Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis.

STUDY QUESTION: Does the molecular and metabolic profile of human mural granulosa cells (GCs) correlate with oocyte fate? SUMMARY ANSWER: A close relation between the metabolic profile of mural GCs and the fate of the corresponding oocyte was revealed by the analysis of selected biomarkers defined by GC Fourier transform infrared microspectroscopy (FTIRM) analysis. WHAT IS KNOWN ALREADY: In ART, oocyte selection is mainly based on the subjective observation of its morphological features; despite recent efforts, the success rate of this practice is still unsatisfactory. FTIRM is a well-established vibrational technique recently applied to evaluate oocytes quality in several experimental models, including human. STUDY DESIGN, SIZE, DURATION: GCs retrieved from single-follicle aspirates were obtained with informed consent from 55 women undergoing controlled ovarian stimulation for IVF treatment. GCs were analysed by FTIRM to retrospectively correlate their spectral features with the fate of the companion oocytes. The study has been conducted between March 2016 and September 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were selected according to the following inclusion criteria: age <40 years; non-smokers; no ovarian infertility diagnosis (only tubal, idiopathic and male infertility); regular ovulatory menstrual cycles (25-30 days) with FSH < 10 IU/I on Day 3 of the menstrual cycle; sperm sample with a total motility count after treatment ≥300.000; number of retrieved oocytes ≥8. Based on the clinical outcome of the corresponding oocyte, GCs were retrospectively classified into the following experimental groups: clinical pregnancy (CP), fertilization failure (FF), embryo development failure (EDF) and implantation failure (IF). All samples were analysed by the FTIRM technique. The spectral biomarker signature of different oocyte fates was derived by several feature selection procedures ('Leave-one-out' method on factorial discriminant analysis (FDA), variable characterization method and logistic regression method with the multinomial Logit model). ANOVA, permutational multivariate ANOVA, FDA and canonical analysis of principal co-ordinates statistical tools were also applied to validate the identified spectral biomarkers. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 284 GCs samples were retrieved and retrospectively classified as FF: (N = 92), EDF (N = 113), IF (N = 56) and CP (N = 23). From the spectral profiles of GCs belonging to CP, FF, EDF and IF experimental groups, 17 spectral biomarkers, were identified by several feature selection procedures (P < 0.0001). These biomarkers were then validated by applying multivariate tools, to evaluate their ability to segregate GCs samples into the four experimental groups. FDA showed a clear separation along the F1-axis (62.75% of discrimination) between GCs from oocytes able (CP, IF groups) or not (FF, EDF groups) to develop into embryos; the F2-axis (24.14% of discrimination) segregated the embryos that gave pregnancy (CP) from those that failed implantation (IF). The confusion matrix (total percentage of correctness = 80.25%) obtained from this analysis pinpointed that GCs from oocytes unable to develop into embryos (FF, EDF) were better characterized than those from oocytes able to give viable embryos (CP, IF). ANOVA (P < 0.05) analysis pinpointed that: each experimental group showed specific macromolecular traits, ascribable to different biological and metabolic characteristics of GCs; these metabolic features were likely associated with different oocytes fates, but not to patient characteristics, since from the same patient we obtained GCs with different metabolic profiles. LIMITATIONS, REASONS FOR CAUTION: The study is based on a small sample size but provides proof of concept that the GCs' metabolic profile is associated with the companion oocyte fate. The generated model should be further tested on a larger cohort of patients, classified in a similar manner, to assess the potential predictive value of this approach. Ultimately, validity of the proposed approach should be tested in a RCT. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, the FTIRM analysis of human GCs has demonstrated an approach to better understand the molecular crosstalk between follicular cells and oocytes and has identified potential spectral biomarkers for improving human IVF success rate. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by GFI 2014 grant. The authors declare that there is no conflict of interest.

Dermatofibrosarcoma protuberans in an adolescent: a case report and review of the literature.

Classically, dermatofibrosarcoma protuberans (DFSP) is a disease of adults. The world literature revision shows that several pediatric cases have been reported so far; this might suggest that the number of infants with the condition might be larger than that estimated previously. Here, we report the 183rd case of histologically confirmed DFSP in young age. A 14-year-old white male patient came under our care for a slowly growing, pale brownish lesion on the neck skin. A biopsy specimen showed a DFSP. Subsequently, a wide surgery excision with 3 cm of resection margins including the underlying fascia was performed. To date, the patient has been in follow-up for 6 years without evidence of recurrent disease. The clinical features and treatment of DFSP diagnosed in childhood and adolescence reported in the published literature are reviewed to provide new insights about this rare entity. The aim is to emphasize the importance of biopsy for histologic evaluation in the cases that show a persistent or a large cutaneous plaque or nodule without pathognomonic clinical features that permit a clinical diagnosis. An accurate knowledge of the disease is the prerequisite for a wider recognition and appropriate treatment.

