Q-switched Nd:YAG laser protects human keratinocytes from oxidative stress and inflammation via AhR-Nrf2 pathway.

In recent years, some treatments for esthetic and pathologic skin conditions have increasingly been based on the use of non-ablative neodymium-doped yttrium aluminum garnet (Nd:YAG) laser due to its greater penetration ability than other types of lasers, few contraindications, minimal side effects, no damage for epidermidis and the rapid recovery of the treated patients. The skin is frequently exposed to many stressors such as radiation, toxic substances, metabolites, foods, mechanical insults, and allergen exposition that cause oxidative damage and have a decisive influence on the aging process. The imbalance between reactive oxygen species, reactive nitrogen species, and the malfunctioning of the antioxidant defense system promotes the establishment of an excessive inflammatory process, which can induce various diseases including cancer and neurodegenerative disorders. The present study investigated the cytoprotective function of Q-switched Nd:YAG laser against stress aging and cell injury in HaCaT cells. We evaluated the effect of the laser on antioxidant defenses, inflammation, metalloproteinases' expression, and the AhR-Nrf2 pathway. Q-switched Nd:YAG is able to upregulate the AhR pathway and the expression of IL-6 and TGF-ß, which are involved in wound repair process, and to downregulate the expression of MMP-2 and 9, so preventing the collagen degradation. Q-switched Nd:YAG can stimulate the cellular antioxidant defenses by activating the AhR-Nrf2 system.

Biopolymer- and Natural Fiber-Based Biomimetic Tissues to Realize Smart Cosmeceuticals and Nutraceuticals Using an Innovative Approach.

More sustainable and smart cosmeceuticals and nutraceuticals are necessary due to the ecological transition. In this study, a pullulan-based water solution containing chitin nanofibril-nano-lignin (CN-LG) complexes that encapsulate fish collagen polypeptide, allantoin and nicotinamide was electrospun onto a nonwoven substrate made of bamboo fibers to obtain a smart nanostructured bilayer system for releasing active molecules onto the skin or other body tissues. Infrared spectroscopy was used to characterize the composition of the bilayer system before and after rapid washing of the sample with distilled water and liquids mimicking physiological fluids. The viability of keratinocytes was studied as well as the antioxidant activity, protective activity towards UV light, metalloproteinase release of aged fibroblasts and the inhibitor activity against collagen degradation. Immunomodulatory tests were performed to investigate the anti-inflammatory activity of the bilayer system as well as its indirect antimicrobial activity. The results indicate that the bilayer system can be used in the production of innovative sustainable cosmeceuticals. In general, the adopted strategy can be extended to several smart treatments for fast release that can be commercialized as solid products, thus avoiding the use of preservatives and water.

Oreoch-1: A Peptide from Oreochromis niloticus as a Potential Tool against Staphylococci.

Staphylococci, including Staphylococcus aureus and Staphylococcus epidermidis, are important human pathogens associated with potentially life-threatening infections. Their great biofilm-producing ability and the development of resistance mechanisms often account for therapeutic failure. Hence, the scientific community has devoted intensive efforts to the development of antimicrobial compounds active against both planktonic and sessile bacterial populations. Contextually, antimicrobial peptides (AMPs) are natural peptides produced by the innate immunity of every organism, representing a potential new therapeutic solution against human microbial pathogens. Our work focused on the in vitro activity of Oreoch-1, an AMP from the gills of Nile tilapia (Oreochromis niloticus), against standard and clinical S. aureus and S. epidermidis strains. Firstly, the cytotoxicity profile of Oreoch-1 was determined in human colon carcinoma cells. Secondly, its antibacterial spectrum was explored against staphylococcal strains to set up the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Our results highlighted an antibacterial activity in the range 6.25-25 µM, with a general bacteriostatic effect. Therefore, the biofilm-inhibitory property was assessed against S. aureus ATCC 25923 and S. epidermidis ATCC 35984, indicating a significant reduction in S. aureus biomass at sub-MIC concentrations. Overall, our study indicates Oreoch-1 as a promising new therapeutic weapon against staphylococcal infections.

V-Gel® Guided Endotracheal Intubation in Rabbits.

