Dynamic Hippo pathway activity underlies mesenchymal differentiation during lung alveolar morphogenesis.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblast-associated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis.

Active Induction of a Multiple Sclerosis-Like Disease in Common Laboratory Mouse Strains.

Experimental autoimmune encephalomyelitis (EAE) is a neuroinflammatory disease with facets in common with multiple sclerosis (MS). It is induced in susceptible mammalian species, with rodents as the preferred hosts, and has been used for decades as a model to investigate the immunopathogenesis of MS as well as for preclinical evaluation of candidate MS therapeutics. Most commonly, EAE is generated by active immunization with central nervous system (CNS) antigens, such as whole CNS homogenate, myelin proteins, or peptides derived from these proteins. However, EAE actually represents a spectrum of diseases in which specific combinations of host/CNS antigen exhibit defined clinical profiles, each associated with unique immunological and pathological features. Similar to MS, EAE is a complex disease where development and progression are also modulated by environmental factors; therefore, the establishment of any given EAE variant can be challenging and requires careful optimization. Here, we describe protocols for three EAE variants, successfully generated in our laboratory, and provide additional information as to how to maintain their unique features and reproducibility.

Cerebellar pathology in multiple sclerosis and experimental autoimmune encephalomyelitis: current status and future directions.

Recent decades have witnessed significant progress in understanding mechanisms driving neurodegeneration and disease progression in multiple sclerosis (MS), but with a focus on the cerebrum. In contrast, there have been limited studies of cerebellar disease, despite the common occurrence of cerebellar symptoms in this disorder. These rare studies, however, highlight the early cerebellar involvement in disease development and an association between the early occurrence of cerebellar lesions and risk of worse prognosis. In parallel developments, it has become evident that far from being a region specialized in movement control, the cerebellum plays a crucial role in cognitive function, via circuitry connecting the cerebellum to association areas of the cerebrum. This complexity, coupled with challenges in imaging of the cerebellum have been major obstacles in the appreciation of the spatio-temporal evolution of cerebellar damage in MS and correlation with disability and progression. MS studies based on animal models have relied on an induced neuroinflammatory disease known as experimental autoimmune encephalomyelitis (EAE), in rodents and non-human primates (NHP). EAE has played a critical role in elucidating mechanisms underpinning tissue damage and been validated for the generation of proof-of-concept for cerebellar pathological processes relevant to MS. Additionally, rodent and NHP studies have formed the cornerstone of current knowledge of functional anatomy and cognitive processes. Here, we propose that improved insight into consequences of cerebellar damage in MS at the functional, cellular and molecular levels would be gained by more extensive characterization of EAE cerebellar pathology combined with the power of experimental paradigms in the field of cognition. Such combinatorial approaches would lead to improved potential for the development of MS sensitive markers and evaluation of candidate therapeutics.

Clinical utility of expanded non-invasive prenatal screening compared with chromosomal microarray analysis in over 8000 pregnancies without major structural anomaly.

OBJECTIVES: To evaluate the theoretical added value of two types of non-invasive prenatal screening (NIPS) expansions in pregnancies without major structural anomalies over the commonly used NIPS for chromosomes 13, 18, 21, X and Y (5-NIPS) and to compare them with the added value of chromosomal microarray analysis (CMA). METHODS: This was a retrospective cohort study based on CMA results of all pregnancies with normal ultrasound (including pregnancies with soft markers and with abnormal maternal serum screening) that had undergone amniocentesis between January 2013 to February 2022 and were registered in the database of the Rabin Medical Center genetic laboratory. We calculated the theoretical yield of 5-NIPS and compared the added value of expanded 5-NIPS for common microdeletions (1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.3-5p15.1, 15q11.2-15q13.1 and 22q11.2) and genome-wide NIPS (including variants > 5 Mb) with the added value of CMA in the overall cohort and in subgroups according to indication for invasive testing. RESULTS: Among the 8605 examined pregnancies, 122 (1.4%) clinically significant CMA results were demonstrated. Of these, 44 (36.1%) were theoretically detectable on 5-NIPS, with the rates of 1.56% in 642 pregnancies with abnormal maternal serum screening, 0.63% in 318 pregnancies with soft markers, 0.62% in 4378 women with advanced maternal age (≥ 35 years) and 0.15% in 3267 women younger than 35 years. In addition to aneuploidies detectable on 5-NIPS, three (0.03%) cases detectable on 5-NIPS expanded for common microdeletions and nine (0.10%) cases detectable on genome-wide NIPS (excluding common microdeletions) were identified in the overall cohort. The added value of expanded NIPS tools over 5-NIPS was significantly lower compared with that of CMA, for the overall cohort and subgroups. CONCLUSIONS: 5-NIPS and even genome-wide NIPS would miss 63.9% and 54.1% of clinically significant CMA findings, respectively. The added value of 5-NIPS expanded to detect common microdeletions over 5-NIPS is about 0.035%, and the overall added value of genome-wide NIPS aimed at large CNVs is about 0.14%, both much lower compared with the added value of CMA (0.91%). These findings should assist healthcare practitioners in guiding couples towards informed decision-making regarding the choice between prenatal invasive testing and NIPS. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Cooperative breeding behaviors in the Hawaiian Stilt (Himantopus mexicanus knudseni).

