Hormonal stimulation reduces numbers and impairs function of human uterine natural killer cells during implantation.

STUDY QUESTION: How does an altered maternal hormonal environment, such as that seen during superovulation with gonadotropins in ART, impact human uterine immune cell distribution and function during the window of implantation? SUMMARY ANSWER: Hormonal stimulation with gonadotropins alters abundance of maternal immune cells including uterine natural killer (uNK) cells and reduces uNK cell ability to promote extravillous trophoblast (EVT) invasion. WHAT IS KNOWN ALREADY: An altered maternal hormonal environment, seen following ART, can lead to increased risk for adverse perinatal outcomes associated with disordered placentation. Maternal immune cells play an essential role in invasion of EVTs, a process required for proper establishment of the placenta, and adverse perinatal outcomes have been associated with altered immune cell populations. How ART impacts maternal immune cells and whether this can in turn affect implantation and placentation in humans remain unknown. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out between 2018 and 2021 on 51 subjects: 20 from natural cycles 8 days after LH surge; and 31 from stimulated IVF cycles 7 days after egg retrieval. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial biopsies and peripheral blood samples were collected during the window of implantation in subjects with regular menstrual cycles or undergoing superovulation. Serum estradiol and progesterone levels were measured by chemiluminescent competitive immunoassay. Immune cell populations in blood and endometrium were analyzed using flow cytometry. uNK cells were purified using fluorescence-activated cell sorting and were subjected to RNA sequencing (RNA-seq). Functional changes in uNK cells due to hormonal stimulation were evaluated using the implantation-on-a-chip (IOC) device, a novel bioengineered platform using human primary cells that mimics early processes that occur during pregnancy in a physiologically relevant manner. Unpaired t-tests, one-way ANOVA, and pairwise multiple comparison tests were used to statistically evaluate differences. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were comparable for both groups. As expected, serum estradiol levels on the day of biopsy were significantly higher in stimulated (superovulated) patients (P = 0.0005). In the setting of superovulation, we found an endometrium-specific reduction in the density of bulk CD56+ uNK cells (P < 0.05), as well as in the uNK3 subpopulation (P = 0.025) specifically (CD103+ NK cells). In stimulated samples, we also found that the proportion of endometrial B cells was increased (P < 0.0001). Our findings were specific to the endometrium and not seen in peripheral blood. On the IOC device, uNK cells from naturally cycling secretory endometrium promote EVT invasion (P = 0.03). However, uNK cells from hormonally stimulated endometrium were unable to significantly promote EVT invasion, as measured by area of invasion, depth of invasion, and number of invaded EVTs by area. Bulk RNA-seq of sorted uNK cells from stimulated and unstimulated endometrium revealed changes in signaling pathways associated with immune cell trafficking/movement and inflammation. LIMITATIONS, REASONS FOR CAUTION: Patient numbers utilized for the study were low but were enough to identify significant overall population differences in select immune cell types. With additional power and deeper immune phenotyping, we may detect additional differences in immune cell composition of blood and endometrium in the setting of hormonal stimulation. Flow cytometry was performed on targeted immune cell populations that have shown involvement in early pregnancy. A more unbiased approach might identify changes in novel maternal immune cells not investigated in this study. We performed RNA-seq only on uNK cells, which demonstrated differences in gene expression. Ovarian stimulation may also impact gene expression and function of other subsets of immune cells, as well as other cell types within the endometrium. Finally, the IOC device, while a major improvement over existing in vitro methods to study early pregnancy, does not include all possible maternal cells present during early pregnancy, which could impact functional effects seen. Immune cells other than uNK cells may impact invasion of EVTs in vitro and in vivo, though these remain to be tested. WIDER IMPLICATIONS OF THE FINDINGS: These findings demonstrate that hormonal stimulation affects the distribution of uNK cells during the implantation window and reduces the proinvasive effects of uNK cells during early pregnancy. Our results provide a potential mechanism by which fresh IVF cycles may increase risk of disorders of placentation, previously linked to adverse perinatal outcomes. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the University of Pennsylvania University Research Funding (to M.M.), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P50HD068157 to M.M., S.S., and S.M.), National Center for Advancing Translational Sciences of the National Institutes of Health (TL1TR001880 to J.K.), the Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania, the Children's Hospital of Philadelphia Research Institute (to S.M.G.), and the National Institute of Allergy and Infectious Diseases (K08AI151265 to S.M.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

A randomized comparison trial of culturally adapted HIV prevention approaches for Native Americans reducing trauma symptoms versus substance misuse: The Healing Seasons protocol.