Background: General anesthesia in rabbits is associated with higher morbidity and mortality relative to other mammalian species commonly anesthetized. Unique challenges related to endotracheal intubation (ETI) in rabbits contribute to this risk. Objective: To improve the safety of ETI in rabbits, we developed two new ETI methods using a supraglottic airway device (v-gel®) to facilitate ETI and compared them to traditional "blind" technique. We hypothesized that relative to blind ETI, v-gel® guided ETI provides more successful placement of the endotracheal tube (ETT) in a shorter time. Outcomes included number of intubation attempts, time for achievement of ETI, endoscopic findings, and serial arterial blood gas (ABG) analysis. Study Design: Prospective, randomized, and crossover study. Methods: Ten female, New Zealand White rabbits aged 1-2 years old, weighing 4.3 ± 0.4 kg, were anesthetized four times. Each time, ETI was performed with one of the following techniques: Method 1: v-gel® guided, polypropylene catheter facilitated, intubation using a cuffed ETT; Method 2: v-gel® guided intubation using an uncuffed ETT directly inserted through the device airway channel; Method 3 and 4: Blind intubation with uncuffed or cuffed ETT. Upper airway endoscopy was performed before intubation attempts and after extubation. Serial ABG analysis was performed during the peri-intubation process. Results: V-gel® guided techniques allowed successful ETI on the initial attempt for 9/10 subjects using Method 1 and 10/10 using Method 2. Relative to the v-gel® guided techniques, the blind techniques required more intubation attempts. A median of 2 attempts (range 1-4, p < 0.007) were required for the uncuffed ETT, and a median of 4 (range 1-4, p < 0.001) attempts were performed for the cuffed ETT. The time to perform successful ETI was positively correlated with the number of attempts (ρ = 0.82), while successful ETI was negatively correlated with number of attempts (ρ = -0.82). Endoscopic findings showed mild to moderate laryngeal trauma. In the absence of oxygen supplementation, ABG analysis demonstrated low PaO2, while PaCO2 remained consistent. Conclusions: Facilitated ETI using the v-gel® guided techniques allows for the rapid establishment of a secure airway to provide ventilatory support for rabbits undergoing general anesthesia.

Oral Microbiota and Salivary Levels of Oral Pathogens in Gastro-Intestinal Diseases: Current Knowledge and Exploratory Study.

Various bi-directional associations exist between oral health and gastro-intestinal diseases. The oral microbiome plays a role in the gastro-intestinal carcinogenesis and fusobacteria are the most investigated bacteria involved. This paper aims to review the current knowledge and report the preliminary data on salivary levels of Fusobacterium nucleatum, Porphyromonas gingivalis and Candida albicans in subjects with different gastro-intestinal conditions or pathologies, in order to determine any differences. The null hypothesis was "subjects with different gastro-intestinal diseases do not show significant differences in the composition of the oral microbiota". Twenty-one subjects undergoing esophagastroduodenoscopy or colonscopy were recruited. For each subject, a salivary sample was collected before the endoscopy procedure, immediately stored at -20 °C and subsequently used for genomic bacterial DNA extraction by real-time PCR. Low levels of F. nucleatum and P. gingivalis were peculiar in the oral microbiota in subjects affected by Helicobater pylori-negative chronic gastritis without cancerization and future studies will elucidate this association. The level of C. albicans did not statistically differ among groups. This preliminary study could be used in the future, following further investigation, as a non-invasive method for the search of gastrointestinal diseases and associated markers.

The Role of Bacterial Colonization of the Suture Thread in Early Identification and Targeted Antibiotic Treatment of Surgical Site Infections: A Prospective Cohort Study.

Background: The aim of the present study is to investigate the role of the colonization of suture thread to identify patients at risk of developing a surgical site infection (SSI) after clean surgical procedures. Methods: Patients who underwent elective clean surgery procedures at the Surgery Unit of the AOU-University of Campania Luigi Vanvitelli in a 21-month period were prospectively enrolled. For each patient, a synthetic absorbable thread in Lactomer 9-1 was inserted into the surgical site at the end of surgery and microbiologically evaluated after 48 h. Antibiotic prophylaxis was chosen according to international guidelines. Results: A total of 238 patients were enrolled; 208 (87.4%) of them were subjected to clean procedures without the placement of prosthesis, and 30 (12.6%) with prosthesis. Of the 238 patients, 117 (49.2%) underwent an antimicrobial prophylaxis. Overall, 79 (33.2%) patients showed a bacterial colonization of the thread: among the 208 without the implantation of prosthesis, 19 (21.8%) of the 87 with antibiotic prophylaxis and in 58 (47.9%) of the 121 without it; among the 30 patients with the implantation of prosthesis, only two patients showed a colonized thread. The patients with antibiotic prophylaxis developed a colonization of the thread less frequently than those without it (17.9% vs. 47.9%, p < 0.001). SSI was observed in six (2.5%) patients, all of them showing a colonized thread (7.6% vs. 0%, p < 0.001). The bacteria identified in colonized threads were the same as those found in SSIs. Conclusions: Our study presents a new method that is able to precociously assess patients who have undergone clean procedures who may develop SSI, and identify the microorganism involved.

