United States

Background: Prior studies demonstrate that chronic prenatal opioid exposure leads to an increased risk for preterm labor and fetal growth restriction, both conditions associated with placenta insufficiency. Current literature also suggests that chronic prenatal opioid exposure negatively impacts the placenta (and fetus) during a critical developmental period where unyielding foundational processes for organ growth are initiated. Not only is there evidence of abnormal biochemical signaling between the placenta and fetus, but there is also histological evidence of chronic hypoxia in placentas chronically exposed to opioids. In a worsening opioid epidemic where identifying avenues for future therapeutics has become critical, the placenta may be an ideal therapeutic target for improving fetal health outcomes related to exposure. The goal of this study was to investigate histological differences in human placental tissue following chronic prenatal opioid exposure.

Methods: Placental tissue was collected from 7 opioid-exposed and 17 unexposed placentas from full-term pregnancies. Analyses were completed for 7 unexposed, control placentas by matching age and smoking history to exposed placentas. All participants in the opioid-exposed group were exclusively taking buprenorphine at some point during their third trimesters. Placental tissue samples were fixed in neutral buffered, 10% formalin, embedded in paraffin wax, and stained with hematoxylin and eosin prior to blinded histopathological analysis by an experienced pathologist. A generalized linear mixed model with a binomial outcome, paired t-tests, and linear mixed effects models were utilized to measure the frequency of a grade 2 or higher score (grade 3 or higher, villous morphology), total grade, or average grade.

Results: No statistically significant differences were observed in vascular pathology and inflammation between the opioid-exposed group and their respective matched controls. The control group placentas were found to exhibit significantly higher frequencies of grade 3, 4 or 5 villous morphology compared to the opioid-exposed placental tissue. When villous morphology was measured by total and average grade, no statistically significant differences were appreciated. 

Conclusion: The findings from this study may suggest that chronic opioid exposure, specifically chronic exposure to the partial opioid agonist buprenorphine, may not have unfavorable histopathological consequences as compared to unexposed placentas. The findings of this study may be limited by small sample size and an inability to control for other substances being used (including tobacco and illicit drugs) in the exposed group throughout pregnancy. Further investigation of microvascular changes as well as signaling changes in the placenta following chronic prenatal opioid exposure is warranted.

AMA Research Challenge 2023

Histopathology of placental tissue after chronic opioid exposure

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Background
Vaginal agenesis (VA) refers to the congenital absence of a vaginal canal often linked with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Current VA treatments include mechanical dilation and surgical vaginoplasty. Our vaginal expansion sleeve (VES) is a novel, transiently implanted device which lengthens the vaginal canal over time and is less complex and invasive than surgery or conventional dilation therapy which may improve patient compliance. Our study evaluated histologic effects of VES on rat vaginal tissue following VA.

Methods
Novel VES were made using a proprietary woven cylindrical material with helicoid trusses and flat resin caps. VES were used at 25-30mm in length depending on initial vaginal canal lengths and all VES pre-contracted to 50% of their original length. VES were then inserted and sutured into the vaginal canals of Sprague-Dawley rats (n=6) and allowed to spontaneously re-expand post-deployment. After one week, VES were removed and new vaginal lengths were measured. Rat vaginas were harvested en-bloc for histologic analysis. Test (n=6) and control (n=4) formalin-fixed paraffin-embedded tissues were stained with hematoxylin and eosin (H&E), Masson’s trichrome, and anti-Desmin immunochemical stain and evaluated.

Results
In 7days, mean vaginal canal lengths increased from 20.0 ± 2.4 mm to 23.8 ± 1.2 mm post-VES (n=6, p <.001), a 19% increase. H&E staining revealed increased submucosal eosinophilic inflammation in 5:6 of samples compared to 1:4 control tissues with an average 30.2 eosinophils per HPF (range: 16 - 38; median: 35). The test tissues also showed increased scattered neutrophils and lymphocytes in the mucosa and submucosa, with one test tissue having the longest VES (30mm) containing lymphocytic and neutrophilic inflammation. Desmin immunostaining and Masson’s trichrome staining both revealed a thinner muscularis with more infiltrative fibrous tissue between muscle fibers in the test tissues compared to control tissues.

