Transcriptional profiling of mucus production in rhesus macaque endocervical cells under hormonal regulation.

OBJECTIVE: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the rhesus macaque (Macaca mulatta). INTERVENTION: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. MAIN OUTCOME MEASURES: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. RESULTS: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. CONCLUSION: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways altered by sex-steroids in cervical mucus production.

Functional evaluation of the cystic fibrosis transmembrane conductance regulator in the endocervix†.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride/bicarbonate ion channel in epithelial cells. Mutations in CFTR cause cystic fibrosis, a disease characterized by thickened mucus secretions and is associated with subfertility and infertility. CFTR function has been well characterized in vitro and in vivo in airway and other epithelia studies. However, little is known about CFTR function in the cervix in health and its contribution to cyclic regulation of fertility from endocervical mucus changes. Contributing to this research gap is the lack of information on the effect of sex steroid hormones on CFTR expression in cervical epithelial cells across the menstrual cycle. Herein, we demonstrate the hormonal regulation of CFTR expression in endocervical cells both in vitro and in vivo, and that conditionally reprogrammed endocervical epithelial cells can be used to interrogate CFTR ion channel function. CFTR activity was demonstrated in vitro using electrophysiological methods and functionally inhibited by the CFTR-specific inhibitors inh-172 and GlyH-101. We also report that CFTR expression is increased by estradiol in the macaque cervix both in vitro and in vivo in Rhesus macaques treated with artificial menstrual cycles. Estrogen upregulation of CFTR is blocked in vivo by cotreatment with progesterone. Our findings provide the most comprehensive evidence to date that steroid hormones drive changes in CFTR expression. These data are integral to understanding the role of CFTR as a fertility regulator in the endocervix.

Hormonal regulation of non-cystic fibrosis transmembrane conductance regulator ion channels in the endocervix.

OBJECTIVE: To characterize ion channel expression and localization in the endocervix under different hormonal conditions using a nonhuman primate primary endocervical epithelial cell model. DESIGN: Experimental. SETTING: University-based, translational science laboratory. INTERVENTIONS: We cultured and treated conditionally reprogrammed primary rhesus macaque endocervix cells with estradiol and progesterone and measured gene expression changes for several known ion channel and ion channel regulators of mucus secreting epithelia. Using both rhesus macaque endocervical samples and human samples, we localized channels in the endocervix using immunohistochemistry. MAIN OUTCOME MEASURES: The relative abundance of transcripts was evaluated using real-time polymerase chain reaction. Immunostaining results were evaluated qualitatively. RESULTS: Compared with controls, we found that estradiol increased gene expression for ANO6, NKCC1, CLCA1, and PDE4D. Progesterone down-regulated gene expression for ANO6, SCNN1A, SCNN1B, NKCC1, and PDE4D (P≤.05). Immunohistochemistry confirmed endocervical cell membrane localization of ANO1, ANO6, KCNN4, LRR8CA, and NKCC1. CONCLUSIONS: We found several ion channels and ion channel regulators that are hormonally sensitive in the endocervix. These channels, therefore, may play a role in the cyclic fertility changes in the endocervix and could be further investigated as targets for future fertility and contraceptive studies.

Transcriptional profiling of mucus production and modification in rhesus macaque endocervical cells under hormonal regulation.

Objective: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the Rhesus Macaque (Macaca mulatta). Design: Experimental. Setting: Translational science laboratory. Intervention: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. Main Outcome Measures: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. Results: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. Conclusion: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways that are altered by sex-steroids in cervical mucus production.

Case report: training neck and head control in children with chronic paralysis due to acute flaccid myelitis.

Background: Acute flaccid myelitis (AFM) occurs rarely in children and adolescents when damage to spinal motor neurons rapidly causes flaccid paralysis of limb, trunk, and neck muscles and potentially respiratory failure. When neck muscles are weakened or paralyzed, a child loses head control, severely compromising engagement with their environment. Compensation for lack of head control is achieved with external support devices attached to a wheelchair, but there is no indication in the AFM literature of therapeutic efforts to restore head control. In this case series, we explore the possibility of the recovery of head control when children with AFM received activity-based restorative therapies (ABRTs) guided by principles targeting motor control. Case description: Three children, two male and one female, aged 6, 9, and 7, with a history of AFM-onset at 5, 7, and 4 years respectively, enrolled in an activity-based restorative therapies outpatient program targeting activation of the neuromuscular system below the lesion. Each of them lacked head control, was either ventilator-dependent or had a tracheostomy, and was a power wheelchair user via hand/foot control. Methods: Activity-based restorative therapies were provided 5 days/week: 1.5 h of activity-based locomotor training and 1.5 h of activity-based neuromuscular electrical stimulation. Results: An approach to addressing head/neck control developed iteratively across disciplines, from complete compensation with passive external head support to emerging head control during diverse tasks, e.g., sitting, reaching, driving a power chair, sit-to-stand, standing, stepping on a treadmill, and walking. Key principles identified and employed were (a) passive facilitation, (b) external head support, (c) posterior head support, (d) graded manual facilitation, and (e) independent head control. Discussion: The recovery of head control in children with paralysis due to AFM may be accelerated when executing a step-wise progression to effectively target and challenge head control in parallel with activity-based restorative therapies. In treating three children with a chronic lack of head control, a therapeutic strategy was iteratively developed guided by scientific principles, e.g., segmental assessment of control, to promote recovery of head control. While this strategy is encouraging, gaps in sensitive and responsive measurement instruments and treatment technologies persist in guiding assistance, challenging, and promoting independent head control.