Cooperative breeding, which is commonly characterized by nonbreeding individuals that assist others with reproduction, is common in avian species. However, few accounts have been reported in Charadriiformes, particularly island-nesting species. We present incidental observations of cooperative breeding behaviors in the Hawaiian Stilt (Himantopus mexicanus knudseni), an endangered subspecies of the Black-necked Stilt (Himantopus mexicanus), during the 2012-2020 nesting seasons on the Hawaiian islands of O'ahu and Moloka'i. We describe two different behaviors that are indicative of cooperative breeding: (a) egg incubation by multiple adults; (b) helpers-at-the-nest, whereby juveniles delay dispersal and reproduction to assist parents and siblings with reproduction. These observations are the first published accounts of cooperative breeding in this subspecies and merit further investigation, as cooperative breeding may improve population viability of the endangered, endemic Hawaiian Stilt.

Mammal-exclusion fencing improves the nesting success of an endangered native Hawaiian waterbird.

Invasive predator control is often critical to improving the nesting success of endangered birds, but methods of control vary in cost and effectiveness. Poison-baiting or trapping and removal are relatively low-cost, but may have secondary impacts on non-target species, and may not completely exclude mammals from nesting areas. Mammal-exclusion fencing has a substantial up-front cost, but due to cost savings over the lifetime of the structure and the complete exclusion of mammalian predators, this option is increasingly being utilized to protect threatened species such as ground-nesting seabirds. However, non-mammalian predators are not excluded by these fences and may continue to impact nesting success, particularly in cases where the fence is designed for the protection of waterbirds, open to an estuary or wetland on one side. Thus, there remains a research gap regarding the potential gains in waterbird nesting success from the implementation of mammal-exclusion fencing in estuarine systems. In this study, we compared the nesting success of endangered Hawaiian Stilts (Ae'o; Himantopus mexicanus knudseni) within a mammal-exclusion fence to that of breeding pairs in a nearby wetland where trapping was the sole means for removing invasive mammals. We predicted success would be greater for breeding pairs inside the exclusion fence and the hatchlings inside the enclosure would spend more time in the nesting area than hatchlings at the unfenced site. During a single breeding season following construction of a mammal-exclusion fence, we used motion-activated game cameras to monitor nests at two sites, one site with mammal-exclusion fencing and one site without. Clutch sizes and hatch rates were significantly greater at the fenced site than the unfenced site, but time spent by chicks in the nesting area did not differ between sites. These results add to the mounting body of evidence that demonstrates the effectiveness of mammal-exclusion fencing in protecting endangered birds and suggests it can aid endangered Hawaiian waterbirds toward recovery. These results also suggest that the single greatest predatory threat to the Hawaiian Stilt may be invasive mammals, despite a host of known non-mammalian predators including birds, crabs, turtles, and bullfrogs, as the complete exclusion of mammals resulted in significant gains in nesting success. As additional fences are built, future studies are necessary to compare nesting success among multiple sites and across multiple seasons to determine potential gains in fledging success and recruitment.