Lactobacillus brevis CD2: Fermentation Strategies and Extracellular Metabolites Characterization.

Functional foods and nutraceuticals frequently contain viable probiotic strains that, at certain titers, are considered to be responsible of beneficial effects on health. Recently, it was observed that secreted metabolites might play a key role in this respect, especially in immunomodulation. Exopolysaccharides produced by probiotics, for example, are used in the food, pharmaceutical, and biomedical fields, due to their unique properties. Lactobacillus brevis CD2 demonstrated the ability to inhibit oral pathogens causing mucositis and periodontal inflammation and to reduce Helycobacter pylori infections. Due to the lack of literature, for this strain, on the development of fermentation processes that can increase the titer of viable cells and associated metabolites to industrially attractive levels, different batch and fed-batch strategies were investigated in the present study. In particular, aeration was shown to improve the growth rate and the yields of lactic acid and biomass in batch cultures. The use of an exponential feeding profile in fed-batch experiments allowed to produce 9.3 ± 0.45 × 109 CFU/mL in 42 h of growth, corresponding to a 20-fold increase of viable cells compared with that obtained in aerated batch processes; moreover, also increased titers of exopolysaccharides and lactic acid (260 and 150%, respectively) were observed. A purification process based on ultrafiltration, charcoal treatment, and solvent precipitation was applied to partially purify secreted metabolites and separate them into two molecular weight fractions (above and below 10 kDa). Both fractions inhibited growth of the known gut pathogen, Salmonella typhimurium, demonstrating that lactic acid plays a major role in pathogen growth inhibition, which is however further enhanced by the presence of Lact. brevis CD2 exopolysaccharides. Finally, the EPS produced from Lact. brevis CD2 was characterized by NMR for the first time up to date.

In Vitro Evaluation of Novel Hybrid Cooperative Complexes in a Wound Healing Model: A Step Toward Improved Bioreparation.

The effectiveness of hyaluronic acid (HA), also called as hyaluronan, and its formulations on tissue regeneration and epidermal disease is well-documented. High-molecular-weight hyaluronan (HHA) is an efficient space filler that maintains hydration, serves as a substrate for proteoglycan assembly, and is involved in wound healing. Recently, an innovative hybrid cooperative complex (HCC) of high- and low-molecular-weight hyaluronan was developed that is effective in wound healing and bioremodeling. The HCC proposed here consisted of a new formulation and contained 1.6 ± 0.1 kDa HHA and 250 ± 7 kDa LHA (low molecular weight hyaluronic acid). We investigated the performance of this HCC in a novel in vitro HaCaT (immortalized human keratinocytes)/HDF (human dermal fibroblast) co-culture model to assess its ability to repair skin tissue lesions. Compared to linear HA samples, HCC reduced the biomarkers of inflammation (Transforming Growth Factor-ß (TGF-ß), Tumor Necrosis Factor receptor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)), and accelerated the healing process. These data were confirmed by the modulation of metalloproteases (MMPs) and elastin, and were compatible with a prospectively reduced risk of scar formation. We also examined the expression of defensin-2, an antimicrobial peptide, in the presence of hyaluronan, showing a higher expression in the HCC-treated samples and suggesting a potential increase in antibacterial and immunomodulatory functions. Based on these in vitro data, the presence of HCC in creams or dressings would be expected to enhance the resolution of inflammation and accelerate the skin wound healing process.

Hyaluronic acid and chondroitin sulfate, alone or in combination, efficiently counteract induced bladder cell damage and inflammation.

