United States

Background: Women ≧35 years old are categorized as “advanced maternal age” (AMA) and are at an increased risk for a decline in ovarian reserve and oocyte competence. Therefore, they may seek fertility assistance through IVF. Pre-implantation genetic testing for aneuploidy (PGT-A) is recommended to AMA women undergoing IVF to determine an embryo’s ploidy status. However, PGT-A findings are currently a point of contention due to mosaicism. This study seeks to identify gene expression patterns found in blastocoel fluid from IVF-embryos from mothers of AMA that resulted in successful pregnancy outcomes.

Methods: Blastocoel fluid-conditioned media was collected from day-5 IVF-embryos that underwent PGT-A biopsy. RNA was purified from the fluid and cDNA was synthesized. Genes identified from a previous RNASeq study were selected for further analysis using RT-qPCR. GAPDH, BCL2L12, CUL2, SHARPIN and DRAP1 gene expression were assessed using RT-qPCR in blastocoel fluid samples from AMA mothers and mothers 35.

Results: Preliminary analysis on 170 blastocoel fluid samples (n= 48 for younger individuals and n=122 individuals of AMA), indicates that BCL2L12, SHARPIN, and DRAP1 are upregulated and expressed to a higher extent in AMA samples.

Conclusion: Initial findings suggest that BCL2L12, SHARPIN, and DRAP1 genes might be associated with AMA. BCL2L12 is an anti-apoptotic gene involved in regulated cell death. SHARPIN stimulates the formation of linear ubiquitin which regulates NF-KB signaling and inhibits extrinsic apoptosis. DRAP1 regulates gene transcription, while CUL2 supports ubiquitin ligase activities within cells. Ongoing studies aim to identify gene expression patterns associated with AMA, which could serve as an additional marker along with PGT-A to aid in selecting the most viable embryo for transfer.


AMA Research Challenge 2022

Analysis of apoptotic gene expression in blastocoel fluid of IVF-embryos from individuals of advanced maternal age

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Introduction:
In vitro fertilization (IVF) cycles are monitored for premature progesterone rise, which contribute to implantation failure. Progesterone thresholds prompting recommendation for freeze-all cycles vary between institutions, depending on laboratory assay and patient population. To our knowledge, this has not been evaluated at a military treatment facility.

Methods:
A retrospective chart review was performed; all IVF cycles with fresh embryo transfers between 2016 and 2019 at Brooke Army Medical Center (BAMC) were eligible. Serum progesterone levels were obtained via electrochemiluminescence assay on Roche Cobas 80036 hours or less prior to ovulation trigger. The number of clinical pregnancies (CP, n=190) and live births (LB, n=159) were stratified by progesterone levels.

Results:
345 women who underwent 393 fresh embryo transfers were included in the analyses. Progesterone was significantly lower in patients with a CP (0.693ng/mL ± 0.300) versus those without a CP (0.758ng/mL ± 0.438). Patients with a progesterone <1.3ng/mL are significantly more likely to achieve a CP (49.7% v 28%, p=0.035). On linear regression, when controlling for age and parity, a progesterone level ≥1.3ng/mL is significantly negatively associated with CP (p=0.045). When controlling for age and parity, a progesterone level <1.3ng/mL trends toward a significant association with LB (p=0.1)

Conclusion:
A progesterone <1.3ng/mL was established as a significant threshold for CP, and should be considered in future fresh IVF cycles at BAMC. However, there was lack of statistical significance for LB given this progesterone level, possibly impacted by the low number of patients with higher progesterone (n=25), which resulted in only 6 live births.

Determining a Progesterone Threshold for Fresh IVF Cycles in a Military Facility

Posterior reversible encephalopathy syndrome (PRES) is a neurotoxic condition that can present with seizures, headaches, and radiologic findings of vasogenic edema. Eclampsia and pre-eclampsia can predispose patients to developing PRES in the setting of endothelial dysfunction that could be caused by COVID-19, or another vasogenic mechanism. It is not well understood how COVID-19 can affect the brain but there is evidence that it can cause neurological symptoms. COVID-19 has been implicated in the pathogenesis of eclampsia, PRES and subarachnoid hemorrhage (SAH). PRES has been implicated in the pathogenesis of SAH and eclampsia. However, eclampsia is not well connected to the presence of SAH. In our case, a 33-year-old multiparous woman presented to the hospital and was admitted for supervision of labor complicated by COVID-19 infection and anemia. She delivered a healthy infant. She proceeded to have severe range blood pressures after delivery, and experienced multiple tonic-clonic seizures that were controlled with magnesium sulfate, labetalol, and nifedipine. At this time, she had radiological evidence of SAH and PRES. Our patient had a SAH in the context of PRES, eclampsia and a COVID-19 infection. Her SAH could be due to the vasogenic edema from PRES that could have been triggered by endothelial damage from COVID-19, and vasogenic dysfunction from eclamptic seizures.