Microarray analysis has no additional value in fetal aberrant right subclavian artery: description of 268 pregnancies and systematic literature review.

OBJECTIVES: Fetal aberrant right subclavian artery (ARSA) is a relatively common sonographic finding. Several studies have reported a significant association between ARSA and Down syndrome, as well as 22q11.2 microdeletion. The objective of this study was to assess the risk of abnormal chromosomal microarray analysis (CMA) findings in a large cohort of pregnancies with fetal ARSA as an isolated, as well as a non-isolated, sonographic anomaly. A secondary objective was to review the literature, examining the frequency of chromosomal microarray aberrations in fetuses with isolated ARSA. METHODS: Data from all pregnancies referred for invasive testing and CMA due to sonographic diagnosis of fetal ARSA, between 2013 and 2017, were obtained retrospectively from the computerized database of the Israeli Ministry of Health. The rate of clinically significant CMA findings in these fetuses was compared to that in a local control population of 2752 low-risk pregnancies with normal ultrasound and serum screening results. In addition, a literature search was conducted in PubMed, from inception to February 2018, of original studies in the English language describing the frequency and nature of microscopic and submicroscopic aberrations in fetuses with isolated ARSA. RESULTS: Of 246 pregnancies with isolated ARSA that underwent CMA analysis, a clinically significant finding was detected in one (0.4%) pregnancy (trisomy 21). This rate did not differ significantly from that in the control population (P = 0.1574). Of 22 fetuses with non-isolated ARSA, one (4.5%) additional case of trisomy 21 was noted. The frequency of trisomy 21 in this cohort also did not differ from that in the control population (relative risk, 5.5 (95% CI, 0.8-37.6)). The literature search yielded 13 additional relevant papers, encompassing 333 cases of isolated ARSA. Of 579 cases overall (including those of the present study), 13 (2.2%) cases of trisomy 21 were detected, with no cases of 22q11.2 microdeletion. CONCLUSION: While an association may exist between non-isolated ARSA and Down syndrome, isolated ARSA might better serve as a soft marker for Down syndrome, rather than a routine indication for invasive prenatal testing. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Indications for episiotomy performance - a cross-sectional survey and review of the literature.

Our objective was to assess the reported reasons for episiotomy performance in Israel and to review the relevant professional literature. Using anonymous questionnaires, a survey was conducted among obstetricians and midwives in four northern Israel hospitals, and the accoucheurs were asked to score their agreement with 13 proposed indications for episiotomy. Overall, 84 doctors and 32 midwives completed the questionnaires. 86.1% of the responders reported performing episiotomy in all or most cases of shoulder dystocia, and more than half reported performing it in most cases of vacuum deliveries, fetal macrosomia and advanced perineal tear in previous delivery. Subjective assessment of perineal characteristics constituted a justified reason for episiotomy for 15.8-43.9% of the accoucheurs. In conclusion, there is a wide variation in reported reasons for episiotomy between the obstetricians, and many of these indications are not congruent with international practice guidelines. Uniform protocols and educational programmes are needed to guide episiotomy practice.

Morbidity associated with episiotomy in vacuum delivery: a systematic review and meta-analysis.

