Combined Rotating Ultra-High-Resolution Spectral Domain OCT and Scheimpflug Imaging for In Vivo Corneal Optical Biopsy.

PURPOSE: This article introduces the Pentacam® Cornea OCT (optical coherence tomography). This advanced corneal imaging system combines rotating ultra-high-resolution spectral domain OCT with sub- 2-micron axial resolution and Scheimpflug photography. The purpose of this study is to present the first experience with the instrument and its potential for corneal diagnostics, including optical biopsy. METHODS: In this prospective study, the Pentacam® Cornea OCT was used to image the corneas of seven patients. The novel wide-angle pericentric scan system enables optimal OCT imaging performance for the corneal layer structure over the entire width of the cornea, including the limbal regions. A detailed analysis of the resulting images assessed the synergism between the OCT and Scheimpflug photography. RESULTS: The Pentacam® Cornea OCT demonstrated significantly improved image resolution and ability to individualize corneal layers with high quality. There is a synergism between the OCT high-definition signal to individualize details on the cornea and Scheimpflug photography to detect and quantify corneal scattering. The noncontact exam was proven safe, user-friendly, and effective for enabling optical biopsy. CONCLUSIONS: Pentacam® Cornea OCT is an advancement in corneal imaging technology. The ultra-high-resolution spectral domain OCT and Scheimpflug photography provide unprecedented detail and resolution, enabling optical biopsy and improving the understanding of corneal pathology. Further studies are necessary to compare and analyze the tomographic reconstructions of the cornea with the different wavelengths, which may provide helpful information for diagnosing and managing corneal diseases.

Keratoconus and Corneal Ectasia with Relatively Low Keratometry.

INTRODUCTION: The study aims to demonstrate and estimate the prevalence of clinical corneal ectasia and keratoconus (KC) in patients with relatively low keratometry (low-K KC). METHODS: In a retrospective, analytical, and non-interventionist study, one eye was randomly selected from 1054 patients from the original Tomographic Biomechanical Index (TBIv1) study and the external validation (from Rio de Janeiro, Brazil, and Milan, Italy clinics). Patients were stratified into three groups. Group 1 included 736 normal patients, and groups 2 and 3 included 318 patients with clinical KC in both eyes, divided into low-K KC (90 patients) and high-K KC (228 patients), respectively. All patients underwent a comprehensive ophthalmological evaluation along with Pentacam and Corvis ST (Oculus, Wetzlar, Germany) examinations. Cases with maximum mean zone 3 mm keratometry (Kmax zone mean 3 mm) lower than 47.6 diopters (D) were considered as low-keratometry keratoconus, and cases with Kmax zone mean 3 mm higher than 47.6 D were regarded as high-keratometry keratoconus. RESULTS: Ninety (28.30%) of the 318 KC group presented ectasia with low-keratometric values (low-Kmax). The average age in the normal group was 39.28 years (range 6.99-90.12), in the low-Kmax KC group it was 37.49 (range 13.35-78.45), and in the high-Kmax KC group it was 34.22 years (range 12.7-80.34). Mean and SD values and median (range), respectively, of some corneal tomographic and biomechanical parameters evaluated from the low-Kmax KC group were as follows: Belin-Ambrósio enhanced ectasia display (BAD-D) 3.79 ± 1.62 and 3.66 (0.83-9.73); Pentacam random forest index (PRFI) 0.78 ± 0.25 and 0.91 (0.05-1); corneal biomechanical index (CBI) 0.58 ± 0.43 and 0.75 (0-1); TBI 0.93 ± 0.17 and 1 (0.35-1); and stiffness parameter at A1 (SP-A1) 86.16 ± 19.62 and 86.05 (42.94-141.66). CONCLUSION: Relatively low keratometry, with a Kmax lower than 47.6 D, can occur in up to 28.30% of clinical keratoconus. These cases have a less severe presentation of the disease. Future studies involving larger populations and prospective designs are necessary to confirm the prevalence of keratoconus with low keratometry and define prognostic factors in such cases.

Threat learning in space: how stimulus-outcome spatial compatibility modulates conditioned skin conductance response.

A central question in Pavlovian conditioning concerns the critical conditions that drive the acquisition and maintenance of the stimulus-outcome association. The spatial relationship between the conditioned (CS) and unconditioned (US) stimuli is considered to exert strong effects on learning. However, how spatial information modulates Pavlovian learning remains mostly unexplored in humans. Here, we test how the compatibility between the CS and the US location influences the acquisition, extinction, and recovery (following reinstatement) of Pavlovian conditioned threat. Participants (N = 20) completed a differential threat conditioning task in which visual CSs appeared on the same (compatible) or opposite (incompatible) hemispace as the US delivery (aversive shock to one hand), while their skin conductance response served as an index of learning. Results show that initial threat expectations were biased in favor of compatible CSs before conditioning. Nevertheless, this bias was revised during acquisition to reflect current stimulus-outcome contingencies. Computational modeling suggested that this effect occurred through a higher reliance on positive aversive prediction errors for incompatible CSs, thereby facilitating learning of their association with the US. Additionally, the conditioned response to incompatible CSs was associated with initially slower extinction and a greater recovery after threat reinstatement. These findings suggest that spatial information conveyed by stimuli and outcomes can be flexibly used to enact defensive responses to the current source of danger, highlighting the adaptive nature of Pavlovian learning.

