Histone deficiency and hypoacetylation in the aging retinal pigment epithelium.

Histones serve as a major carrier of epigenetic information in the form of post-translational modifications which are vital for controlling gene expression, maintaining cell identity, and ensuring proper cellular function. Loss of histones in the aging genome can drastically impact the epigenetic landscape of the cell leading to altered chromatin structure and changes in gene expression profiles. In this study, we investigated the impact of age-related changes on histone levels and histone acetylation in the retinal pigment epithelium (RPE) and retina of mice. We observed a global reduction of histones H1, H2A, H2B, H3, and H4 in aged RPE/choroid but not in the neural retina. Transcriptomic analyses revealed significant downregulation of histones in aged RPE/choroid including crucial elements of the histone locus body (HLB) complex involved in histone pre-mRNA processing. Knockdown of HINFP, a key HLB component, in human RPE cells induced histone loss, senescence, and the upregulation of senescence-associated secretory phenotype (SASP) markers. Replicative senescence and chronological aging in human RPE cells similarly resulted in progressive histone loss and acquisition of the SASP. Immunostaining of human retina sections revealed histone loss in RPE with age. Acetyl-histone profiling in aged mouse RPE/choroid revealed a specific molecular signature with loss of global acetyl-histone levels, including H3K14ac, H3K56ac, and H4K16ac marks. These findings strongly demonstrate histone loss as a unique feature of RPE aging and provide critical insights into the potential mechanisms linking histone dynamics, cellular senescence, and aging.

Unraveling Histone Loss in Aging and Senescence.

As the global population experiences a notable surge in aging demographics, the need to understand the intricate molecular pathways exacerbated by age-related stresses, including epigenetic dysregulation, becomes a priority. Epigenetic mechanisms play a critical role in driving age-related diseases through altered gene expression, genomic instability, and irregular chromatin remodeling. In this review, we focus on histones, a central component of the epigenome, and consolidate the key findings of histone loss and genome-wide redistribution as fundamental processes contributing to aging and senescence. The review provides insights into novel histone expression profiles, nucleosome occupancy, disruptions in higher-order chromatin architecture, and the emergence of noncanonical histone variants in the aging cellular landscape. Furthermore, we explore the current state of our understanding of the molecular mechanisms of histone deficiency in aging cells. Specific emphasis is placed on highlighting histone degradation pathways in the cell and studies that have explored potential strategies to mitigate histone loss or restore histone levels in aging cells. Finally, in addressing future perspectives, the insights gained from this review hold profound implications for advancing strategies that actively intervene in modulating histone expression profiles in the context of cellular aging and identifying potential therapeutic targets for alleviating a multitude of age-related diseases.

Evaluation of Pleurotus Mushroom Effects on Histopathological Changes in Organs of Diabetic Rats.

Diabetes mellitus (DM) is a metabolic disorder that can be categorized mainly into type 1 and type 2. Diabetes type 1 is caused due to ß-cell destruction, whereas type 2 is caused by the resistance of cell receptors. Many therapies are available for the management of diabetes, but they have some side effects, and as a result of this, people are attracted to natural treatments. Pleurotus mushrooms are well documented for their medicinal attributes and their role in the treatment of diseases like cancer, infectious disease, neurodiseases, and inflammatory disease. The protective mechanism of the Pleurotus fossulatus (P. fossulatus) mushroom and its detailed histological study on kidneys and the liver in diabetic conditions were unexplored. The present study evaluated the effects of P. fossulatus aqueous extract on histological changes in the diabetic rat model. Male Wistar albino rats were used to create the diabetic model by using streptozotocin (STZ) intraperitoneal (IP) injection. The animals were separated into five different groups, with six animals in each. Only group I, animals that did not receive STZ, was considered a normal control. Group II was a diabetic control and received normal saline, and group III was a drug control and received metformin as a standard drug. Groups IV and V were dosing groups, which received the aqueous extract of P. fossulatus in 250 mg/kg and 500 mg/kg of body weight concentrations, labeled as T1 and T2 groups, respectively. The T1 and T2 groups clearly showed their potential to reverse the histopathological changes in the kidney and liver. However, the T2 group was more effective than the T1 group, as results indicate that functions of the glomerulus and its structural deformity were restored to their near-natural form in the T2 group. In the case of the liver, the histological changes like the dilatation of sinusoids, more numbers of the Kupffer cell formation, and necrosis were restored in the T2 group. All these results proved the potential of P. fossulatus against the side effects of diabetes. It could protect the organs from developing diabetic nephropathy (DN) and liver-related diseases like cirrhosis and nonalcoholic fatty liver disease (NAFLD).

Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.

Computational exploration of the dual role of the phytochemical fortunellin: Antiviral activities against SARS-CoV-2 and immunomodulatory abilities against the host.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections generate approximately one million virions per day, and the majority of available antivirals are ineffective against it due to the virus's inherent genetic mutability. This necessitates the investigation of concurrent inhibition of multiple SARS-CoV-2 targets. We show that fortunellin (acacetin 7-O-neohesperidoside), a phytochemical, is a promising candidate for preventing and treating coronavirus disease (COVID-19) by targeting multiple key viral target proteins. Fortunellin supports protective immunity while inhibiting pro-inflammatory cytokines and apoptosis pathways and protecting against tissue damage. Fortunellin is a phytochemical found in Gojihwadi kwath, an Indian traditional Ayurvedic formulation with an antiviral activity that is effective in COVID-19 patients. The mechanistic action of its antiviral activity, however, is unknown. The current study comprehensively evaluates the potential therapeutic mechanisms of fortunellin in preventing and treating COVID-19. We have used molecular docking, molecular dynamics simulations, free-energy calculations, host target mining of fortunellin, gene ontology enrichment, pathway analyses, and protein-protein interaction analysis. We discovered that fortunellin reliably binds to key targets that are necessary for viral replication, growth, invasion, and infectivity including Nucleocapsid (N-CTD) (-54.62 kcal/mol), Replicase-monomer at NSP-8 binding site (-34.48 kcal/mol), Replicase-dimer interface (-31.29 kcal/mol), Helicase (-30.02 kcal/mol), Papain-like-protease (-28.12 kcal/mol), 2'-O-methyltransferase (-23.17 kcal/mol), Main-protease (-21.63 kcal/mol), Replicase-monomer at dimer interface (-22.04 kcal/mol), RNA-dependent-RNA-polymerase (-19.98 kcal/mol), Nucleocapsid-NTD (-16.92 kcal/mol), and Endoribonuclease (-16.81 kcal/mol). Furthermore, we identify and evaluate the potential human targets of fortunellin and its effect on the SARS-CoV-2 infected tissues, including normal-human-bronchial-epithelium (NHBE) and lung cells and organoids such as pancreatic, colon, liver, and cornea using a network pharmacology approach. Thus, our findings indicate that fortunellin has a dual role; multi-target antiviral activities against SARS-CoV-2 and immunomodulatory capabilities against the host.

Biomolecules of mushroom: a recipe of human wellness.

The Indian system of medicine - Ayurveda says "When diet is wrong, medicine is of no use. When diet is correct, medicine is of no use". In this context, mushroom constitutes one of the major resources for nutraceuticals. Biomolecules of mushrooms have attracted the attention of researchers around the globe due to their proven healthy attributes. They have a plenitude of health-giving properties and these range from immunomodulatory, antiviral, antibacterial, antifungal, antioxidant, anti-inflammatory, antitumor, anticancer, anti-HIV, antidiabetic, anticholesterolic to antiarthritic activities.Mushrooms contain both primary and secondary metabolites. The primary metabolites provide energy while the secondary metabolite exhibits medicinal properties. Hence, the mushroom can be a recipe for human wellness and will play a significant role in fighting COVID-19 pandemics and other infectious diseases.The key findings suggested in this paper refer to the exploration of health and the healing traits of biomolecules of mushrooms. This article reviews the current status of the medicinal attributes of mushrooms and their biomolecules in different diseases such as cardiovascular, diabetes, reproductive diseases, cancer, and neurodegenerative diseases. The global malnutrition-related morbidity and mortality among children under five and lactating women presents a frightening picture and also a black spot on the human face. Malnutrition is responsible for more ill-health than any other cause. Mushrooms as a rich source of bioactive compounds can be claimed as "Best from the Waste" since they grow on the most abundant organic wastes of the Earth, the lignocellulosic substrate, and 'Best of the Rest' because they are excellent nutraceutical resources.

Investigation of soft and living matter using a micro-extensional rheometer.

