% and 50% to 60%, respectively. But their total yields decreased in more than 10% w/w sugar concentration in both basal media, because of substrate inhibition. The fermentation properties (C(La), P(La), mu, and eta) were found to be in the order of: FCR > tapioca > glucose for all concentrations of the three substrates. The fermentation properties for FCR and tapioca were higher than those for glucose, in TLW or TSB, because S. bovis in a medium containing starch (FCR and tapioca) has more amylase activity than in a medium containing glucose. The nutrients in FCR with poor nutrient basal medium (TLW) more strongly affected the fermentation properties than those in FCR with rich nutrient basal medium (TSB). The proposed fermentation medium of FCR slurried with TLW is worth studying in order to reduce production cost of biodegradable plastics.