Eclampsia complicated by subarachnoid hemorrhage in the context of posterior reversible encephalopathy and COVID-19 infection

Background: Ovarian cysts frequently resolve on their own, but little information on this resolution is available for use in clinical practice. The objective of this study was to characterize the resolution of incident ovarian cysts in relation to cyst diameter, structure, age, body habitus, and menopausal status. These categorizations are important for decisions on whether to continue monitoring the cyst or intervene surgically.

Methods: A total of 2,638 women with incident cysts were selected who had undergone 51,356 TVUS examinations in the University of Kentucky Ovarian Cancer Screening Program. The variables that were collected in this study were: age, BMI, cyst diameter, cyst structure, family history of ovarian cancer, and use of hormone replacement therapy. All images were reviewed by a physician. Methods of analysis include t-tests, chi-square, and Cox regression analysis of cyst resolution times.

Results: Women with a history of 1 or more cysts were included so that 2465 unilocular and 1420 septated cysts were available for analysis. Septated cysts (mean + SEM, 3.8+0.03), had a larger diameter than unilocular cysts (3.4+0.03), P<0.05. The resolution of incident septated cysts was faster than unilocular cysts. Unilocular cysts resolved the quickest in women 70+ years of age. Resolution was the most prolonged in women <40 years. No difference in resolution was observed between pre- and post-menopausal status nor BMI in the resolution of unilocular or septated cysts.

Conclusion: Septated cysts were significantly more likely to resolve than unilocular cysts in univariate and multivariate analyses. As the cyst diameter increased, cysts were found to decrease in time to resolution in univariate and multivariate analyses. With HRT use, cysts were significantly less likely to resolve in univariate analyses but were not significantly affected in multivariate analysis. As the patients aged, it was determined they were more likely to have cyst resolution in univariate and multivariate analyses. Menopausal status and body mass index were not seen to have an influence on the time to resolution of unilocular and septated cysts. For the clinician, this study establishes expectations for cyst resolution based on a primary estimation of cyst diameter in a population where no malignancy was observed.

Findings on ovarian cyst resolution for the practicing physician based on cyst diameter

Abstract Title: Hypoplastic Left Heart Syndrome: Prevalence of Anatomic Subtypes and Coexisting Genetic and Noncardiac Anatomic Anomalies Among Neonates at the University of Florida

Background
Hypoplastic left heart syndrome (HLHS) is a form of congenital heart disease (CHD) characterized by the underdevelopment of the left ventricle. It affects 2-3 of 10,000 live births and accounts for 2.7% of CHD. The severity of HLHS depends on the degree of left ventricular inlet and/or outlet obstruction arising from patterns of structural malformations including stenosis and atresia of the mitral and aortic valves. The objective of this study is to determine the prevalence of anatomic subtypes of HLHS and to assess fetal co-morbidities among fetuses with HLHS at the University of Florida (UF). 

Methods
Retrospective chart review of neonates born with HLHS at UF between 1/1/2006 and 12/31/2019. Data pertinent to maternal history, fetal workups, prenatal sonography, labor and delivery, neonatal hospital course, and pre- and postnatal echocardiograms were collected. Descriptive statistics were used.

Results
A total of 51 cases were included. Of all 51 cases, there were various combinations of valvular abnormalities as follows: mitral atresia and aortic atresia (MA/AA) in 19/51 (37.3%), mitral stenosis and aortic atresia (MS/AA) in 14/51 (27.4%), mitral stenosis and aortic stenosis (MS/AS) in 5/51 (9.8%), and variant subtypes in 13/51 (25.5%) cases. Noncardiac congenital anatomic abnormalities, fetal growth abnormalities, and genetic abnormalities were present in 11/51 (21.6%), 2/51 (3.9%), and 4/51 (7.8%), respectively. Genetic testing was performed in a majority of patients 49/51 (96.1%) with prenatal genetic testing performed in 20/51 (39.2%) patients and postnatal genetic testing in 46/51 (90.2%). Genetic testing revealed 4/49 (8.2%) identifiable genetic abnormalities and 11/49 (22.4%) variances of unknown significance. 