Native Americans (NA) experience interrelated risks of trauma exposure, substance use, and HIV risk behaviors that put them at increased risk for HIV infection. Despite these known risk factors, there are very few published randomized trials testing interventions to reduce trauma-related symptoms and substance misuse among NA. METHODS: The Healing Seasons study is a randomized comparsion trial of two counseling strategies, Narrative Exposure Therapy (NET) addressing PTSD or Motivational interviewing with cognitive behavioral therapy skills training (MIST) addressing substance misuse as a means to prevent HIV among NA. Using a community-based participatory research approach, we adapted both evidence-based interventions to be specific to the risk contexts and realities of NA and to include psychoeducational and skill-building components that include cultural-specific stories, virtues, and traditional treatment strategies. Participants, 16 years and older, were recruited from a Pacific Northwest tribal community, screened over the phone, enrolled in person, and randomized in equal numbers to NET or MIST. We stratified by age (16-29 years and 30 or older) and gender (male or female identified) to ensure balance between treatment arms. The primary outcomes were number of sex partners and frequency of sexual acts (with and without condoms), sex under the influence of substances, frequency of substance use, and PTSD severity. DISCUSSION: Behavioral interventions for NA are needed to prevent HIV risk behaviors when faced with trauma symptoms and substance misuse. This study will provide evidence to determine feasibility and efficacy of addressing related risk factors as part of counseling-based HIV prevention intervention to reduce sexual risk among this population. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT03112369, registered April 12, 2017.

Repetitive Transcranial Magnetic Stimulation of the Primary Somatosensory Cortex Modulates Perception of the Tendon Vibration Illusion.

The effect of repetitive transcranial magnetic stimulation on kinesthetic perception, when applied to the somatosensory cortex, was examined. Further, the facilitatory and inhibitory effects of repetitive transcranial magnetic stimulation using different stimulation frequencies were tested. Six female (M age = 32.0 years, SD = 6.7) and nine male (M age = 32.9 years, SD = 6.6) participants were asked to perceive the tendon vibration illusion of the left wrist joint and to replicate the illusion with their right hand. When comparing changes in the corresponding movement amplitude and velocity after three different repetitive transcranial magnetic stimulation protocols (sham, 1 Hz inhibitory, and 5 Hz facilitatory repetitive transcranial magnetic stimulation), the movement amplitude was found to decrease with the inhibitory repetitive transcranial magnetic stimulation, while the movement velocity respectively increased and decreased with the facilitatory and inhibitory repetitive transcranial magnetic stimulation. These results confirmed the modulating effects of repetitive transcranial magnetic stimulation on kinesthetic perception in a single experimental paradigm.

Seroepidemiology of varicella-zoster virus in Korean adolescents and adults using fluorescent antibody to membrane antigen test.

We conducted a cross-sectional seroepidemiological study in 2012-2013 to determine the seroprevalence of varicella-zoster virus (VZV) in adolescents and adults living in Korea, where varicella vaccination has been recommended universally at age 12-15 months since 2005. Residual serum samples were collected from 1196 healthy adults and adolescents aged ⩾10 years between November 2012 and March 2013. The fluorescent antibody to membrane antigen (FAMA) test and enzyme-linked immunosorbent assay (ELISA) were performed to determine the seroprevalence of VZV. The seroprevalences of VZV were compared between six age groups: 10-19, 20-29, 30-39, 40-49, 50-59, and ⩾60 years. The seroprevalence of VZV in the entire study cohort was 99·1% according to the FAMA test and 93·1% as determined by ELISA. The seroprevalences of the six age groups were as follows: 96·0%, 99·5%, 99·5%, 99·5%, 100%, and 100%, respectively, by the FAMA test, and 83·3%, 93·0%, 93·0%, 97·5%, 94·5%, and 97·5%, respectively, by ELISA. Seroprevalence increased significantly with age (P < 0·001); moreover, the seroprevalence in subjects aged 10-19 years was significantly lower than in other age groups (P < 0·001), as measured by both the FAMA test and ELISA. Thus, strategies to increase protective immunity against VZV in teenagers are necessary.

Liquid plug propagation in flexible microchannels: A small airway model.

In the present study, we investigate the effect of wall flexibility on the plug propagation and the resulting wall stresses in small airway models with experimental measurements and numerical simulations. Experimentally, a flexible microchannel was fabricated to mimic the flexible small airways using soft lithography. Liquid plugs were generated and propagated through the microchannels. The local wall deformation is observed instantaneously during plug propagation with the maximum increasing with plug speed. The pressure drop across the plug is measured and observed to increase with plug speed, and is slightly smaller in a flexible channel compared to that in a rigid channel. A computational model is then presented to model the steady plug propagation through a flexible channel corresponding to the middle plane in the experimental device. The results show qualitative agreements with experiments on wall shapes and pressure drops and the discrepancies bring up interesting questions on current field of modeling. The flexible wall deforms inward near the plug core region, the deformation and pressure drop across the plug increase with the plug speed. The wall deformation and resulting stresses vary with different longitudinal tensions, i.e., for large wall longitudinal tension, the wall deforms slightly, which causes decreased fluid stress and stress gradients on the flexible wall comparing to that on rigid walls; however, the wall stress gradients are found to be much larger on highly deformable walls with small longitudinal tensions. Therefore, in diseases such as emphysema, with more deformable airways, there is a high possibility of induced injuries on lining cells along the airways because of larger wall stresses and stress gradients.

