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Objectives To investigate long-term outcomes of ultrasound-guided intrauterine device (IUD) insertion. The rate of and reasons for IUD discontinuation were reviewed, including the prevalence of uterine fibroids. Methods A retrospective cohort of patients who underwent transabdominal ultrasound (TAUS)-guided IUD insertion was reviewed. Information collected included age, body mass index, insertion date, indication for IUD use, indication for using TAUS guidance, and presence of uterine fibroids. The date of and reason for discontinuation were recorded. Results One hundred sixteen patients with successful TAUS-guided IUD placement were identified. Of these, 51 patients (43.97%) no longer had an IUD in place. An overall actual-to-expected use ratio was calculated to be 63.49%. The most common reason for IUD discontinuation was spontaneous expulsion (11.21%). The prevalence of fibroid uteri was 27.6% in our cohort. The actual-to-expected use ratio of those with a fibroid uterus was calculated to be 43.28%, which was significantly lower than that for a nonfibroid uterus (73.80%; P = .002). There were 27 of 104 patients with IUD use of less than 1 year, and fibroids were present 44.4% of the time. Comparatively, of the 77 patients that had IUD continuation of greater than 1 year, only 24.7% had fibroids (P = .022). The yearly continuation rates over 5 years were 74.04%, 55.84%, 41.67%, 35.14%, and 32.0% respectively. Of the 18 patients who received TAUS-guided insertion for a previous IUD expulsion, 33.3% had another spontaneous expulsion. Conclusions Uterine fibroids and a previous expulsion appear to be the most likely predictors of IUD discontinuation, particularly within 1 year after insertion.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS−/− CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10–19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500‐L and 1000‐L bioreactors replicated laboratory results using 5‐L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic’s efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Objective As life expectancy increases, the number of women reporting adverse genito-urinary symptoms (genitourinary syndrome of menopause; GSM) from menopause, including vaginal dryness and sexual pain, also will increase. Current objective measurements of vaginal atrophy such as maturation index require vaginal swabs and are invasive; at present, no minimally invasive measurements exist. The purpose of this study was to assess whether total vaginal wall thickness (TVT) and total vaginal mucosa thickness (TMT) as measured by transabdominal ultrasound could qualify as additional objective markers of vaginal wall thinning which could be related to menopausal status. Design Women presenting for pelvic ultrasound had a transabdominal ultrasound scan performed to measure TVT and TMT at the level of the bladder trigone. In addition, a transvaginal endometrial lining thickness was measured. Results The ultrasound measurement data from 76 participants showed that there was a significant difference in the mean value for TVT and endometrial lining between pre- and post-menopausal women. The same difference in mean was not observed for TMT. Conclusion TVT may be a reliable measure of vaginal thinning, which worsens with estrogen decline. These preliminary data also suggest that TMT does not have the same correlation as the TVT measurement. A larger sample is needed to further assess the usefulness and sensitivity of these measures and whether there is clinical and/or research usefulness in obtaining vaginal wall measurements by transabdominal ultrasound.

Transabdominal and transvaginal sonography are used to measure bladder wall and detrusor thickness. Only transvaginal sonography has been used to measure the vaginal wall thickness. We describe the use of transabdominal sonography to measure the total vaginal wall thickness and total vaginal mucosal thickness at the bladder trigone. The mean bladder wall thickness and SD from published data were within the 95% confidence interval of our data. Total vaginal and mucosal thicknesses are reliable measurements, which require specific evaluation in a postmenopausal population. They could be used to quantify vaginal atrophy and could correlate to symptoms of atrophy and response to treatment. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 45:461-464, 2017.