Rheological properties of a material often require to be probed under extensional deformation. Examples include fibrous materials such as spider-silk, high-molecular weight polymer melts, and the contractile response of living cells. Such materials have strong molecular-level anisotropies which are either inherent or are induced by an imposed extension. However, unlike shear rheology, which is well-established, techniques to perform extensional rheology are currently under development and setups are often custom-designed for the problem under study. In this article, we present a versatile device that can be used to conduct extensional deformation studies of samples at microscopic scales with simultaneous imaging. We discuss the operational features of this device and present a number of applications.

Effect of Pleurotus fossulatus Aqueous Extract on Biochemical Properties of Liver and Kidney in Streptozotocin-Induced Diabetic Rat.

BACKGROUND: The antidiabetic effects of the Pleurotus fossulatus on liver and kidney were unexplored. The present study assessed the vital effects of P. fossulatus aqueous extract (PFA-extract) on liver and kidney function in diabetic rats model. METHODS: The albino Wistar rats were divided into five groups with six animals in each. Group, I, II, and III were normal, diabetic, and drug control, respectively, and groups IV and V were test groups. As treatment dose, group II received saline, and group III (drug control) received metformin in 100 mg/kg of body weight as a standard drug. Whereas, group IV (D1) and V (D2) were test groups, received PFA-extract in 250 mg/kg, and 500 mg/kg of body weight, respectively. The changes in body weight, blood glucose level (BGL), lipid profile, liver, and kidney biochemical parameters were evaluated. RESULTS: The PFA-extract dose at 500 mg/kg in D2 groups showed very good effects. The body weight was recovered by 97.9% and blood glucose level (BGL) was reduced to 53% in the D2 group. For the lipid profile, total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) were estimated in blood plasma and their values were 92.31±3.788, 80.85±8.962, and 31.35±1.781, respectively. PFA-extract improved the liver function for which aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were evaluated and recorded to their normal range in D2 group which were 132.01±5.553, 58.63±4.390, and 143.26±2.423, respectively. For the kidney function test, creatinine (CRT), blood urea nitrogen (BUN), and uric acid were evaluated and in the D2 group, it was significantly reduced ie, 0.656±0.0707, 15.13±1.463, and 6.27±0.325, respectively. CONCLUSION: These attributes of PFA-extract make it a potential natural agent to provide protection to the liver and kidney and also reduce the BGL and control the total lipid in type 1 diabetes condition.

The axonal actin-spectrin lattice acts as a tension buffering shock absorber.

Axons span extreme distances and are subject to significant stretch deformations during limb movements or sudden head movements, especially during impacts. Yet, axon biomechanics, and its relation to the ultrastructure that allows axons to withstand mechanical stress, is poorly understood. Using a custom developed force apparatus, we demonstrate that chick dorsal root ganglion axons exhibit a tension buffering or strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon to show that the recently discovered membrane-associated actin-spectrin scaffold plays a prominent mechanical role. Finally, using a theoretical model, we argue that the actin-spectrin skeleton acts as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to release excess mechanical stress. Our results revise the current viewpoint that microtubules and their associated proteins are the only significant load-bearing elements in axons.

Strain softening and stiffening responses of spider silk fibers probed using a Micro-Extension Rheometer.

Spider silk possesses unique mechanical properties like large extensibility, high tensile strength, super-contractility, etc. Understanding these mechanical responses requires characterization of the rheological properties of silk beyond the simple force-extension relations which are widely reported. Here we study the linear and non-linear viscoelastic properties of dragline silk obtained from social spider Stegodyphus sarasinorum using a Micro-Extension Rheometer that we have developed. Unlike continuous extension data, our technique allows for the probing of the viscoelastic response by applying small perturbations about sequentially increasing steady-state strain values. In addition, we extend our analysis to obtain the characteristic stress relaxation times and the frequency responses of the viscous and elastic moduli. Using these methods, we show that in a small strain regime (0-4%) dragline silk of social spiders shows a strain softening response followed by a strain stiffening response at higher strains (>4%). The stress relaxation time, on the other hand, increases monotonically with increasing strain for the entire range. We also show that the silk stiffens while ageing within the typical lifetime of a web. Our results demand the inclusion of the kinetics of domain unfolding and refolding in the existing models to account for the relaxation time behavior.

Role of edible mushroom as a potent therapeutics for the diabetes and obesity.