Dermoscopic, histological and immunohistochemical evaluation of cancerous features in acquired melanocytic nevi that have been repeatedly exposed to UVA or UVB.

Previous studies have reported that repeated solar and artificial UVB (280-320 nm) and UVA (320-400 nm) exposures can modify acquired melanocytic nevi (AMN). We therefore investigated the clinical, dermoscopic, histological and immunohistochemical changes in AMN exposed to UVB and UVA radiation. Twenty healthy volunteers with at least three AMN on the trunk were enrolled in the present study and randomized into two groups to receive equally effective doses of narrow-band (NB)-UVB or UVA1. Three exposures per week were delivered for a total of 4 weeks. During exposures, one AMN was left unprotected, a second one was shielded with an opaque adhesive tape and the third nevus was covered with a commercial sunscreen. After the irradiation cycle, the AMN were surgically removed and underwent histological and immunohistochemical assessment of melanocyte/melanogenesis-related proteins (MART-1, tyrosinase, HMB-45), cell cycle activation markers (Ki-67, topoisomerase IIalpha, p53, Cdk2) and transcription factors (microphthalmia-associated transcription factor, STAT3). Nevi that were exposed to NB-UVB or UVA1 also showed statistically significant increase in size and changes in their dermoscopic features, including overall darkening, increased pigment network expression, formation of branched streaks, and increased number and size of brown globules and dots. AMN that had been covered with opaque tape or sunscreen did not show changes in size or dermoscopic features following UVA1 or NB-UVB exposure. Histological and immunohistochemical analysis did not show any significant change in exposed AMN in comparison with AMN shielded with an opaque adhesive tape or covered with the sunscreen.

Cutaneous melanoma in patients in treatment with biological therapy: review of the literature and case report.

Herein we report a case of a melanoma arising in a patient receiving adalimumab and methotrexate for rheumatoid arthritis. A limited number of studies reported melanoma growth in patients undergoing treatment with biologics. This case report with a brief review of literature suggests that patients under treatment with biologics should be counseled to identify new pigmented lesions or changes in preexisting nevi. Clinicians' collaboration will facilitate recognition and timely diagnosis of early melanoma. If there is any doubt, excision for histological evaluation should be considered. Pending new studies, careful observation is encouraged.

Vibrational characterization of granulosa cells from patients affected by unilateral ovarian endometriosis: New insights from infrared and Raman microspectroscopy.

Endometriosis is a chronic gynaecological disease characterised by the presence of endometrial cells in extra-uterine regions. One of the main factors impacting on the fertility of women affected by endometriosis is the poor oocyte quality. Granulosa Cells (GCs) regulate oocyte development and maintain the appropriate microenvironment for the acquisition of its competence; hence, the dysregulation of these functions in GCs can lead to severe cellular damages also in oocytes. In this study, luteinized GCs samples were separately collected from both ovaries of women affected by Unilateral Ovarian Endometriosis and analysed by infrared and Raman microspectroscopy. The spectral data were compared with those of GCs from women with diagnosis of tubal, idiopathic or male infertility (taken as control group). The coupling of these two spectroscopic techniques sheds new light on the alteration induced by this pathology on GCs metabolism and biochemical composition. In fact, the study revealed similar biochemical modifications in GCs from both ovaries of women affected by unilateral ovarian endometriosis, such as the alteration of the protein pattern, the induction of oxidative stress mechanisms, and the deregulation of lipid and carbohydrate metabolisms. These evidences suggest that unilateral endometriosis impairs the overall ovarian functions, causing alterations not only in the ovary with endometriotic lesions but also in the contralateral "healthy" one.

What criteria for the definition of oocyte quality?

Although the spermatozoon provides an essential contribution to the generation of a new individual, the developmental fate of the embryo is principally dictated by the oocyte. Oocyte competencies are acquired throughout oogenesis, via the interaction with somatic cells. The ability to reinitiate the meiotic process and undergo preimplantation development is progressively determined during the antral phase. It is known that these changes involve the nuclear and cytoplasmic compartments, respectively, but the underlying cellular and molecular mechanisms are still poorly understood. Analysis of various aspects of oocyte morphology (cytoplasm, zona pellucida, and polar body) via conventional phase-contrast microscopy has generated contrasting evidence on the possibility of establishing reliable criteria for the prediction of developmental potential. The introduction of a newly developed microscopy technique based on the detection of polarized light generated by birefringent cell structures has offered the possibility of visualizing noninvasively the meiotic spindle, whose presence is critical for fertilization and later developmental stages. However, further studies are needed to standardize and interpret the information accessible through such a technique. Although unable to preserve cell viability and therefore provide a method by which to select oocytes with superior developmental competence, invasive techniques can make a fundamental contribution to defining objective criteria of oocyte quality. In particular, immunofluorescence analysis, which is able to identify critical anomalies of the meiotic spindle and cytoskeleton organization that can account for oocyte quality, is an important method for assessing the efficiency of in vitro maturation systems.