Introduction It has previously been suggested in the literature that ultrasound measurement of total vaginal wall thickness (TVT) differs significantly between pre- and postmenopausal women, indicating that it may be a useful and noninvasive objective assessment to correlate the degree of vaginal atrophy to patient-reported symptoms. Aim The purpose of this cross-sectional pilot study was to determine whether TVT in postmenopausal women, as measured with transabdominal ultrasound, is associated with patient-reported dyspareunia and symptoms related to genitourinary symptomatology. Methods Postmenopausal women presenting for pelvic ultrasound had TVT and total mucosal thickness (TMT) measured via transabdominal ultrasound. A questionnaire also was administered assessing menopausal status, relevant medical history, and self-report of dyspareunia and other symptoms related to the genitourinary syndrome of menopause (GSM). This questionnaire was derived from the Vulvovaginal Symptom Questionnaire, which has been validated in the literature. Main Outcome Measure The main outcome measures included the average TVT and TMT for postmenopausal women reporting any symptom of GSM and average TVT and TMT of women reporting no symptoms of GSM. Results Data from 44 postmenopausal women showed no significant association between transabdominal ultrasound-measured TVT or TMT and patient report of dyspareunia or other genitourinary symptoms. Data were stratified by individual GSM symptoms, sexual symptoms as an aggregate, and individual sexual symptoms. Neither of these subgroups showed a statistically significant difference in TVT or TMT between symptomatic and asymptomatic women. Clinical Implications Although no statistically significant data were derived from this study, we propose that future studies investigating the longitudinal relationship between TVT and GSM symptomatology may show an association between total vaginal thickness measurement change over time as determined by ultrasound with the presence of patient-reported dyspareunia and other GSM symptoms. Strengths & Limitations This study is limited by its small sample size as well as the patient population, which was restricted to postmenopausal women with a clinical indication for ultrasound. A major strength of this investigation is that it is the first study to look at the relationship between sexual pain and other GSM symptoms and TVT using transabdominal ultrasound, which is a readily available, non-invasive tool in most clinical settings. Conclusion Based on the results of this small pilot study, transabdominal pelvic ultrasound cannot be used at this time to objectively quantify the presence of sexual pain or other GSM symptoms; however, future studies should continue to investigate the longitudinal relationship between these 2 variables.

Children who are educated using a Waldorf Steiner approach demonstrate superior expressive drawing skills (Rose et al., 2011) but little is known about how art is taught within this educational system. Four Waldorf Steiner primary school teachers participated in semi-structured interviews designed to explore the Waldorf Steiner educational philosophy, their training and the ways in which they approach art in the classroom. A social constructionist thematic analysis identified two themes – teacher's experience of art and the teacher and child's approach to art. Within these themes the importance of adequate training which stresses the value of art and gives teachers opportunity to engage in art activities was emphasised. Such training was linked to an effective teaching approach which placed importance on teaching skills and encouraging children to develop their understanding of art through discussion.

Objectives The purpose of this study was to identify clinical indications in which endometrial sampling was performed under transabdominal ultrasound (US) guidance and to evaluate the outcomes of this approach in an academic practice. Methods We retrieved data from the electronic medical record for all patients referred to the gynecologic US unit for transabdominal US-guided endometrial sampling from January 2011 to June 2017. Data retrieved included age, parity, previous cesarean delivery or pelvic surgery, indication for endometrial biopsy, US abnormalities, clinical reasoning for US-guided sampling, and pathologic reports. Results A total of 113 patients were referred for US-guided sampling between January 2011 and June 2017. We identified the following reasons for US-guided biopsy referral: failed blind biopsy attempt, fibroids, uterine position or anomaly, need for targeted sampling, cervical stenosis, and other indications. Ninety-five of the 113 patients (84%) underwent successful US-guided endometrial sampling. Forty of the 113 patients were referred after a failed blind biopsy, with 83% subsequently undergoing successful US-guided endometrial sampling. Of the 30 patients referred for fibroids, 29 (97%) underwent successful US-guided sampling. High success rates were also noted for transabdominal US guidance referrals for the uterine position or anomaly (86%) and the need for targeted sampling (83%). Conclusions Our results suggest that endometrial sampling performed under US guidance could be considered for patients with a failed blind approach, fibroids, uterine anomalies, and interest in targeted sampling. In such cases, US-guided sampling could be considered before surgical options.

From 2011 to 2015, a total of 67 patients were referred for IUD insertion guided with transabdominal sonography (TAS). Fifty-six of the 67 patients had successful IUD insertion under TAS guidance. The clinical indications for referral included fibroids, uterine position, previous history of IUD expulsion, and limited tolerance of pelvic examination. Reasons for failed TAS-guided IUD insertion included patient discomfort, cervical stenosis, and inability to remove and replace an existing device. Ultrasound guidance could help broaden the patient population that may benefit from the therapeutic value of an IUD.

Transabdominal and transvaginal sonography are used to measure bladder wall and detrusor thickness. Only transvaginal sonography has been used to measure the vaginal wall thickness. We describe the use of transabdominal sonography to measure the total vaginal wall thickness and total vaginal mucosal thickness at the bladder trigone. The mean bladder wall thickness and SD from published data were within the 95% confidence interval of our data. Total vaginal and mucosal thicknesses are reliable measurements, which require specific evaluation in a postmenopausal population. They could be used to quantify vaginal atrophy and could correlate to symptoms of atrophy and response to treatment. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 45:461-464, 2017.