Conclusion
The most common structural subtype of HLHS was MA/AA. Approximately 1/5 of the fetuses had major extracardiac congenital anatomic abnormalities. Genetic anomalies were present in 8.2% of the fetuses although only 39.2% received prenatal genetic testing. This highlights the importance of offering prenatal genetic testing to pregnancies in which HLHS is suspected. A thorough prenatal evaluation including assessment of anatomic subtype and identification of anatomic and genetic comorbidities is essential because it enables extensive patient counseling and ensures the involvement of a multidisciplinary team to plan perinatal management for optimal neonatal outcome.

Hypoplastic Left Heart Syndrome: Prevalence of Anatomic Subtypes and Coexisting Genetic and Noncardiac Anatomic Anomalies Among Neonates at the University of Florida

In Utero Effects of Opioids on Fetal Growth

Title
Pathology Findings and Prognostic Outcomes with Endometrial Cancer Diagnosis via Myosure 

Contributing Authors 
Diana Fan, Kelly Larimore, Rachel Pasquesi, Jacob Dodd, Manisha Kullar, Jeffrey Gavard, Shannon Grabosch 

Introduction
Endometrial cancer is the most common gynecologic malignancy in the United States. It commonly presents in post-menopausal women with uterine bleeding. Workup begins with an endometrial sampling to pathologically determine the presence and progression of cancer. Sampling is typically done with endometrial biopsy and/or dilation and curettage. The Myosure device is an endometrial tissue removal system performed under hysteroscopic visualization with superior sample collection compared to dilation and curettage. However, this mechanical morcellator may denude the endometrium or affect the ability to render an accurate pathologic assessment prior to definitive surgical intervention. Specifically, studies have shown that morcellators similar to the Myosure device can spread cancerous cells. Therefore, our objective is to investigate whether the final pathology or recurrence rate of endometrial cancer is affected by the use of the Myosure device. 

Methods
The Myosure device was introduced to our institution in January 2013. Patients diagnosed with endometrial cancer and who received definitive hysterectomy from January 2013 to March 2021 were identified and reviewed. Demographic information, past medical history, predisposing factors, method of diagnosis, initial and final pathology, treatment, and prognostic outcomes were identified and reviewed. 618 patients were identified. 493 patients were included in the final analysis. 125 were excluded due to incomplete records, neoadjuvant therapy, or no definitive hysterectomy. Chi-Square and Fisher’s Exact tests were used for categorical variables. Continuous variables were assessed using the Mann-Whitney U and Kruskal-Wallis tests. All analyses were performed using SPSS v28.0. 

Results 
Diagnosis was made by EMB, DC, and MYO for 249, 151, and 93 women respectively. The majority were endometrioid type with grade 1 histology. Demographics were well-matched between MYO vs EMB/DC other than a higher age at diagnosis (66 vs 63, p < 0.05). Pre-operative ultrasound was performed more in the MYO group vs EMB/DC (81.5% vs 63.0%, p < 0.001). Final histology and tumor grade were well-matched, with a predominance of endometrioid histology in both cohorts. There were markedly fewer patients with final Stage IB disease and more patients with Stage III disease in the MYO group. There was a trend towards higher lymphovascular invasion (LVSI) in the MYO group as well. In all patients with Stage III disease, histology and tumor grade were well-matched, but the MYO cohort had significantly greater reports of LVSI (90.5% vs 62.9%, p < 0.05). The recurrence rate was even across both cohorts. There were higher rates of recurrence in the MYO group with Stage III disease.

Conclusion
The Myosure device does not appear to significantly impact final pathology or recurrence rate of endometrial cancer. Despite some interesting patterns reported, there was no statistically significant data to suggest definitive effects. Overall, the Myosure device appears to provide a safe procedure for endometrial cancer diagnosis without affecting final pathology or prognostic outcomes.