Diabetes and obesity are the most frequently found disease worldwide. Several factors are responsible for obesity, i.e., imbalance in energy expenditure, environmental factors, feeding habit, lifestyle, etc., which can also be responsible for type 2 diabetes mellitus. There are several synthetic drugs available to combat these diseases which have some side effects on sufferers. Therefore, people are shifting towards inexpensive, effective, widely available natural and herbal medicines. Edible mushrooms, which have been used from ancient time to cure these diseases, contain anti-oxidant, fibers, triterpenoids, alkaloid, and other phytochemicals. Comatin, ß-glucan, Tremellastin, and Lentinan KS-2 are active chemicals of mushrooms which show great effect on diabetes mellitus and obesity by modulating either cellular function or biochemical pathways. Here, in this review, we have discussed the potential role of edible mushrooms and its biochemicals in control of diabetes and obesity. Using Bioinformatics, we can find the specific targets of theses biochemicals, so that these can be more effective.

Molecular mechanistic insight of hepatitis B virus mediated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer which occurs in liver and severity of this cancer makes it the sixth most prevalent cancer and second leading cause of death among all cancers. The load of hepatitis-B virus (HBV) in serum is one of the important risk factors for the HCC. Several other factors also contribute to the HBV associated malignant hepatoma (HCC) i.e. HBV mutation, integration and condition of the host. Transformation of the liver to HBV-associated HCC usually accompanies long-run symptoms i.e. inflammation and cirrhosis of the liver and infective agent load could be a vigorous prognosticator for each incidence and progression of this carcinoma. One of the prominent factors i.e. HBV X supermolecule (HBx) interferes with many signal pathways that are related to the proliferation and apoptosis of hepatic cells. Besides, HBx C-terminal truncation is also responsible for HCC. Longtime HBV infection causes risk of HCC; thus most of the study related to HBV (85%) is limited to HBV endemic regions. In this review, we have outlined the molecular mechanisms that come from other than HBV endemic places which can be innovative approaches to treat HCC.

Whole mitochondrial genome analysis in South Indian patients with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a mitochondrial DNA (mtDNA) associated neurodegenerative disorder of retinal ganglion cells. In this study, whole mitochondrial genome sequencing of 75 LHON patients and 40 controls was performed to identify the mutation frequency and haplogroup background of South Indian population. Analysis of mtDNA revealed 559 different variants in LHON patients, including 7 pathogenic mutations, 30 private, and 22 other disease associated variants. A significantly higher (p=0.0008) overall variation load per individual was noted among LHON patients versus controls. We reported for the first time, the association of M haplogroup (p=0.028) with LHON in this cohort.

Evaluation of genetic polymorphisms in clusterin and tumor necrosis factor-alpha genes in South Indian individuals with pseudoexfoliation syndrome.

PURPOSE: The aim of this study was to explore the potential association of genetic variants across clusterin (CLU) and tumor necrosis factor-alpha (TNF-α) genes in South Indian individuals with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG). MATERIALS AND METHODS: A total of 523 individuals including 299 unrelated cases (150 PEXS and 149 PEXG) and 224 age- and ethnically-matched healthy controls were recruited for genetic analysis. Six single-nucleotide polymorphisms (SNPs) including, five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) and one promoter SNP (rs1800629) of TNF-α were genotyped in all study subjects. Genotyping of CLU SNPs were performed using the TaqMan allelic discrimination assay while TNF-α SNP was genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Association analysis was performed by determining the distributions of genotype and allele frequencies, Hardy-Weinberg equilibrium, and chi-square p values and odds ratios as implemented in the Golden Helix SNP & Variation Suite (SVS). RESULTS: Five CLU SNPs did not show any significant differences in allele frequencies between patients and control subjects (rs3087554, p = 0.919, OR = 1.01, 95% CI: 0.77-1.33; rs2279590, p = 0.432, OR = 1.12, 95% CI: 0.84-1.51; rs9331931, p = 0.310, OR = 1.24, 95% CI: 0.81-1.89; rs11136000, p = 0.072, OR = 1.31, 95% CI: 0.97-1.76; rs9331888, p = 0.911, OR = 1.01, 95% CI: 0.78-1.31). The investigation of TNF-α SNP established a significant association with PEXS and PEXG (p = 0.042, OR = 0.61, 95% CI: 0.38-0.99). However, this association did not remain significant after Bonferroni correction. CONCLUSIONS: Our data suggest that genetic variants in CLU and TNF-α genes do not play a major role in the development of PEXS and PEXG in the South Indian population.