BACKGROUND: The role of episiotomy in vacuum deliveries is controversial. OBJECTIVES: To perform a meta-analysis of the literature examining this subject. SEARCH STRATEGY: The search was conducted in four databases. SELECTION CRITERIA: Two investigators independently selected original research examining the effects of episiotomy on any neonatal and maternal outcomes during vacuum delivery. DATA COLLECTION AND ANALYSIS: The effect estimates were presented as odds ratios (ORs) with 95% confidence intervals (95% CIs). MAIN RESULTS: Fifteen articles were included, encompassing a total of 350 764 vacuum deliveries. A non-significant relationship was shown between mediolateral episiotomy and obstetric anal sphincter injuries (OASIS) in nulliparous women (OR 0.68, 95% CI 0.43-1.07; six studies), whereas an increased risk was demonstrated in parous women (OR 1.27, 95% CI 1.05-1.53; two reports). A higher risk of OASIS with median episiotomy use was shown in nulliparous (OR 5.11, 95% CI 3.23-8.08; two studies) as well as in parous (OR 89.4, 95% CI 11.8-677.1; one study) women. Lateral episiotomy was related to lower OASIS risk in nullipara (OR 0.59, 95% CI 0.49-0.70; single paper). Mediolateral episiotomy was linked to increased rates of postpartum haemorrhage (OR 1.82, 95% CI 1.16-2.86) and analgesia use (OR 2.10, 95% CI 1.39-3.17; two reports). Overall, the quality of evidence was rated as low to very low. AUTHOR'S CONCLUSIONS: Mediolateral and median episiotomy in parous woman may increase the rate of OASIS at vacuum delivery, whereas lateral episiotomy in nulliparous women could be associated with a decreased risk of OASIS. The suboptimal quality of the available evidence necessitates high-quality well-designed randomised trials. TWEETABLE ABSTRACT: Episiotomy in vacuum delivery does not appear to be of benefit, and might even increase maternal morbidity.

Análisis de mutaciones en regiones regulatorias del gen CYP21A2 como causa de la deficiencia de 21-hidroxilasa: correlación genotipo-fenotipo / Mutation analisys in regulatory regions of CYP21A2 gen as a cause of 21-hydroxylase deficiency

INTRODUCCIÓN En trabajos previos se detectaron dos variantes de secuencias en regiones regulatorias, en pacientes con deficiencia de 21-hidroxilasa no clásicos (NC) de la población estudiada, aunque su posible patogenicidad no fue examinada. Paralelamente, se observó que muchos pacientes NC poseen sólo un alelo con mutación o ninguno. OBJETIVOS Ensayar in vitro la funcionalidad de variantes regulatorias del gen y realizar una correlación genotipo-fenotipo de los pacientes con diagnóstico de hiperplasia suprarrenal congénita (HSC) NC. MÉTODOS Mutagénesis dirigida de un vector con la región regulatoria clonada río arriba del gen de luciferasa, transfección en líneas adrenales en cultivo y medición de la actividad del gen reportero. Para la correlación con el fenotipo se agruparon los genotipos de 271 pacientes en 5 categorías: a) 2 alelos leves; b) 1 alelo leve y 1 severo; c) sólo 1 alelo leve; d) sólo 1 alelo severo; e) sin mutaciones. Para cada grupo se analizó la edad de aparición de la sintomatología y los valores de 17-OHP basal y post ACTH, androstenediona, testosterona y dehidroepiandrosterona. RESULTADOS Se obtuvo una disminución significativa (p<0,001) en la capacidad de estimulación de la transcripción entre el vector salvaje (100%) y el mutado A>G (65,5 ± 5,1%). En la correlación genotipo-fenotipo, sólo se observaron diferencias significativas para los valores de 17-OHP basal y post ACTH en los pacientes con 2 alelos mutados respecto del resto. DISCUSIÓN Por primera vez se demuestra la presencia de una variante en una de las regiones regulatorias del gen CYP21A2, que modula en forma negativa su tasa transcripcional. Los resultados obtenidos de la correlación genotipo-fenotipo sugieren que el valor hormonal utilizado como corte incluiría a pacientes portadores como afectados y que el estudio molecular sería necesario para un correcto asesoramiento genético en individuos con hiperandrogenismo.

Defectos genómicos como causa de cardiopatías congénitas / Genomic abnormalities as a cause of congenital heart defects