Interstitial cystitis and/or bladder pain syndrome (IC/BPS) are characterized by discomfort, abdominal pain, and pelvic pain, and they are often associated with chronic diseases. Pathological conditions related to IC/BPS can occur due to a defect in the integrity of the bladder lining. This defect has been ascribed to damage to the glycosaminoglycan (GAG) layer of the urinary epithelium. In addition, the incipient cascade of inflammation events might prompt extracellular matrix degradation. Several medical devices based on GAG instillation were proposed to re-establish epithelial integrity by GAGs binding to proteoglycans or interacting with structural urothelium. However, to date, only in vitro studies have investigated the GAG, hyaluronic acid (HA). In the present study, TNFα treatment was used to mimic IC/BPS-induced damage in bladder cells in an in vitro model. Highly purified fermentative HA and pharmaceutical grade bovine chondroitin sulfate (CSb), alone or in combination, were evaluated for the ability to counteract bladder cell damage. We evaluated NF-κB with western blots, and we analyzed interleukin 6 and 8 expression at the transcriptional and protein levels with quantitative RT-PCR, western blotting, and ELISA. We also evaluated the expression of an antibacterial peptide, human ß-defensin-2. We confirmed our results in a 3D bladder epithelium model. Our results demonstrated that inflammatory status was reduced in the presence of HA, CSb, and the combination of both (HA/CSb 1.6%/2% w/v). This result suggested that these GAGs might be suitable for treating IC/BPS. All the assayed biomarkers showed that HA/CSb treatment modulated cells towards a more physiological status. Finally, we compared two commercial products suggested for the IC/BPS treatments and found that the product with more Ca++, showed enhanced anti-inflammatory activity and provided superior mucoadhesivity.

Chitin Nanofibrils and Nanolignin as Functional Agents in Skin Regeneration.

Chitin and lignin, by-products of fishery and plant biomass, can be converted to innovative high value bio- and eco-compatible materials. On the nanoscale, high antibacterial, anti-inflammatory, cicatrizing and anti-aging activity is obtained by controlling their crystalline structure and purity. Moreover, electropositive chitin nanofibrlis (CN) can be combined with electronegative nanolignin (NL) leading to microcapsule-like systems suitable for entrapping both hydrophilic and lipophilic molecules. The aim of this study was to provide morphological, physico-chemical, thermogravimetric and biological characterization of CN, NL, and CN-NL complexes, which were also loaded with glycyrrhetinic acid (GA) as a model of a bioactive molecule. CN-NL and CN-NL/GA were thermally stable up to 114 °C and 127 °C, respectively. The compounds were administered to in vitro cultures of human keratinocytes (HaCaT cells) and human mesenchymal stromal cells (hMSCs) for potential use in skin contact applications. Cell viability, cytokine expression and effects on hMSC multipotency were studied. For each component, CN, NL, CN-NL and CN-NL/GA, non-toxic concentrations towards HaCaT cells were identified. In the keratinocyte model, the proinflammatory cytokines IL-1α, IL-1 ß, IL-6, IL-8 and TNF-α that resulted were downregulated, whereas the antimicrobial peptide human ß defensin-2 was upregulated by CN-LN. The hMSCs were viable, and the use of these complexes did not modify the osteo-differentiation capability of these cells. The obtained findings demonstrate that these biocomponents are cytocompatible, show anti-inflammatory activity and may serve for the delivery of biomolecules for skin care and regeneration.

Induction of Different Apoptosis Pathways by Two Proteus mirabilis Clinical Isolates Strains in Prostatic Epithelial Cells.

Bacterial prostatitis is believed to be the leading cause of recurrent urinary tract infections (UTIs) in men under 50 years of age and occurs both as an acute febrile disease responsive to antibiotics and as a chronic infection that is often unresponsive to antibiotic treatment. Proteus mirabilis is more commonly associated with UTIs in these abnormalities, especially in patients undergoing catheterisation. This pathogen is able to colonise the host's tissues and to cause disease thanks to the production of many virulence factors such as fimbriae, flagella, immune avoidance, host-damaging factors, and the ability to form crystalline biofilms. In addition, Proteus lipid A may exhibit apoptotic activity and induce desquamation of epithelial cells. The aim of this work was to evaluate the ability of two clinically isolated strains of P. mirabilis that are phenotypically different, named PM1 of PM2, respectively, to induce apoptosis in human prostatic adenocarcinoma PC-3. Our results demonstrate that PM1 and PM2 are able to activate two different apoptotic pathways, and this different behaviour is confirmed by the expression level of the ZapA gene, molecular fingerprinting and different spectrum of antibiotic resistance. The identification and knowledge of relations between the microorganism and host may provide the basis for new solutions to clinical problems with regard to diagnosis and therapy.

Anti-Inflammatory, Immunomodulatory, and Tissue Repair Activity on Human Keratinocytes by Green Innovative Nanocomposites.