The presence of aggregated forms of proteins can be problematic for therapeutics due to the potential for immunogenic and pharmacokinetic issues. Although downstream processing can remove the aggregated forms, inhibiting aggregate formation upstream during the cell culture stage could reduce the burden on downstream processing and potentially improve process yields. This study first examined the effects of environmental factors (temperature, pH, and dissolved oxygen) and medium components (bivalent copper ion, cysteine, and cystine) on the aggregation of two different recombinant fusion proteins expressed by Chinese hamster ovary (CHO) cells. Any strategy to reduce protein aggregation upstream during cell culture must also consider potential effects on critical upstream parameters such as cell growth, harvest titer, and protein sialylation levels. Manipulating the culture temperature shift and cystine concentration in the medium were both identified as effective and practical strategies for reducing protein aggregation in both CHO-cell expression systems. Furthermore, a combination of both strategies was more effective in reducing protein aggregation levels compared to either approach individually; and without any negative effects on harvest titer and protein sialylation. This study demonstrates a practical methodology for decreasing protein aggregation during upstream processing and emphasizes the importance of process understanding to ensure production of recombinant glycoprotein therapeutics with consistent product quality.

Dextran sulfate has been reported to reduce cell aggregation and increase culture viability in suspension cultures of CHO cells. However, only a few of these reports have focused on the effects of dextran sulfate at the cellular level, especially with respect to cell viability. The goal of this study was to examine whether dextran sulfate inhibits CHO cell death through interference with the cellular apoptotic pathway. Apoptosis was induced using staurosporine, and the addition of dextran sulfate was shown to prevent apoptosis, as evident by a reduction in DNA laddering. Dextran sulfate also affected other events in the apoptotic pathway following the addition of staurosporine. Specifically, dextran sulfate decreased pro-caspase-9 activation, cleaved caspase-3 levels, cytochrome c release into the cytosol and mitochondrial transmembrane potential collapse. This data indicates that the mechanism by which dextran sulfate decreases staurosporine-induced apoptosis involves the mitochondrial pathway as early as cytochrome c release, but without caspase-8 activation. This study advances the understanding of the anti-apoptotic action of dextran sulfate on CHO cells and provides a rationale for the application of dextran sulfate in CHO cell culture processes.

The objective of this study was to determine the effects of removal of amniotic fluid in cases of symptomatic severe polyhydramnios on Doppler waveform indices of the uterine and umbilical arteries and flow velocities of the uterine arteries. Nine women underwent therapeutic amniocentesis during ten pregnancies for symptomatic polyhydramnios due to Beckwith-Wiedemann Syndrome (n = 1), esophageal atresia (n = 2), chorioangioma (n = 1), twin-twin transfusion syndrome (n = 3), a presumed autosomal recessive syndrome (n = 2), and an unbalanced double translocation (n = 1; partial dup 3q and partial del 9p syndrome). An average of 2.78 +/- 0.9 (range 1-4) 1 of fluid were removed at each procedure between the gestational ages of 18 and 34 weeks (mean of 28 weeks). The systolic/diastolic (S/D) ratio, pulsitility index (PI), and resistance index (RI) of the uterine and umbilical arteries were obtained before and after the procedure using color and pulsed Doppler. After angle correction, the peak systolic velocity (PSV) and mean velocity (MV) in centimeters/second (cm/s) of the uterine arteries were also determined. The presence or absence of a uterine artery waveform notch was determined. Dominant uterine arteries were defined as those with lower impedance indices or higher flow velocities. Statistical analysis was performed with the Wilcoxon signed-rank test. Significance was set at P0.05. There was a significant increase in the median value of the uterine artery MV (43.8 vs. 81.1 cm/s, P = 0.005) and PSV (74.2 vs. 125.5 cm/s, P = 0.007) after amniocentesis. The uterine S/D (3.0 vs. 1.84, P = 0.007), PI (1.12 vs. 0.68, P = 0.008), and RI (0.60 vs. 0.45, P = 0.005) impedance indices significantly decreased following amniocentesis. When uterine arteries were categorized as dominant vs. nondominant, there were greater improvements in impedance indices and flow velocities in the nondominant uterine arteries. There were three cases of unilateral and one case of bilateral early diastolic notches of the uterine artery waveforms which either resolved (n = 4) or improved (n = 1). There was no effect on the umbilical artery impedance indices. Therapeutic amniocentesis significantly improved uterine artery impedance indices and resulted in improved flow velocities, while there was no effect on umbilical artery waveform indices. The procedure resulted in the disappearance or improvement of the uterine waveform notch. Our findings suggest that in cases of severe polyhydramnios abnormal uterine artery velocimetry may not be due to lack of trophoblastic invasion of the spiral arteries but to increased intrauterine pressure secondary to polyhydramnios.