Conclusion
VESs achieved significant vaginal lengthening. A positive correlation was observed between the VES length (and therefore tension generated in the vagina) and the degree of inflammation. The eosinophilia observed in the test tissues may indicate a localized, specific immune reaction to the VES device. The Desmin immunostain and Masson’s trichrome stain is consistent with muscularis remodeling. Future studies with longer durations of VES placement as well as serial implants with VESs of increasing lengths can inform longer-term remodeling of test tissues and help further characterize immune response associated with VES.

Histologic Changes in Rat Vaginal Tissue Following Vaginal Expansion Sleeve (VES) Implantation: Implications for Vaginal Agenesis Treatment

Background
Premature ovarian failure, whether occurring spontaneously or iatrogenically, especially as a result from chemotherapy, is a challenging clinical problem in Reproductive Endocrinology and Infertility and Oncology. These patients have no autologous fertility-restoration options available if preemptive egg or embryo freezing was not successfully completed. Lack of endogenous hormone production has sequelae beyond reproduction, including accelerated bone loss, increased risk of cardiovascular disease, and sexual dysfunction. While hormone replacement therapies exist, long-term outcomes from such treatments, especially when initiated at a young age, remain unclear. These issues are heightened in cancer survivors, who have lower baseline fertility rates and success with assisted reproduction.
Methods
A methodology for the creation of oocytes via reprogrammed granulosa cells was first performed in mouse and, to the greatest extent possible, recapitulated in human cells. Granulosa cells (GCs) were reprogrammed to generate induced pluripotent stem cells (IPSCs) as previously described in our lab. Human GCs and follicular fluid were obtained from the IVF lab after oocyte removal during egg retrieval procedures. Cells were labeled with GFP and differentiated into ovarian lineages using an oocyte derivation media containing follicular fluid. Stem cell and differentiated cell status was verified via RT-PCR, immunocytochemistry, alkaline-phosphatase reaction, steroidogenic activity, and the formation of embryoid bodies and teratomas. Mouse GFP-labeled differentiated IPSCs were orthotopically implanted into one ovary of subfertile immunocompromised mice which also had chemotherapeutically-induced ovarian failure to assess for the restoration of hormone production and fertility.
Results
Differentiated IPSCs expressed ovarian markers (i.e. AMHR2, ZP1) and were able to produce physiologically relevant concentrations of the reproductive steroid hormones (i.e. estradiol/progesterone) in vitro. Interestingly, the addition of follicular fluid increased the steroidogenic potential of the cells in mice. Mice treated with alkylating chemotherapy to induce ovarian failure had ovarian follicular atresia and breast atrophy, which was reversed within 72 hours of transplanting terminally differentiated GFP-IPSCs into the thighs or ovaries of these mice. In subfertile nude mice, the orthotopic implantation of terminally differentiated IPSCs enhanced fertility, resulting stem cell-derived neogametogenesis and delivery of F1 and F2 litters with GFP expression.
Conclusion
New autologous methods of restoring fertility and ovarian function would be indispensable for patients with prematurity ovarian insufficiency or failure. Our work demonstrates that other populations of cells within the body may be reprogrammed for the purpose of endocrine health and fertility restoration in such patients.

The Generation of Stem Cell-Derived Functional Oocytes from Reprogrammed Granulosa Cells

Background: Cardiovascular disease (CVD) affects 1-4% of pregnancies in the U.S. and is the primary contributor to morbidity and mortality in pregnancy and post-partum. While it has been established that adverse pregnancy outcomes (APOs) increase future maternal CVD risk, the factors predicting these pregnancy complications are less studied. Our primary objective is to report preliminary data from our cardio-obstetrics registry on the association between pre-conception cardiovascular risk factor burden and APO development. Given the relative sparsity of cardio-obstetrics patient data, we also aim to contribute patient demographics.