The use of raw materials obtained by waste and processed through innovative industrial methodologies has generated an industry of about a trillion dollars in a short time, and in the near future will provide resources and services for the conservation and sustainable use of natural resources in order to ensure a better and fairer welfare for the human race. The production of nano-fiber chitin non-woven tissue is in accordance with the Organization for Economic Co-operation and Development (OECD) and European Union (EU) bio-economic programs: 100% biodegradable, ecological, and therefore useful in decreasing dependence on fossil fuel resources. The aim of our study is the evaluation of different formulations of a non-woven tissue obtained from electrospinning of a mixture of nanochitin fibrils, lignin, and poly (ethylene) oxide (PEO) on the restoration of damaged tissues. Wound repair is a complex process that involves epithelial and immune cells and includes the induction of metalloproteinases, inflammatory mediators, and angiogenic factors. Our in vitro results have shown that all of the realized chitin nanofibrils-bio-lignin non-woven tissues tested as nontoxic for human keratinocytes (HaCat) cells. Furthermore, the bio-composites that included bio-lignin at 0.1% have been able to modulate the expression of pro-inflammatory cytokines (Tumor Necrosis Factor-α, IL-1α, and IL8), lipopolysaccharide (LPS)-induced, and matrix metalloproteinases (MMPs) and human beta-defensin 2 (HBD-2) expression in HaCat cells, suggesting an anti-inflammatory and immunomodulatory role. Taken together, our results suggest that our chitin nanofibrils-bio-lignin non-woven tissue represents a skin-friendly tool that is able to favor a correct and fast wound repair.

ß-Defensins: Work in Progress.

Defensins are a group of antimicrobial peptides (AMPs) found in different living organisms, and are involved in the first line of defense in the innate immune response against pathogens. The increase in the resistance of bacteria to conventional antibiotics and the need for new antibiotics has stimulated interest in the use of AMPs as new therapeutic agents. The inducible nature of human defensin genes suggests that it is possible to increase the endogenous production by utilizing small molecules of various origins to enhance, even selectively, the expression of these peptides. In the light of their role in immunomodulation, angiogenesis, wound healing, inflammation and cancer, as well as their antimicrobial activity, it is possible induce their expression or create analogs with increased specific activity or various degrees of selectivity, or obtain human defensins with genetic engineering to optimize the potency and safety in order to reduce cytotoxicity and potential proinflammatory activity and susceptibility to protease and salt. Restoring the balance between immunostimulating and immunosuppressive molecules may be an important strategy to correct expression defects in specific diseases.

Radically resected pituitary adenomas: prognostic role of Ki 67 labeling index in a monocentric retrospective series and literature review.

Ki-67 Labeling Index is an immunocytochemical marker of cell proliferation. The correlation of Ki-67 expression with pituitary adenomas recurrence has been investigated and is highly debated. Aim of this study was to evaluate whether Ki-67 correlates with recurrence even in patients with an apparently completely removed pituitary adenoma. We retrospectively reviewed the database of the Hypothalamic-Pituitary Disease Unit at the Catholic University of Rome, collected between 2003 and 2011. Inclusion criteria were: patients who underwent surgery at the Department of Neurosurgery with an apparently complete removal of a pituitary adenoma; Ki-67 histological evaluation by the same operator and values of <3%. All patients underwent endocrine evaluation of the hypothalamic-pituitary function, ophthalmologic and neuro-radiological examinations, during the preoperative period and follow-up. Out of 490 patients recorded on the database of the Hypothalamic-Pituitary Disease Unit at the Catholic University of Rome, 191 cases met the inclusion criteria. Recurrence was observed in 49 cases (25.7% of the patients who had undergone radical excision). Optional cut-off value was identified at Ki-67 values of 1.50%. This was associated with worse disease-free survival time, even after correction for age at treatment, gender, positivity to p53, functional classification and Knosp grading. Ki-67 labeling index may be useful in postoperative management, even in patients who underwent radical PA removal. We suggest a Ki-67 cut-off value of 1.5% to plan an adequate clinical follow-up.

Treatment with octreotide LAR in clinically non-functioning pituitary adenoma: results from a case-control study.