Pigmentation of axillary sentinel nodes from extensive skin tattoo mimics metastatic melanoma: case report.

BACKGROUND: The relationship between the occurrence of skin diseases and skin tattoos remains unclear. Dermatologic disorders have been reported to occur in about 2% of cases. In addition, tattoo pigment can migrate to the regional lymph nodes through the lymphatic vessels and subsequently mimic metastatic disease from melanoma. METHODS: A 23-year-old Caucasian man presented with a pigmented lesion on the left scapular region, which had slowly enlarged over time. The patient exhibited an extensive tattoo on the left upper arm, left shoulder, and part of the upper back. His medical history was unremarkable. The pigmented lesion was excised. Histology confirmed malignant melanoma. Ultrasound examination of the abdomen, neck, and inguinal and axillary lymph nodes and a total body computed tomography scan showed no sign of disease. A re-excision with 2-cm margins and sentinel lymph node biopsy (SLNB) were performed. Two grossly enlarged, black sentinel lymph nodes (SLNs) highly suggestive of melanoma metastases were removed. RESULTS: No evidence of melanoma metastasis was found in any of the sampled tissues. Large amounts of pigment were present within the subcapsular space and sinusoid areas of the two clinically suspicious lymph nodes. Immunohistochemical analysis was negative. CONCLUSIONS: Sentinel lymph node biopsy is widely performed in cutaneous melanoma. Histologic confirmation of any enlarged, pigmented SLN is essential prior to radical surgery, especially when pigmented SLNs are found near a tattoo. Tattoo pigments may deposit in the regional lymph nodes and may clinically mimic metastatic disease. A history of tattooing should be considered in all melanoma patients eligible for SLNB. In a finding of darkly pigmented nodes during SLNB, radical lymphadenectomy should be withheld until immunohistologic confirmation of metastasis in the SLN is obtained.

A critical reappraisal of off-label indications for topical photodynamic therapy with aminolevulinic acid and methylaminolevulinate.

Photodynamic therapy (PDT) with methylaminolevulinate (MAL) has demonstrated high efficacy, minimal side effects, and improved cosmetic outcome in the treatment of its EU-approved clinical indications, i.e. actinic keratoses (AKs), basal cell carcinoma, and Bowen's disease. In addition, PDT with MAL or 5-aminolevulinic acid (ALA) is approved in the US for the treatment of AK. However, besides anti-tumoral activity, PDT has also demonstrated various anti-inflammatory and anti-infectious effects as well as those on the metabolism of connective tissue, keratinization of normal keratinocytes and maturation processes of sebaceous glands and hair follicles. These findings have expanded the spectrum of possible applications of PDT that now encompasses infectious (viral, bacterial, fungal, protozoal) disorders, epidermal and dermal inflammatory diseases, tumors of lymphocytes, adnexal diseases and premature skin aging due to sun exposure. However, the findings obtained so far must be considered in most off-label indications only preliminary and more detailed studies are required to clarify the role of PDT beyond the treatment of non-melanoma skin cancer. In addition, possible advantages over standard treatments remain to be clarified.

Predictive factors for embryo implantation potential.

In spite of recent improvements in IVF, pregnancy rates have not increased significantly and one of the major problems remains the high multiple pregnancy rate. Better criteria are therefore necessary to establish the viability of a transferable embryo. Early prognosis of the developmental fate of the oocyte would help in selecting the best embryos to transfer, but non-invasive selection at the oocyte stage (extracytoplasmic and intracytoplasmic morphology) has proved to be of little prognostic value. Recently, it has been shown that follicular vascularization appears to be predictive of oocyte developmental fate, making it a good first-step approach for selection. Observation of pronuclei patterns at the zygote stage appears to offer an additional prognostic tool, correlating well with IVF outcome. Morphological evaluation of the embryo at days 2-3 remains the most used and valid method of selection, even though it is not sufficient to select embryos with the higher implantation potential. Blastocyst culture is another possible strategy for selecting the best embryos with reduced risk of aneuploidies, though not all major chromosomal aberrations are excluded by prolonged in-vitro culture. In summary, selecting the best embryo for transfer is a decision that should be based on choices made during the different stages of assisted reproductive technologies.