Minimally Invasive Treatment in Benign Prostatic Hyperplasia (BPH).

Review efficacy and safety of minimally-invasive treatments for Low Urinary Tract Symptoms (LUTS) in patients affected by Benign Prostate Hyperplasia (BPH). We performed a systematic review of the literature from 1993 to 2022 leveraging original research articles, reviews, and case-studies published in peer-reviewed journals and stored in public repositories. Prostate artery embolization (PAE), transurethral needle ablation (TUNA), transurethral microwave thermotherapy (TUMT), high intensity focused ultrasound (HIFU), laser treatments and Cryoablation are valid and safe alternatives to the gold standard (surgery) in the treatment of LUTS in patients affected by BPH, with fewer undesired effects being reported.

The effect of inflorescence display size and flower position on pollination success in two deceptive and one rewarding orchid.

Inflorescence display size and flower position on the inflorescence play important roles in plant reproduction, in the formation of fruits and are primarily linked to pollinator behaviour. We used three orchids to determine how visitation rates and choice of pollinator depend on number and position of the flowers along the inflorescence. We measured reproductive success in (1) natural conditions, (2) hand-pollination experiments and (3) an experimental design, by modifying composition of inflorescences in populations of two deceptive orchids, Orchis anthropophora and O. italica, and one rewarding orchid, Anacamptis coriophora subsp. fragrans. There were no differences in natural fruit production in relation to flower position on the inflorescence (i.e. upper versus lower part), suggesting no preference of pollinators for different parts of the inflorescence. Hand-pollination experiments highlighted low pollen limitation in A. coriophora subsp. fragrans but high limitation in O. italica and O. anthropophora. Reproductive success of deceptive orchids in experimental plots decreased significantly when flowers on the upper half of the inflorescence were removed leading to reduced floral display, while reproductive success of the nectariferous species did not differ significantly. Our data highlight that in the examined orchids there is no clear relationship between fruit formation and flower position along inflorescences. Thus we can affirm that, for orchids, the entire inflorescence plays a dominant role in insect attraction but the part of the flower spike does not influence the choice of the insect. This implies that all flowers have the same possibility of receiving visits from pollinators, and therefore each flower has the same opportunity to set fruit.

Biomechanics in Keratoconus Diagnosis.

Purpose: To prospectively review the importance of biomechanical assessment in the screening, diagnosis, prognosis, individualized planning, and clinical follow-up for ectatic corneal diseases.Methods: We demonstrate two commercially available devices to assess the corneal biomechanics in vivo, the Ocular Response Analyzer (ORA, Reichester, NY, USA) and the Corvis ST (Oculus, Wetzlar, Germany). Novel devices have been demonstrated to provide in vivo biomechanical measurements, including Brillouin optical microscopy and OCT elastography. Conclusion: The integration of biomechanical data and other data from multimodal refractive imaging using artificial intelligence demonstrated the ability to enhance accuracy in diagnosing ectatic corneal diseases.

Pain perception in paediatric patients: evaluation of computerised anaesthesia delivery system vs conventional infiltration anaesthesia in paediatric patients.

Aim: The aim of the present study is to compare the effectiveness of a computerised system (QuickSleeper) compared to traditional syringe in injection of local anaesthetic focusing on the perception of anxiety and pain in paediatric patients. Methods: Study design: 100 children aged between 3 and 15 years in need of two dental treatments that required local anaesthesia were selected and treated randomly but alternately with computerised and traditional local anaesthesia. After each anaesthetic injection, patient's anxiety was measured using the Venham test. Results: Electronic anaesthesia showed statistically significant better results than traditional anaesthesia according to the Venham pain scale, in both mandibular and maxillary sites. Statistics: Data were analysed using the paired Wilcoxon test. Conclusion: The computer-assisted anaesthesia system resulted in a significantly lower pain perception score and yielded to helpful, cooperative behaviour. For this reason, it is an advantageous alternative to traditional injection anaesthesia and can avoid invasive treatments and trauma for young patients.

In silico framework to inform the design of repair constructs for peripheral nerve injury repair.

Peripheral nerve injuries affect millions of people per year and cause loss of sensation and muscle control alongside chronic pain. The most severe injuries are treated through a nerve autograft; however, donor site morbidity and poor outcomes mean alternatives are required. One option is to engineer nerve replacement tissues to provide a supportive microenvironment to encourage nerve regeneration as an alternative to nerve grafts. Currently, progress is hampered due to a lack of consensus on how to arrange materials and cells in space to maximize rate of regeneration. This is compounded by a reliance on experimental testing, which precludes extensive investigations of multiple parameters due to time and cost limitations. Here, a computational framework is proposed to simulate the growth of repairing neurites, captured using a random walk approach and parameterized against literature data. The framework is applied to a specific scenario where the engineered tissue comprises a collagen hydrogel with embedded biomaterial fibres. The size and number of fibres are optimized to maximize neurite regrowth, and the robustness of model predictions is tested through sensitivity analyses. The approach provides an in silico tool to inform the design of engineered replacement tissues, with the opportunity for further development to multi-cue environments.