Surgical cure cannot be achieved in most patients with invasive non-functioning pituitary macroadenoma (NFPA). Short-term residual tumor treatment with somatostatin analogs has produced disappointing results. This prospective case-control study assessed the efficacy of chronic treatment with long acting octreotide (octreotide LAR) on tumor volume in patients harboring post-surgical NFPA residue. The study population comprised 39 patients with NFPAs not cured by surgery. All patients underwent somatostatin receptor scintigraphy at least 6 months after the last surgery. Patients with a positive pituitary level octreoscan at (n = 26) received octreotide LAR (20 mg every 28 days) for ≥ 12 months (mean follow-up 37 ± 18 months) (Treated group). Moreover, a fragment of tumor tissue from patients in the treated group was retrospectively collected to assess the immunohistochemical expression of somatostatin receptor subtypes (SSTRs). The patients with a negative octreoscan (n = 13) formed the control group (mean follow-up 37 ± 16 months). Hormonal, radiological and visual field parameters were periodically assessed. In the treated group, all tumors expressed at least one SSTR subtype. The SSTR5 subtype was the most abundant, followed by SSTR3. The tumor residue increased in five of 26 patients (19%) in the treated group and in seven of 13 controls (53%). Visual field and pituitary function did not change in any patient. This study indicates that SSTR5 and SSTR3 are the most frequently expressed SSTR subtypes in NFPAs and supports a potential role of SSTR subtypes in stabilization of tumor remnant from NFPAs.

Efficacy of the combined cabergoline and octreotide treatment in a case of a dopamine-agonist resistant macroprolactinoma.

Prolactinomas in males can be voluminous macroadenomas invading the surrounding structures. Medical therapy with dopamine agonists (the treatment of choice for these tumours) may be ineffective in the case of pharmacological resistance. In such cases, even surgical and/or radiation therapy cannot be curative due to the invasive potential of the adenoma. Hence, the appropriate therapeutic approach for these tumours is still a relevant clinical problem for endocrinologists. We report the history of an adolescent male who was diagnosed with a large invasive macroprolactinoma in 2002. He had severe bitemporal hemianopsia and hypopituitarism; prolactin levels at diagnosis were higher than 8,000 ng/ml. Medical therapy with cabergoline was initiated and resulted in decreased prolactin levels but not complete normalisation (maximal tolerated dose 3 mg/day). However, due to the worsening of the visual defect, the patient was operated in July 2004 through the trans-nasal approach and 2 years later through both the transcranial and the transphenoidal approaches. After the second surgery, a significant reduction of tumour mass was obtained. Immunohistochemistry for somatostatin receptors (sstr) subtypes showed a positive staining with the anti-sstr5 antibody. A scintigraphy with 111In-pentetreotide (Octreoscan) revealed a very intense tracer uptake in the sellar region. The administration of long-acting octreotide was initiated. After 12 months of therapy, prolactin levels normalised for the first time. Pituitary MRI did not reveal any tumor progression during a 2-year follow-up. This is a case of an invasive dopamine-resistant macroprolactinoma that was successfully controlled by extensive surgery and combined treatment with cabergoline and octreotide. The expression and functionality of sstr should be investigated in these tumours since a combined therapy with cabergoline and octreotide may be a good therapeutic course of action for select cases.

The effect of treatment with growth hormone on fertility outcome in eugonadal women with growth hormone deficiency: report of four cases and review of the literature.

OBJECTIVE: To highlight the clinical role of standard GH replacement treatment on fertility and pregnancy outcomes in four infertile eugonadal women with GH deficiency (GHD). DESIGN: Case report. SETTING: Department of endocrinology and infertility clinic, tertiary-care university hospital. PATIENT(S): Four normogonadotrophic, normoprolactinemic patients with long-standing infertility, affected by GHD. In two patients (aged 30 and 34 years) GHD was diagnosed after a brain injury. The third patient (age 30 years) had a primary empty sella, documented by magnetic resonance imaging of the pituitary. The last patient (age 28 years) underwent transsphenoidal surgery for Ratke's cyst. The LH and FSH responses to GnRH were normal in all four patients. Two of the four patients also had secondary hypoadrenalism and hypothyroidism. INTERVENTION(S): Patients received recombinant human GH replacement therapy (0.9-1.8 mg/week) for 6-12 months until pregnancy was first indicated by biochemical markers (beta-hCG) and later confirmed by transvaginal sonography. The GH therapy was discontinued after confirmation of pregnancy. MAIN OUTCOME MEASURE(S): Pregnancy. RESULT(S): All patients remained off treatment throughout pregnancy; they had uneventful pregnancies and term deliveries. The babies were healthy and normal in terms of length and weight. CONCLUSION(S): Our case studies confirm the important clinical role of the GH-insulin-like growth factor I system in oocyte fertilization and the beginning of pregnancy in a selected population of eugonadotrophic infertile women.