The coordination between cell proliferation and terminal differentiation is of critical importance during the development of multicellular organisms. The metazoan SWI/SNF-related protein complexes function in both of these processes in a broad range of tissues by altering gene expression at specific promoters through ATP-dependent chromatin remodeling (32). The Saccharomyces cerevisiae SWI/SNF complex is a ∼2-MDa multisubunit assembly capable of ATP-dependent chromatin remodeling both in vitro and in vivo (53). Highly related complexes have also been identified in flies (23, 48) and mammals (73). Among the SWI/SNF complex proteins, none appears to be capable of sequence-specific DNA binding; thus, the precise mechanism for targeting the SWI/SNF complexes to their respective gene targets in vivo is not well understood. The complex can interact with transcription factors to facilitate SWI/SNF function at specific promoters (53). Interactions have been observed with steroid hormone receptors; EKLF (erythroid Kruppel-like factor), which activates the β-globin locus; C/EBPβ, which activates transcription of myeloid genes (35); c-Myc (13, 65); and HRX (56), a homolog of Drosophila melanogaster Trithorax (TRX) that regulates expression of the homeotic genes. Depending on the chromatin context of the binding site, some transcription factors may initially bind to their recognition site and then recruit SWI/SNF to alleviate chromatin repression at the promoter, or conversely, the factor may require SWI/SNF activity for binding. While there is presently very little known about what types of protein interactions might promote such recruitment, several complex subunits may function coordinately to mediate these associations (46). Further, mounting evidence suggests that the various SWI/SNF and related complexes might function in both gene activation and repression (22, 64). Studies of the fly Brm and mammalian hBrm/Brg1 complexes have revealed widespread and important biological roles in development (45, 66). In addition to transcriptional activation, the complex functions in regulating cell cycle progression through interactions with the retinoblastoma (Rb) protein and/or cyclin-cdk complexes (7). The purified Brm complex contains at least eight proteins, including BRM, MOR, SNR1, and OSA (15, 48). While brm, mor, and osa were identified in genetic screens as dosage-dependent activators of homeotic gene transcription, counteracting the repressive effects of the Polycomb group of genes (31), none of the other Brm complex genes was identified in any genetic screen. The snr1 gene (SNF5-Related-1) was identified (23) by molecular means based on its similarity to the human INI1/SMARCB1/hSNF5 gene, which encodes a core subunit of the hBrm and Brg1 complexes. Both snr1 and INI1 are strongly conserved with SNF5, which encodes an essential component of the yeast SWI/SNF complex, and SNR1, INI1, and SNF5 are required for full function of their respective complexes in vivo and in vitro (23, 38, 39, 55). While brm, mor, and osa have dosage-sensitive mutant phenotypes in combination with other gene mutations that can be examined in the imaginal tissues, such as leg and wing disks (8, 9, 24, 67), the existing snr1 null mutations do not display significant dosage sensitivity in most genetic assays, apparently inconsistent with SNR1 serving as a core subunit of the Brm complex and making analysis of this essential gene very difficult. Thus, the snr1 gene product is either in excess of that required to assemble the Brm complex, has unidentified functions independent of the complex, or has a limited role at a subset of target genes and thus is dispensable for some Brm complex functions. To help distinguish among these possibilities, we carried out a large-scale noncomplementation chemical mutagenesis screen to identify conditional mutant alleles of snr1 (77), and we report here the molecular and genetic characterization of snr1E1, which exhibits recessive lethality at 29°C and a variety of temperature-dependent phenotypes. Our genetic and biochemical characterizations of snr1E1 reveal that snr1 has essential functions in the development of many tissues including the peripheral nervous system, eye, and wing veins, and surprisingly, it is also required for sustained adult viability. Further, our analyses provide strong links among snr1 function, chromatin remodeling, and the regulation of cell proliferation, confirming its predicted role in restricting cell growth during normal development.