INTRODUCCIÓN Las cardiopatías congénitas (CC) son causadas por el desarrollo anómalo del corazón durante el período embrio-fetal y representan las anomalías congénitas más frecuentes. Si bien la etiología de las CC es heterogénea, los factores genéticos juegan un rol preponderante tanto en casos esporádicos como hereditarios. OBJETIVOS Caracterizar las causas genéticas asociadas a CC conotroncales (CCC) en una muestra de afectados de diferentes regiones argentinas. MÉTODOS En el período comprendido entre mayo de 2013 y mayo de 2014, se incluyó a 80 pacientes provenientes de cuatro hospitales de diferentes jurisdicciones argentinas: Resistencia (Chaco), La Plata (Provincia de Buenos Aires) y las ciudades capitales de las provincias de Neuquén y Salta. Se recogieron muestras para estudios de cariotipo y el análisis de la deleción 22q11 por FISH (Fluorescence in situ hybridization). Asimismo, se analizó la presencia de anomalías genómicas mediante la utilización de dos kits de MLPA (Multiplex Ligation Probe Amplification)- Resultados Al realizar el cariotipo, no se observaron anomalías cromosómicas entre los niños analizados. Sin embargo, el 39% de los afectados poseía alguna anomalía genómica. En 16 pacientes se halló la deleción 22q11. El 50% de los niños con interrupción de arco aórtico presentó esta deleción, que se halló con mayor frecuencia entre aquellos pacientes que presentaban al menos otra anomalía mayor asociada. En ningún paciente con transposición de grandes vasos se halló la deleción 22q11. En 14 pacientes se observó otra anomalía genómica diferente (la más frecuente, desbalances en 17p). DISCUSIÓN Este estudio relevó por primera vez la presencia de anomalías genómicas como causa de CCC a partir de afectados de diferentes regiones argentinas. Se identificó la presencia de la deleción 22q11 en el 21% de los afectados con CCC, y en el 18% de los pacientes se observó la presencia de otros desbalances. El estudio, además, permitió estimar la eficiencia de diferentes metodologías de análisis

Estudios de la región 5'UTR del gen FMR-1 en pacientes con falla ovárica prematura / Studies of the 5' - UTRTR region in the FMR-1 gene in patients withe Premature Ovarian Failure

La falla ovárica prematura (FOP) es un síndrome de patogénesis multicausal que afecta aproximadamente al 1% de las mujeres en edad reproductiva. Numerosos estudios asocian el estado de premutación (amplificación del número de tripletes CGG entre 50/55 y 200 repeticiones) en el gen FMR-1 y FOP. Alrededor de un 4% de las pacientes FOP presentan alelos con premutación. La amplificación del número de tripletes por encima de 200 repeticiones causa el Síndrome de Fragilidad del X (SFX). El objetivo del presente trabajo fue estudiar la región 5´ no codificante del gen en un grupo de pacientes FOP de Argentina. La región de interés se amplificó por PCR a partir de muestras de ADN de 100 pacientes FOP y 145 mujeres controles. Los alelos de las pacientes y controles fueron agrupados en 7 categorías de acuerdo al número de tripletes obtenidos. Se observó que el número de repeticiones más frecuente se encuentra en el rango de 26 a 30 tripletes, tanto en pacientes como en controles. En el grupo de pacientes FOP, 5/197 (2.6%) alelos no relacionados estudiados presentaron un número de tripletes CGG mayor a 50, mientras que sólo 1 de 290 (0.34%) para el grupo control. Todas las pacientes FOP con valores de tripletes CGG mayor a 50 presentaron amenorrea secundaria. Estos resultados están en concordancia con lo comunicado para otras poblaciones acerca de la existencia de una asociación entre la premutación del gen FMR-1 y el desarrollo de FOP. Asimismo, los resultados obtenidos refuerzan la importancia de la genotipificación del gen FMR-1 en las pacientes FOP, a los efectos de estimar el riesgo de su descendencia para el SFX.

Premature ovarian failure (POF) is a syndrome of multicausal pathogenesis that affects 1% of women before the age of 40. Several studies associate the premutation state (CGG repeats increased in number between 50/55 and 200) in the FMR-1 gene and POF. About 4% of POF women have alleles in the FMR-1 gene in the permutation range. An increase above 200 in the number of triplets in this gene causes the Fragile X Syndrome (FXS). The purpose of the present study was to analyze the 5´untranslated region of the FMR-1 gene in a group of patients from Argentina. The region of interest was amplified by PCR from DNA samples of 100 POF patients and 145 control women. Alleles from controls and patients were grouped in 7 categories according to the number of triplets obtained. We observed that the most frequent number of repeats ranged from 26 to 30 triplets, in both patient and control groups. In the POF group, 5 out of 197 (2.6%) not related alleles presented a number of CGG triplets higher than 50, while only 1 out of 290 (0.34%) was present in controls. All POF patients with a number of CGG repeats higher than 50 presented secondary amenorrhea. These results are in accordance with previous reports from other populations showing an association between the premutation state in the FMR-1 gene and POF development. In addition, these results reinforce the importance of genotyping POF patients to estimate the risk of their offspring for Fragile X Syndrome.