Methods: Utilizing retrospective chart review, we created a registry of patients evaluated at USF’s cardio-obstetrics program October 7, 2021-November 1, 2022. Charts were queried for demographics, pre-conception risk factors, and maternal-fetal outcomes. Risk factors included BMI>25 kg/m2, chronic hypertension, hyperlipidemia, pre-existing diabetes, tobacco use, sleep apnea, chronic kidney disease, autoimmune disease, advanced maternal age, and history of a prior APO. APOs were defined as intrauterine growth restriction (IUGR), hypertensive disorder of pregnancy (HDP), and pre-term birth (delivery <37 weeks). Statistical analysis included chi-square test and Fisher’s exact test for categorical variables and Mann-Whitney U test for continuous variables.

Results: Our analysis included 63 women, most commonly presenting with valvular or structural heart disease (33.3%), cardiomyopathy (28.6%), and arrythmias (15.9%). Throughout pregnancy and post-partum, 42 (66.7%) women did not experience any APO, while 21 (33.3%) did; 19 (30.2%) developed an HDP, 3 (4.8%) experienced IUGR, and 12 (19.0%) delivered pre-term. Race, ethnicity, age, gravidity, parity, gestational age at first cardio-obstetrics visit, and individual risk factors were not significantly associated with APO development. Analysis of risk factor burden showed the number of risk factors a woman had pre-conception was not a statistically significant predictor APO development (p=0.139). While the risk factor burden did not affect the overall incidence of APOs, patients who gave birth pre-term did have a significantly higher number of risk factors (p<0.001; 3.08 ± 1.00 pre-term, 1.69 ± 1.19 not pre-term), as did patients with chronic hypertension with superimposed HDP (p<0.001; 3.43 ± 0.79 with, 1.77 ± 1.21 without). 

Conclusion: Cardiovascular risk factor burden was not associated with APO development in pregnant women with CVD. Women who deliver pre-term may have a higher risk factor burden than those who deliver at term. Since pre-term birth is associated with future CVD in women independent of its cause, optimization of preconception cardiovascular health could help mitigate these risks in an already vulnerable high-risk population.

Cardiovascular Risk Factor Burden and Adverse Pregnancy Outcomes in Women with Cardiovascular Disease


Background:

The two main classes of AMTs commercially available today are “cryopreserved” and “dehydrated.”

Many studies have indicated that the process of cryopreservation is effective at maintaining both the quantity and activity of biological signaling proteins found within the amniotic tissue. One notable drawback of cryopreserved amniotic membranes is that they carry a higher monetary cost due to their stringent production and storage requirements.

Dehydrated amniotic membranes are a cheaper alternative to their cryopreserved counterparts. This is primarily because they have simpler storage requirements, as they can be kept at room temperature. However, studies have also shown that dehydrated amniotic membranes are less effective at maintaining the availability and activity of important biological factors that influence healing and tissue regeneration. Yet, it remains unclear if these findings have any significant real-world implications in a clinical setting. Accordingly, our study seeks to identify and compare the benefits and shortcomings of each type of membrane. 

Methods: 

We completed a retrospective, non-inferiority trial involving 36 patients who formed 2 populations. Group 1 received a cryopreserved membrane while Group 2 received a dehydrated membrane. The duration of therapy was 1 week. We recorded each patient's indications, number of amniotic membrane transplants (AMTs), adjunct medications, tolerability, visual acuity, and ocular pressure pre/post treatment. A positive outcome at the 1-month post-AMT visit was defined as improvement in ≥3 symptoms of eye irritation, resolution of primary indication for AMT, improvement in visual acuity, and improvement in intraocular pressure. If the criteria was not met, the outcome was considered to be negative. This trial was non-randomized nor blinded.


Results:
51 AMTs from 36 patients were included. At both the 1 week and 1 month interval, patients who received a cryopreserved amniotic membrane transplant (CAMT) self-reported a greater number of side-effects when compared to patients who received a dehydrated membrane (DAMT). Those who received a CAMT also had greater vision improvement and a higher self-reported feeling of wellness at both intervals when compared to patients who received a DAMT [P =.03]. Patients who received a DAMT were less likely to require repeated treatments [P = .001]. 