Pathology Findings and Prognostic Outcomes with Endometrial Cancer Diagnosis via Myosure

Objective: Robin sequence (RS) is a craniofacial anomaly characterized by a small jaw (micrognathia) with associated posterior displacement of the tongue, which in turn leads to airway obstruction. With advances in fetal imaging, fetuses with micrognathia may now be detected prenatally. This study aims to determine if differences in outcomes exist between infants with Robin sequence who had micrognathia identified on prenatal ultrasound and those who were not noted to have micrognathia until after delivery. 

Study Design: A retrospective case series with chart review was performed to examine a cohort of children with Robin sequence who received care at an urban tertiary medical center between 01/01/2010 and 12/31/2020. Demographic information as well as outcome metrics such as length of stay, number of hospital transfers, age at airway intervention, rates of oral feeding upon discharge home, and whether the child received early intervention services were assessed. 

Results: There were 47 infants with Robin sequence included in the analysis. 40.4% (n=19) were noted to have micrognathia / retrognathia on prenatal ultrasound, and 47.4% (n=9) of these 19 pregnancies were referred to a maternal fetal medicine program and subsequent prenatal consultation with a craniofacial team. As compared to those infants who were not diagnosed with micrognathia until after birth, the 19 infants identified prenatally were significantly less likely to require transfer from birth hospital (p= 0.02) and were more likely to be enrolled in early intervention services (p= 0.02) during infancy. Subgroup analysis of the 19 fetuses noted to have micrognathia suggests that those who were referred to a maternal fetal medicine program and underwent prenatal craniofacial team consultation had fewer hospital transfers and shorter overall lengths of stay when airway intervention was required. 

Conclusions: Prenatal recognition of micrognathia is associated with fewer hospital transfers for the infant prior to discharge home compared to diagnosis of Robin sequence after birth. When Robin sequence is suspected on ultrasound, prenatal consultation with a maternal fetal medicine program and an affiliated craniofacial team may facilitate transfer of perinatal care to a tertiary medical center, thereby avoiding subsequent separation of mother and baby should airway problems develop after delivery and transfer of the newborn be required.

Prenatal consultation for micrognathia improves timely diagnosis and management of Robin sequence

Background
Depression and anxiety are highly prevalent in American society. Patients undergoing infertility treatment are at especially high risk for depression and anxiety, and these effects may linger for decades. 

Objective
To determine the risk for anxiety and depression in women seeking IVF for primary infertility and analyze potential corresponding factors. 

Methods
Patients were English-speaking women with a diagnosis of primary infertility seeking first-time IVF recruited from a clinic in Spokane, WA between 4/2020 and 5/2022.
A total of 55 patients (aged 21-44) met criteria and consented to the study. They completed the initial Generalized Anxiety Disorder-7 scale (GAD-7) and Patient Health Questionnaire-9 (PHQ9) prior to treatment via phone. A positive screen for anxiety was a score ≥8, and a positive screen for depression was ≥5.

Results
A total of 22 patients screened positive for either anxiety or mild depression. 13 patients (23%) screened positive for anxiety, and 16 (29%) screened positive for mild depression. 
9 of the 13 patients who screened positive for anxiety, also screened positive for mild depression.
Patients who screened positive for anxiety were on average 31 years old (SD=3.7), while those who screened positive for mild depression were on average 31.5 (SD= 5.0).
29 patients had previously completed an intrauterine insemination, with 9 (31%) screening positive for symptoms of mild depression and 7 (24%) screening positive for anxiety. 

Conclusions
Within the general population 11.3% report regular feelings of anxiety and 4.5% report regular feelings of depression; however, our study showed a 23% prevalence of anxiety and a 29% prevalence of mild depression. There was no significant increased risk for anxiety or depression in those who had previously undergone intrauterine insemination, and the analysis of age as a corresponding factor was limited by the small sample size in this study.
Given the high rates of anxiety and depression seen in patients undergoing IVF compared to the general population, this patient population may benefit from additional mental health support and routine screening throughout treatment.

Screening for Anxiety and Depression in Women Undergoing First-time in Vitro Fertilization (Ivf) for Primary Infertility

Abstract Title
A New Case and Comprehensive Review of the Ophthalmic Manifestations of 172 Individuals with Branchio-Oculo-Facial Syndrome

Background
Branchio-oculo-facial syndrome (BOFS) is an autosomal dominant condition attributable to TFAP2A gene variants. Patients classically present with head and neck anomalies, ocular, and facial malformations. In 2008, TFAP2A gene mutations were first associated with BOFS. To help characterize the most common eye manifestations associated with the syndrome, we present a new case and the results of a comprehensive literature review where we identified the common genetic variants and ophthalmic findings among previously reported patients.