Gas-liquid two-phase flow patterns in rectangular polymeric microchannels: effect of surface wetting properties.

Here we map gas-liquid two-phase flow regimes observed in polymeric microchannels with different wetting properties. We utilized video and confocal microscopy to examine two-phase flow patterns produced by parallel injection of air and water through a Y-shaped junction into a rectangular microchannel made of poly(dimethylsiloxane) (PDMS). We observed seven flow regimes in microchannels with hydrophobic walls, whereas only two flow patterns were identified in hydrophilic microchannels. Our study demonstrates that surface wettability has a profound influence on the spatial distribution of air and water moving in microchannels.

Staple line covering procedure after thoracoscopic bullectomy for the management of primary spontaneous pneumothorax.

BACKGROUND: Thoracoscopic bullectomy together with a pleural adhesive procedure is generally accepted as the standard for the definitive treatment of primary spontaneous pneumothorax (PSP). The purpose of this study was to evaluate whether the results of a thoracoscopic bullectomy followed by coverage of the staple line with cellulose mesh and fibrin glue could be comparable with those of adhesive procedures described in the literature. METHODS: Between May 2000 and February 2003, we performed 227 thoracoscopic surgeries on 219 patients with PSP using a single technique. After the bullectomy, the staple line was covered with cellulose mesh and fibrin glue. The postoperative status was evaluated with a mean follow-up of 46 months. RESULTS: The mean patient age was 24.3 years and 90.9 % of the 219 patients were male. Recurrent pneumothorax (37.4 %) was the most common operative indication, followed by persistent air leakage of more than 5 days (28.2 %). The mean duration of postoperative chest tube drainage was 1.6 days and the mean postoperative hospital stay was 3.8 days. Six patients experienced surgical complications (2.2 %); there was air leakage of more than 3 days in two cases, a small apical dead space in one case, a fever-associated wound problem in one case, and a reoperation due to air leakage of more than 7 days in two cases. Eleven patients (4.8 %) suffered a recurrence of pneumothorax during the follow-up period. Of these, nine cases required readmission and three (1.3 %) of these cases required a reoperation. CONCLUSIONS: Given the nature of a meticulous thoracoscopic bullectomy followed by coverage with cellulose mesh and fibrin glue, good surgical results can be expected without the need for a pleural adhesive procedure.

In vitro activities of quinupristin/dalfopristin and eight other antimicrobial agents against 360 clinical isolates from Korea.

The emergence of multi-drug resistant gram-positive cocci such as methicillin-resistant (MR) staphylococci, vancomycin-resistant (VR) enterococci, and vancomycin-intermediate resistant S. aureus (VISA) has given new urgency to the development of new antimicrobial agents. One of these is quinupristin/dalfopristin (Q/D). We decided to determine the susceptibility of gram-positive cocci isolated at two university hospitals in Seoul to Q/D and compare the results with eight other antimicrobial agents. We investigated 120 isolates of S. aureus including 49 MRSAs and one VISA, 120 isolates of coagulase negative staphylococci (CNS), 64 E. faecalis and 56 E. faecium, including seven strains of VR E. faecium. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for several antimicrobials, including vancomycin and Q/D, were determined by broth microdilution. All S. aureus including VISA were susceptible to Q/D. Q/D MIC90 for both methicillin-susceptible S. aureus (MSSA) and MRSA was 0.25 g/mL. 49 (87.5%) of 56 E. faecium including six of seven VR E. faecium were susceptible to Q/D. E. faecalis were not susceptible to Q/D (only 1.5% susceptible), but were inhibited by ampicillin (94% susceptible) or vancomycin (95%). CNS was susceptible to Q/D (96% susceptible) and vancomycin (100% susceptible). One of 38 staphylococci and two of 17 E. faecium were tolerant to Q/D. In conclusion, Q/D showed excellent activity against all species of gram-positive cocci including MRSA, VISA, and VR E. faecium except E. faecalis, and may provide a valuable option for the treatment of infections caused by these emerging nosocomial pathogens of gram-positive cocci.

Survival improvement with combined radio-chemotherapy in the primary central nervous system lymphomas.