Systemic polidocanol from intravenous or pressurized intrauterine administration produces reversible cardiovascular toxicity.

Objective: Fatal allergic responses and cardiac arrhythmias have been reported with the intravenous (IV) administration of polidocanol. We sought to identify the physiologic mechanism of systemic cardiovascular response after transcervical (TC) and IV administration of polidocanol. Methods: We continuously monitored blood pressure (BP) and heart rate using an arterial line during IV and intraperitoneal (IP) administration of polidocanol solution (PS) and polidocanol doxycycline solution in female rats and TC and IP administration of polidocanol foam (PF) and PDF (TC only) in female baboons. We performed TC procedures using a catheter with (pressurized) and without (nonpressurized) balloon inflation. Baboons also underwent monitoring during IV PS administration with and without pretreatment with antihistamines. We performed cardiac echo and electrocardiograms during selected experiments. We defined a refractory hypotension as a sustained decrease of more than 30% from baseline that prevented delivery of the target dose. Results: We found a dose-related increase in the proportion of baboons that developed refractory hypotension during TC administration of 5% PDF and PF, an effect confined to pressurized administration. The infusion of 0.5% PS in rats induced a rapid and dramatic refractory hypotension. The inclusion of doxycycline did not improve or deteriorate these outcomes, and doxycycline solution or saline (control) alone did not affect BP. All five female baboons that received up to 20 mL of 1% PS (200 mg) developed refractory hypotension. Pretreatment with diphenhydramine, ranitidine, or both did not block the refractory hypotension induced by IV administration of 1% PS (100 mg). In contrast, only one of the six female baboons treated with IP PF 400 mg developed a decrease of more than 30% in BP, and this response was not sustained. Cardiac echocardiography done in four baboons during TC treatment demonstrated a decrease in cardiac output as the physiologic mechanism of hypotension. We did not observe important changes on the electrocardiograms. Conclusions: Adverse cardiovascular effects of polidocanol treatment occur owing to a direct myocardial effect of polidocanol and not as a result of a hypersensitivity reaction. Pressurized TC administration of PF results in refractory hypotension owing to endometrial vascular uptake of polidocanol and not as a result of uptake from peritoneal surfaces.

Case Report: Capitalizing on Development and Activity-Dependent Plasticity, an Interaction With Pediatric-Onset Spinal Cord Injury.

Background: Spinal cord injury (SCI) in infancy halts typical development secondary to paralysis/paresis and the limited ability to engage with the environment. Traditional therapies further restrict a child via bracing, equipment, and medications. In contrast, activity-based restorative therapies (ABRT) promote activation of the neuromuscular system below the level of injury and affords a more typical sensorimotor experience. Case Description: A premature male infant exhibiting hypotonia, poor head control, and extremity weakness was diagnosed at age 5 months with a remote incomplete upper cervical SCI based on magnetic resonance imaging (MRI), presumed to have occurred perinatally. From 4 to 15 months of age, he received physical, occupational and speech therapies. Enrolled in an ABRT program at 15 months, he was unable to sit, pull-to-stand, stand, or walk and had upper extremity impairments. Results of the Bayley-III Scales of Infant and Toddler Development revealed gross and fine motor scores consistent with a 4-month-old. Methods: Activity-based restorative therapies was provided 5 day/week: 1.5 h of activity-based locomotor training and 1 h of activity-based occupational therapy. Results: Activity-based restorative therapies are reported for 177 sessions and are on-going. Improvements are noted in trunk control, standing, walking, grasp, in-hand manipulation, and associated kinematics. Bayley-III fine motor score improved to that of a 16-month-old and gross motor score to that of a 7-month-old. Discussion: While the two treatment periods (i.e., 4-15 months old and 15-24 months) were each ∼9 months, the child's accelerated progress toward typical development during the latter, ABRT period is noteworthy. In comparison to the period of traditional therapies in which paralysis was compounded by a restrictive environment and compensation, ABRT provided a potentially rich sensorimotor experience with an emphasis on active weight-bearing and proper kinematics to activate the neuromuscular system below the lesion in an age-appropriate, task-specific context of activities. Improved physical capacity enabled exploration more typically associated with development at this age expanding the positive impact to other developmental domains.

Impact of Activity-Based Therapy on Respiratory Outcomes in a Medically Complex Child.