Methods
A search of the PubMed database with the term branchio-oculo-facial syndrome generated 378 articles. We included our patient and identified 172 total patients from the literature. Among these, 102 patients received molecular confirmation. Results were delineated by the following groups: clinical diagnosis only, those with confirmed molecular testing, and total numbers among both groups.

Results
We report a girl with a de novo pathogenic mutation in the TFAP2A gene consistent with BOFS and had similar ophthalmic findings to those reported in the literature, which included cataract, coloboma, hypertelorism, strabismus, and epicanthal folds. The most common pathogenic variants among our patient and individuals with molecular confirmation among past reports were p.R255G, p.A256V, p.R254W, and p.G251E. Common eye abnormalities associated with the syndrome in total numbers among both groups were nasolacrimal duct stenosis (n = 98, 57%), coloboma (n = 76, 46%), anophthalmia/microphthalmia (n = 64, 37%), and cataracts (n = 27, 16%).

Conclusion
Eye abnormalities presented were rather similar when comparing groups, for example, nasolacrimal duct stenosis (60% vs 55% in clinical only and molecular confirmation groups, respectively) and anophthalmia and/or microphthalmia (39% vs 36%) were two common findings among both groups with similar percentages. This supports that the cases identified by clinical suspicion alone without molecular confirmation were likely also accurate. Our patient also had epicanthal folds, a finding previously reported in only 3 patients. This finding could be an underreported ocular adnexa abnormality associated with the phenotype. This analysis provides a comprehensive review of common genetic variants and ophthalmic findings to characterize the most common eye manifestations associated with BOFS. Our report provides incentive to further investigate TFAP2A variants and identify genotype-phenotype correlations.

A New Case and Comprehensive Review of the Ophthalmic Manifestations of 172 Individuals with Branchio-Oculo-Facial Syndrome

Background
Glaucoma is the second leading cause of blindness, affecting 80 million patients globally including 3 million patients in the USA. Elevated intraocular pressure (IOP), caused by the blockage of aqueous humor (AH) outflow from the anterior chamber (AC), is an important risk factor in glaucoma. After medical treatments fail to control IOP, a glaucoma drainage device (GDD) is implanted. Unfortunately, current GDDs have major drawbacks: (1) large sizes requiring extensive surgical dissection, (2) uncontrolled release of AH risking vision loss, and (3) prolonged inflammation and fibrosis leading to 50% failure rate at 5 years. To address this clinical need, our aim was to develop a new GDD called the Squid Glaucoma microShunt (SGS). 
Methods
A 3D model of the SGS was generated using computer-aided-design software ANSYS. SGS is 90% smaller designed to release AH in a slow, sprinkle-like fashion minimizing the complications. To further reduce inflammation, SGS will be coated with a patented low-cost ultrananocrystalline diamond (UNCD) film exhibiting superb biocompatibility. Finally, a pair of patented micro forceps have been designed to facilitate easy insertion of the SGS promoting a 50% reduction in surgery time enabling surgeons to treat more patients efficiently.
We designed an in-silico ANSYS fluidic simulation to study fluid flow and pressure drop. The efficacy of UNCD was tested by coating it on an Ahmed® Shunt and inserting it in a rabbit eye. The control eye had an Ahmed® shunt without UNCD coating. 
Results
The in-silico ANSYS fluidic simulation showed that the SGS reduced IOP by 50% from 17 mmHg to 9 mmHg. At one year, the rabbit eye with a UNCD coated Ahmed® shunt was quiet with low IOP, while the uncoated eye shunt had failed within days because of intense inflammation and scarring. Several SGS and micro forceps prototypes have been fabricated using a Stratasys® 3D-printer and are awaiting laboratory testing. 
Conclusion
Due to the complications associated with current GDDs, only 40% of glaucoma patients undergo surgical interventions. Our minimally invasive SGS implant would make early surgical intervention an option for glaucoma patients with presumably better outcomes and prevent blindness.

Next from AMA Research Challenge 2022

Determining a Progesterone Threshold for Fresh IVF Cycles in a Military Facility

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