Modification of neutrophil function by plasma of rheumatoid arthritis patients treated with infliximab.

OBJECTIVE: To examine whether the release of superoxide anions from neutrophils of healthy donors was affected when incubated with plasma from infliximab-treated rheumatoid arthritis (RA) patients. METHODS: Fifteen consecutive seropositive RA patients were treated with 3mg/kg infliximab on weeks 0, 2, 6, and 14. Disease activity was assessed by DAS28 score and by IL-6 level. Neutrophils from healthy donors were incubated with plasma drawn before each infliximab treatment. PMA-stimulated superoxide release was measured by the ferricytochrome C reduction method. RESULTS: 53% of the patients had a favorable clinical response. IL-6 levels showed a significant decline at week two, with a gradual increase thereafter. Treatment with infliximab did not change the superoxide production. However, when the group was divided retrospectively to responders (DeltaDAS28 > -1.2) and non-responders (DeltaDAS28 < -1.2), two different patterns were seen, although the pre-treatment levels were similar: Among the responders IL-6 remained low at its 2 weeks level till week 14, while in the non responders IL-6 increased 3 times (P < 0.03) from week 2 to 14. The responders showed mild, but continuous, reduction of superoxide release, while in the non-responders it increased significantly from week 2 on. CONCLUSION: The reduction in IL-6 in RA sera following anti-TNFalpha therapy has little influence on the capacity of these sera to stimulate healthy neutrophils to produce superoxide, suggesting the existence of non-TNFalpha non-IL-6 dependent neutrophil-stimulating mediators in RA sera. The increasing level of IL-6 among the non-responders after initial dramatic decline might represent an escape phenomenon, possibly caused by alternative mediator(s). Clinically, this IL-6 "escape" might be used as a tool for early identification of responders from non-responders.

High-resolution gamma-ray radiography produced by a laser-plasma driven electron source.

An electron beam from a laser-plasma accelerator is converted into a gamma-ray source using bremsstrahlung radiation in a dense material. The gamma-ray beam has a pointlike source size because it is generated by a high quality electron beam with a small source size and a low divergence. Using this gamma-ray source, the radiography of complex and dense objects with submillimeter resolution is performed. It is the first evidence of a gamma-ray source size of a few hundreds micrometers produced with laser-driven accelerators. This size is consistent with results from Monte Carlo simulations.

Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis.

Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles. The aim of the present study was to examine the in vitro effect of a GnRH-a (leuprolide acetate, LA) and its interactions with FSH, dibutyryl cAMP, and growth factors (IGF-I, EGF, and FGF) on follicular apoptosis in early antral ovarian follicles obtained from prepubertal DES- treated rats. Follicles cultured 24 hr in the absence of hormones showed spontaneous onset of apoptotic DNA fragmentation. The presence of FSH suppressed the spontaneous onset of apoptotic DNA fragmentation (75-85%). Quantitative estimation of DNA cleavage from ovarian follicles revealed no significant changes in DNA fragmentation after in vitro LA treatment (1-100 ng/ml). However, coincubation with LA interfered partially with the effects of FSH on apoptosis suppression. This apoptosis suppression was also obtained by treatment with dibutyryl cAMP (80%), and was partially prevented by the presence of LA in the cultures. Follicles were cultured 24 hr with FGF, EGF, or IGF-I, and these factors suppressed DNA fragmentation (70, 60, and 70% respectively), while the presence of LA (100 ng/ml) in the culture medium prevented this effect. In conclusion, we show that the rescue from apoptotic DNA fragmentation produced in early antral follicles by FSH, cAMP, and growth factors, is prevented by coincubation with LA. This GnRH analog would thus interfere in the pathway of FSH, cAMP and/or growth factors by an as yet unknown mechanism.