Mutational analysis and genotype-phenotype correlations in southern Indian patients with sporadic and familial aniridia.

PURPOSE: Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia. METHODS: Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools. RESULTS: Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations. CONCLUSIONS: This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients.

Lysyl oxidase-like 1 gene in the reversal of promoter risk allele in pseudoexfoliation syndrome.

IMPORTANCE: This study was necessary to establish the association between common genetic variants in the lysyl oxidase-like 1 (LOXL1) gene with pseudoexfoliation (PEX) syndrome and emphasize the reversal of promoter risk allele in a South Indian population. OBJECTIVE: To investigate the potential association of genetic variants across the LOXL1 gene in South Indian patients with PEX syndrome and glaucoma. DESIGN, SETTING, AND PARTICIPANTS: A case-control study of individuals from Madurai, India, with PEX syndrome and glaucoma as well as healthy people serving as controls. Three hundred unrelated people with PEX syndrome and 225 age- and ethnically matched controls were recruited for genetic analysis. MAIN OUTCOMES AND MEASURES: Four single-nucleotide polymorphisms in LOXL1 (rs16958477, rs1048661, rs3825942, and rs2165241) were genotyped by direct sequencing in all participants. Regulatory regions and 7 coding exons of LOXL1 were directly sequenced in 50 patients and 50 controls. A case-control association analysis was performed using the Golden Helix SVS suite. RESULTS: An association between 4 LOXL1 single-nucleotide polymorphisms with PEX syndrome and glaucoma was observed (rs16958477, P = 4.77 × 10-6 [odds ratio, 0.50]; rs1048661, P = 4.28 × 10-5 [1.79]; rs3825942, P = 4.68 × 10-30 [9.19]; and rs2165241, P = 1.98 × 10-15 [2.88]). Sequencing of 7 exons and regulatory regions of LOXL1 identified 11 additional sequence variants; only rs41435250 showed an association (P = 3.80 × 10-5 [0.49]) with PEX syndrome and glaucoma. CONCLUSIONS AND RELEVANCE: Genetic variants in LOXL1 are associated with PEX syndrome and glaucoma in the South Indian population. To our knowledge, this is the first study to demonstrate the association of rs41435250 with PEX as well as reversal of the promoter risk allele. Understanding the role of the LOXL1 gene in PEX pathogenesis will facilitate early detection in individuals at risk for this condition.

ABCB6 mutations cause ocular coloboma.

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.

A new commercially viable synthetic route for donepezil hydrochloride: anti-Alzheimer's drug.

An economical new process has been developed for the synthesis of donepezil hydrochloride (1) an anti-Alzheimer's drug. The process involves Darzen reaction of pyridine-4-carboxaldehyde and 2-bromo-5,6-dimethoxy indanone affording epoxide 5,6-dimethoxy-3-(pyridine-4-yl)spiro[indene-2,2'-oxiran]-1(3H)-one (4) as a key intermediate. The one-pot deoxygenation of 4 and hydrogenation of the aryl moiety in high yield improved the overall yield of the process.

Human sperm can fertilize ova inside the Graafian follicles before ovulation.

OBJECTIVE: To report an incidental finding of fertilization of ova inside the ovarian follicles before ovulation. DESIGN: Case report. SETTING: IVF Center, King Fahd Specialist Hospital, Buraidah, Al-Qassim, Saudi Arabia. PATIENT(S): One 31-year-old woman with 8 years of primary infertility. INTERVENTION(S): Long-protocol superovulation and ovum pickup (OPU). MAIN OUTCOME MEASURE(S): The retrieved nine oocytes were incubated and followed up closely. RESULT(S): Three hours after OPU and only after decoronization of oocytes, sperm were seen attached to the zonae pellucidae of six mature oocytes. Three of them were showing signs of fertilization (two pronuclei). Neither conventional IVF nor intracytoplasmic sperm injection was done, and we decided to continue incubation and observation of all oocytes. From the nine total oocytes, fertilization was demonstrated in six with attached sperm, of which four divided. On day 2 after OPU, three embryos were transferred into the uterus. CONCLUSION(S): Human sperm can migrate and ascend through the female genital tract to get through the ovarian tissue. Moreover, under certain conditions, it can penetrate the wall of an intact ovarian follicle to reach the ovum and fertilize it just before ovulation.