The benefits of radio-chemotherapy in HIV-negative primary central nervous system (CNS) lymphomas were analyzed in 40 patients, who received radiotherapy to the brain or craniospinal axis with the total dose of 4460-5940 cGy to the primary tumor. Radiotherapy was followed by systemic chemotherapy, mainly with the cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) regimen, in 16 of the patients. Follow-up ranged from four to 95 months with a median of 15 months. The relapse rate was 72.5%, and 83% of the relapses occurred within the radiation field. Median survival was 19 months and the two-year survival rate was 41%. Survival was significantly influenced by treatment method and radiation dose when measured by univariate analysis; median survival and the two-year survival rate was 29 months and 63% after radio-chemotherapy, while 13.5 month and 29% after radiotherapy alone (p= 0.027), and 22 months and 49% with doses of 50 Gy or more, but 12.5 months and 13% with doses less than 50 Gy (p=0.009). However, statistical significance was lost in multivariate analysis. These results might suggest the short-term efficacy of radio-chemotherapy, however, cautious observation is needed to confirm long-term effects.

Application of infrequent-restriction-site PCR to clinical isolates of Acinetobacter baumannii and Serratia marcescens.

We applied infrequent-restriction-site PCR (IRS-PCR) to the investigation of an outbreak caused by 23 isolates of Acinetobacter baumannii in an intensive care unit from November 1996 to May 1997 and a pseudoepidemic caused by 16 isolates of Serratia marcescens in a delivery room from May to September 1996. In the epidemiologic investigation of the outbreak caused by A. baumannii, environmental sampling and screening of all health care workers revealed the same species from the Y piece of a mechanical ventilator and the hands of two health care personnel. IRS-PCR showed that all outbreak-related strains were genotypically identical and that three strains from surveillance cultures were also identical to the outbreak-related strains. In a pseudoepidemic caused by S. marcescens, IRS-PCR identified two different genotypes, and among them one genotype was predominant (15 of 16 [93.8%] isolates). Extensive surveillance failed to find any source of S. marcescens. Validation of the result of IRS-PCR by comparison with that of field inversion gel electrophoresis (FIGE) showed that they were completely concordant. These results suggest that IRS-PCR is comparable to FIGE for molecular epidemiologic studies. In addition, IRS-PCR was less laborious and less time-consuming than FIGE. To our knowledge, this is the first report of the application of IRS-PCR to A. baumannii and S. marcescens.

Molecular analysis of fluoroquinolone-resistance in Escherichia coli on the aspect of gyrase and multiple antibiotic resistance (mar) genes.

We analyzed the fluoroquinolone resistance mechanism of 28 isolates of ciprofloxacin-resistant E. coli from patients who received ciprofloxacin as a regimen of a selective gut decontamination. Isolates distinctive by infrequent restriction site polymerase chain reaction (IRS-PCR) were subjected to Hinf I restriction fragment length polymorphism analysis, single-stranded conformation polymorphism (SSCP), and nucleotide sequencing of the quinolone resistance determining region (QRDR) in gyrA. Double mutations in QRDR of gyrA (Ser83 Leu and Asp87Asn) were found from most of the strains. Nucleotide sequencing of the marR locus showed that 18 out of 28 (64%) ciprofloxacin-resistant E. coli strains had three types of base change in marR loci: a double-base change at nucleotides 1628 and 1751, or 1629 and 1751: and a single-base change at 1751. However, all the mutated strains showed no tolerance to cyclohexane test, suggesting the mutation in the marR region had no influence on overexpression of the MarA protein. In conclusion, mutation in gyrA was the main mechanism of ciporfloxacin resistance in E. coli from patients with selective gut decontamination. Therefore, mutation in the mar region did not influence the levels of ciprofloxacin resistance in our isolates.

Molecular epidemiological analysis of quinolone-resistant Escherichia coli causing bacteremia in neutropenic patients with leukemia in Korea.

We analyzed an outbreak of Escherichia coli bacteremia in eight patients with leukemia in a hematology-oncology unit from July to September 1994. The antibiograms and genotypic patterns of the isolates were different, thus suggesting that the outbreak did not originate from a single clone. However, all the isolates were resistant to quinolones, which led us to examine the microbiological records from 1992 to 1994. The incidence of quinolone-resistant E. coli bacteremia in the hematology-oncology unit ranged from 81.8% to 94.6% during this period. We then analyzed 36 more isolates recovered from late 1994 to 1995. Field inversion gel electrophoresis patterns of these isolates were also different. Analysis of the quinolone resistance determining region in gyrA revealed that all the isolates had a double mutation in gyrA. In conclusion, quinolone-resistant E. coli could be an emerging threat to neutropenic patients with leukemia who receive a quinolone prophylactically, and attention must be paid to this trend of resistance.