Conclusion:
Cryopreserved membranes are currently more expensive than dehydrated membranes. These cost disparities become amplified given that our data suggests a higher re-treatment rate in the Group 1. Despite these concerns, CAMTs may be viewed as a more effective treatment since patients in this cohort were more likely to have a positive outcome.


Comparing patient tolerability of cryopreserved and dehydrated amniotic membrane transplantations for managing corneal disease

Introduction: This study identifies retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) patients at an inherited retinal disease (IRD) clinic that qualify for ongoing or FDA-approved adeno-associated virus (AAV) gene-replacement therapies. The goal is to demonstrate the benefits of genetic testing at the initial evaluation.

Method: A database for RP and LCA patients was curated and clinicaltrials.gov was used to search all ongoing or approved gene-replacement therapies between 1 January 2022 – 1 January 2023. Patients were evaluated for qualification based on the inclusion/exclusion criteria set by each trial.

Results: 199 RP and 31 LCA patients were included in the study. Our team identified six AAV gene-replacement therapy clinical trials and the FDA-approved Luxturna®. One hundred fifty-five patients underwent genetic testing, and 89 patients had a pathogenic variant identified. A total of 15 patients qualified for one of the proposed trials. Three patients had a biallelic RPE65 mutation, and two of them qualified for Luxturna®. All 11 patients with an RPGR mutation qualified for one of the three clinical trials that focused on this gene. Three patients had a c.2991+1655A>G mutation in CEP290 and two of them qualified one of two clinical trials for this gene.

Conclusion: Overall, ~10% of patients who had genetic testing qualified for one of the reviewed therapies. A total of 15 patients qualified for an AAV gene-replacement therapy. This study highlights the importance of promoting genetic testing for IRD patients, the need for earlier disease evaluation, and the value of continual monitoring of disease progression.


Qualifications for Retinitis Pigmentosa and Leber Congenital Amaurosis Patients for Adeno-Associated Viral Gene-Replacement Therapy Clinical Trials

Background
Meibomian gland disease (MGD) is classically diagnosed using clinical symptoms and eyelid exam. Advancements in meibomian gland imaging can offer an additional, quantitative measure for disease correlation. This study examines whether a patient’s diagnosis of MGD correlates with the severity of meibomian gland dropout assessed through Meibography. 
Methods
Meibography images from 319 lower eyelids of patients with dry eye disease (DED) were collected. Three different graders assigned each image a Meiboscore using a previously validated scale (score 0 = no dropout, score 3 = severe dropout). The mean of the 3 gradings was used to assign a Meiboscore to each eyelid. A retrospective medical records review was performed to determine which cases of DED were made in conjunction with MGD. Diagnosis of MGD was made only based on clinical exam without consideration of Meibography images. 
Results
Of the 319 images included in the study, 46% (148 images) of them came from patients with only DED and 54% (171 patients) came from patients with both DED and MGD. There was no significant difference in age (p=0.73) or gender (p=0.45) between the two groups. The mean Meiboscore for patients diagnosed with DED without associated MGD is 1.81 ± 0.82, while the mean Meiboscore for patients with diagnoses of DED and MGD is 1.63 ± 0.844 (p=0.045, level of significance <0.05). 
Conclusion
Patients diagnosed with DED without diagnosed MGD exhibit significantly more gland dropout than patients with associated MGD. This suggests that patients with DED may be underdiagnosed with MGD. As Meibography becomes more accessible in clinical settings, revision of MGD diagnostics is needed to incorporate Meibography into established criteria. Determining the cause of dry eye disease through meibography can be used in conjunction with techniques like meibum expression and tear analysis to help tailor treatments. For example, can the Meiboscore in combination with other clinical information help us determine if a meibomian gland expression, probing or IPL treatment be most suitable for a particular patient? To decrease the variability in capturing and scoring images, future research should focus on standardizing these measures.