Putative association of a mutant ROM1 allele with retinitis pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous form of retinal degeneration. Several genes and loci have been shown to be involved in the disease, although each of them only accounts for a few cases. Mutations in the gene encoding ROM1, a rod-specific protein, have been putatively associated with several forms of RP. Here we describe a double-mutant allele of this gene, P60T and T108M, present in two affected sibs and also in two healthy members of a Spanish RP family. The same double-mutant allele was previously considered to be responsible for autosomal dominant RP in one family. We now report data that question the potential pathogenicity of these two ROM1 mutations.

Human granulosa cells express HLA-DR antigen and are capable of synthesizing interleukin-1.

To study the origin of interleukin 1 (IL-1) present in human follicular fluid we determined the percentage of macrophages (MO) and cells with HLA-DR antigen (DR+) present in 22 samples of human follicular fluid (FF) from women undergoing in vitro fertilization, and examined the release of IL-1 beta by cultures of purified human granulosa cells (GC). The number of red blood cells (RBC) in the crude preparation was taken as a measure of possible contamination with peripheral blood monocytes (assuming a ratio of one monocyte or MO per 10(4) RBC). For the evaluation of MO and DR+ cells percentages we employed an indirect immunofluorescence technique using specific monoclonal antibodies. Total cells from FF were purified by Ficoll-Hypaque density gradient centrifugation (delta = 1.076 g/l and GC were purified using a gradient delta = 1.065 g/l. This method reduced the contamination with MO to 0-1%. The spontaneous release of IL-1 beta was measured by ELISA. We found that FF contained 9.81 +/- 1.47% of MO but only 7.85% were ovarian MO. In addition the total percentage of DR+ cells was 17.13 +/- 2.35% but only 9.81% corresponded to MO. Therefore about 7.32% of DR+ cells could be GC. Then purified GC (10(4)/0.2 ml/well) were cultured during 24 hours at 37 degrees C in serum free medium (DMEM:F12). IL-1 beta levels were 84 +/- 17 pg/ml and these values were increased by 44% when GC were stimulated with FSH (200 ng/ml). These results suggest that GC produced IL-1 beta and that the synthesis of this cytokine might be regulated by hormones.

Regulation of the steroidogenic response of cultured human granulosa cells: effects of serum and 25-hydroxycholesterol.

OBJECTIVE: To examine the effect of serum and 25-hydroxycholesterol on steroidogenesis in cultured human granulosa cells from women undergoing assisted fertilization. DESIGN: Retrospective. SETTING: Private Fertility Clinic and National Research Institute. PATIENTS: Women undergoing IVF-ET or GIFT programs. RESULTS: In serum-free medium P production decreased significantly with culture time (2, 4, 6, and 8 days: 566 +/- 128, 161 +/- 50, 71 +/- 16, and 36 +/- 7 ng/mL P, respectively; conversion factor to SI unit, 3.180; mean +/- SEM). The addition of 25-hydroxycholesterol (10 micrograms/mL), a substrate for steroidogenesis, did not prevent the decrease in P levels. However, P production was greater in the presence of this substrate at all times. The presence of fetal bovine serum (10% FBS) in the cultures allowed the maintenance of 75% of P production with respect to the initial time considered (at which maximal P values are detected). Cultured granulosa cells treated with 10 ng/mL LH in the presence of FBS showed an increase in the percentage of stimulation with culture time (2, 4, and 7 days: 2.4%; 54.8%, and 55.1%, respectively). This effect was not observed when 25-hydroxycholesterol was added to the cultures. Similar results to that obtained by LH were attained when steroidogenesis was stimulated with 0.1 mM dibutyryl cyclic adenosine 3':5' monophosphate (cAMP). In addition, cAMP production in response to 100 ng/mL LH in the presence of 0.1 mM methyl-isobutyl-xanthine decreased with culture time, showing a time dependency similar to that observed for P. CONCLUSION: Our results demonstrate that the decrease in granulosa cell steroidogenic activity with culture time is inhibited by serum but not by 25-hydroxycholesterol, suggesting that other factors despite LH and cholesterol are necessary to support the luteal function.