INTRODUCTION: Activity-based therapies (ABTs) focus on activating the neuromuscular system below the level of spinal cord injury (SCI) promoting neuromuscular capacity. CASE DESCRIPTION: A 2 year 7 month old with history of prematurity at 29 weeks, neonatal epidural abscess, resultant cervical SCI, respiratory failure, and global developmental delays presented for enrollment in an outpatient activity-based therapy program. Upon presentation to this program, he required nighttime mechanical ventilation via tracheostomy and daytime suctioning. He could not perform any age-appropriate activities and was described by his mother as 'present', neither engaged nor attentive. During and after 7 months of participation in ABTs including locomotor training and neuromuscular electrical stimulation, the patient demonstrated unexpected changes in his respiratory status leading to ventilator weaning with concomitant improvements in head and trunk control, participation, development, and quality of life. DISCUSSION: ABT was not only safe for a medically complex child, but also this intervention had a remarkable effect on unresolved respiratory capacity and a more widespread impact on other functions as well as development. A child with a chronic, severe SCI demonstrated positive and impactful improvements in health, functional status, and quality of life during an episode of ABT.

Sensitivity to change and responsiveness of the Segmental Assessment of Trunk Control (SATCo) in children with spinal cord injury.

PURPOSE: The purpose of this study was to assess the sensitivity and responsiveness of the Segmental Assessment of Trunk Control (SATCo) for evaluating trunk control in children with spinal cord injury (SCI) receiving activity-based locomotor training (AB-LT). METHODS: Prospective study of nine outcomes for consecutively enrolled children in outpatient AB-LT. To evaluate sensitivity to change, linear-mixed models were constructed and adjusted for covariates: age at and time since SCI. To evaluate responsiveness, standardized response means and 95% confidence intervals were estimated per outcome. RESULTS: SATCo scores increased significantly (p < 0.05) regardless of chronicity, initial score, and injury level. The SATCo was the most responsive measure and the only outcome demonstrating a large effect size after 3 months of therapy. CONCLUSIONS: Children with SCI receiving AB-LT improved trunk control regardless of chronicity, initial impairment, or prior experience. SATCo sensitivity and responsiveness support its usefulness in measuring trunk control in children with SCI.

Activity-Based Therapy Targeting Neuromuscular Capacity After Pediatric-Onset Spinal Cord Injury.

Background: Activity-based therapies aim to improve neuromuscular capacity after spinal cord injury (SCI). Objective: The purpose of this prospective study was to report the impact of Activity-based Locomotor Training (AB-LT) on neuromuscular capacity in pediatric patients with SCI. Methods: Participants were enrolled for their first episode of AB-LT for a minimum of 60 daily, 1.5-hour sessions. The Segmental Assessment of Trunk Control (SATCo) and the Pediatric Neuromuscular Recovery Scale (Pediatric NRS) were assessed initially, every 20 sessions, and post 60 sessions. Results: Twenty-six consecutive patients, mean age 5 years (SD = 3), completed a mean 55 sessions (SD = 4) within 63 weekdays (SD = 9). The Pediatric NRS total score improved significantly, adjusted mean 11.4, from initial to post-60 sessions (p < .05) with an average adjusted evaluation-to-evaluation 3.7 change. SATCo scores improved significantly across 60 sessions, mean change 5.2, an estimated 1.7 change between evaluations (p < .05). Age at enrollment and chronicity had no effect; however, initial neuromuscular capacity scores were negatively correlated with change scores (p < .05). Conclusion: Sixty AB-LT sessions significantly improved trunk and neuromuscular capacity in children with SCI, regardless of age or chronicity at enrollment. Patients with lower initial scores made greater improvements than patients with higher initial neuromuscular capacity. Anecdotal parent reports of their child's functional change in the home and community highlight the synergy between quantitative change in neuromuscular capacity and meaningful, improved quality of life and the need for formal investigation of this relationship.

Interrater Reliability of the Pediatric Neuromuscular Recovery Scale for Spinal Cord Injury.

Background: In synergy with the mounting scientific evidence for the capacity of recovery after spinal cord injury (SCI) and training, new evidence-based therapies advancing neuromuscular recovery are emerging. There is a parallel need for outcome instruments that specifically address recovery. The Pediatric Neuromuscular Recovery Scale (Pediatric NRS) is one example with established content validity to assess neuromuscular capacity within task performance. Objective: The objective of this study was to determine interrater reliability of the Pediatric NRS to classify motor capacity in children after SCI. Methods: Pediatric physicians (3), occupational therapists (5), and physical therapists (6) received standardized training in scoring the scale, then rated video assessments of 32 children post SCI, 2-12 years of age, 78% non-ambulatory. Interrater reliability was analyzed using Kendall coefficient of concordance for individual Pediatric NRS items and overall score. Results: The interrater reliability coefficient was determined to be near 1 for the overall Pediatric NRS score (ICC = 0.966; 95% CI, 0.89-0.98). Twelve of 16 individual items exhibited high concordance coefficients (Kendall's W ≥ 0.8) and four items demonstrated concordance coefficients, < 0.8 and > 0.69. Interrater reliability was equivalent among groups defined by age and neurological level, but lower among non-ambulatory individuals. Conclusion: Strong interrater reliability was demonstrated by pediatric clinicians who scored children with SCI using the Pediatric NRS.