Identifying the Underdiagnosis of Meibomian Gland Disease
Through Meibography

Pediatric Keratoconus (KCN) is believed to progress more rapidly to cornea transplant surgery (CTS) than adults. This study evaluated children with KCN to identify potential topographic indicators for evaluation for Corneal Collagen Cross-Linking (CXL) in order to halt or lessen the progression of KCN.

A retrospective study was conducted on 27 patients, ages 12-18, that were diagnosed with KCN based on slit-lamp findings, loss of vision, and corneal topography. A total of 50 eyes were included in this study with CXL performed in 33 eyes and 17 untreated. Cross-linked and untreated eyes were compared on anterior mean keratometry (AKm), posterior mean keratometry (PKm), and thinnest pachymetry (TTP) using a two-tailed t-test.

At initial presentation, treated eyes had a mean AKm of 48.4, PKm of -7.3, and TTP of 454.4, whereas untreated eyes had means of 44.8, -6.6, and 495.8, respectively. Additionally, the mean difference for AKm was 3.59 (P = 0.028, 95% CI ±3.18), PKm was -0.637 (P=0.024, 95% CI ± 0.55), and TTP was -41.46 (P=0.022, 95% CI ± 35.23). None of the cross-linked eyes progressed to CTS.

Children with KCN often present with more advanced disease and are at a greater risk of progression than adults. Earlier treatment with CXL may halt or slow progression of KCN. Our results indicate that pediatric KCN patients presenting with steeper AKm and PKm, as well as thinner corneas, should be considered for CXL.

Topographic Findings in the Eyes of Children Treated with Collagen Cross-Linking for Keratoconus

Background
Ocular funduscopic examinations is rarely performed at bedside by non-ophthalmology providers, resulting in diagnostic errors and worse patient outcomes when the correct diagnosis is delayed. This is particularly a concern in emergency departments (ED) where ophthalmologists are not always available for consultation. The goal of this study is to evaluate the short-term impact of the implementation of a non-mydriatic (without pharmacologic dilation of the pupils) fundus camera in a busy ED based in a metropolitan quaternary care center hospital.

Methods
Quality improvement project involving the systematic prospective collection of data from patients who had ocular fundus photographs taken in the ED for visual complaints, headaches, neurologic symptoms, hypertensive crisis. We collected demographic information, indication for fundus photographs and final diagnosis, who ordered photographs, timing of photographs and whether photographs had an impact on the patients’ length of ED stay and final outcomes.

Results
Since the camera was introduced in our ED 6 weeks ago, 148 patients had photographs taken (ordered by ED in 83.9%, ophthalmology in 8.9%, neurology in 7.2%). The reasons included visual complaint 33.5%, headache 20.5%, papilledema 8.9%, hypertension 1.3%, diabetes 0.6%, other neurologic complaints 34.9%. Photographs could be interpreted remotely, avoiding an ophthalmology or neurology consultation in 17.8%. It took less than 5 minutes to obtain photographs (60.2% high quality; 75.2% able to exclude emergent findings. The photographs accelerated the management/disposition of patients with acute disorders in 29.5%, including 8 acute central retinal arterial occlusions, 3 giant cell arteritis, 18 papilledema from intracranial hypertension.

Conclusion
Incorporating a non-mydriatic ocular fundus camera into the ED workflow can be used as a substitute for the funduscopic examination in most cases, allowing for remote fundus interpretation by ophthalmologists who can provide real time recommendations without seeing patients in person. These positive preliminary results obtained only 6 weeks after implementation of the camera suggest that this strategy reduces the overall hospital stay and accelerates the diagnosis of urgent vision- or life-threatening conditions, resulting in improved patients’ outcomes. Further data will be collected over the next few months and results of the first 3 months will be presented at the conference.

Implementation of a Non-mydriatic Ocular Fundus Camera in a Large Emergency Department

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Intraocular Pressure Reduction comparing Cataract Extraction and Hydrus Microstent Implantation with Cataract Extraction, Canaloplasty, and Hydrus Microstent Implanation

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Histologic Changes in Rat Vaginal Tissue Following Vaginal Expansion Sleeve (VES) Implantation: Implications for Vaginal Agenesis Treatment

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