Cetacean presence and distribution in the central Mediterranean Sea and potential risks deriving from plastic pollution.

The Sardinian and Sicilian Channels are considered hotspots of biodiversity and key ecological passages between Mediterranean sub-basins, but with significant knowledge gaps about marine mammal presence and potential threats they face. Using data collected between 2013 and 2019 along fixed transects, inter and intra-annual cetacean index of abundance was assessed. Habitat suitability, seasonal hot spots, and risk exposure for plastic were performed using the Kernel analysis and the Biomod2 R-package. 661 sightings of 8 cetacean species were recorded, with bottlenose and striped dolphins as the most sighted species. The north-eastern pelagic sector, the coastal waters and areas near ridges resulted the most suitable habitats for these species. The risk analysis identified the Tunis, Palermo, and Castellammare gulfs and the Egadi Island as areas of particular risk of plastic exposure. The study represents a great improvement for cetacean knowledge in this region and contributes to the development of effective conservation strategies.

Chromatographic Fingerprinting Strategy to Delineate Chemical Patterns Correlated to Coffee Odor and Taste Attributes.

Coffee cupping includes both aroma and taste, and its evaluation considers several different attributes simultaneously to define flavor quality and therefore requires complementary data from aroma and taste. This study investigates the potential and limits of a data-driven approach to describe the sensory quality of coffee using complementary analytical techniques usually available in routine quality control laboratories. Coffee flavor chemical data from 155 samples were obtained by analyzing volatile (headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS)) and nonvolatile (liquid chromatography-ultraviolet/diode array detector (LC-UV/DAD)) fractions, as well as from sensory data. Chemometric tools were used to explore the data sets, select relevant features, predict sensory scores, and investigate the networks between features. A comparison of the Q model parameter and root-mean-squared error prediction (RMSEP) highlights the variable influence that the nonvolatile fraction has on prediction, showing that it has a higher impact on describing acid, bitter, and woody notes than on flowery and fruity. The data fusion emphasized the aroma contribution to driving sensory perceptions, although the correlative networks highlighted from the volatile and nonvolatile data deserve a thorough investigation to verify the potential of odor-taste integration.

Deconvolution of hemodynamic responses along the cortical surface using personalized functional near infrared spectroscopy.

In functional near infrared spectroscopy (fNIRS), deconvolution analysis of oxy and deoxy-hemoglobin concentration changes allows estimating specific hemodynamic response functions (HRF) elicited by neuronal activity, taking advantage of the fNIRS excellent temporal resolution. Diffuse optical tomography (DOT) is also becoming the new standard reconstruction procedure as it is more accurate than the modified Beer Lambert law approach at the sensor level. The objective of this study was to assess the relevance of HRF deconvolution after DOT constrained along the cortical surface. We used local personalized fNIRS montages which consists in optimizing the position of fNIRS optodes to ensure maximal sensitivity to subject specific target brain regions. We carefully evaluated the accuracy of deconvolution when applied after DOT, using realistic simulations involving several HRF models at different signal to noise ratio (SNR) levels and on real data related to motor and visual tasks in healthy subjects and from spontaneous pathological activity in one patient with epilepsy. We demonstrated that DOT followed by deconvolution was able to accurately recover a large variability of HRFs over a large range of SNRs. We found good performances of deconvolution analysis for SNR levels usually encountered in our applications and we were able to reconstruct accurately the temporal dynamics of HRFs in real conditions.

Pollen viability of Euro-Mediterranean orchids under different storage conditions: The possible effects of climate change.

The future impact of climate change and a warmer world is a matter of great concern. We therefore aimed to evaluate the effects of temperature on pollen viability and fruit set of Mediterranean orchids. The in vitro and controlled pollination experiments were performed to evaluate the ability of pollinia stored at lower and higher temperatures to germinate and produce fruits and seeds containing viable embryos. In all of the examined orchids, pollen stored at -20 °C remained fully viable for up to 3 years, reducing its percentage germination from year 4 onwards. Pollinia stored at higher temperatures had a drastic reduction in vitality after 2 days at 41-44 °C, while pollinia stored at 47-50 °C did not show any pollen tube growth. The different levels of pollen viability duration among the examined orchids can be related to their peculiar reproductive biology and pollination ecology. The germinability of pollinia stored at lower temperatures for long periods suggests that orchid pollinia can be conserved ex situ. In contrast, higher temperatures can have harmful effects on the vitality of pollen and consequently on reproductive success of the plants. To our knowledge, this is the first report demonstrating the effects of global change on orchid pollen, and on pollen ability to tolerate, or not, higher air temperatures. Although vegetative reproduction allows orchids to survive a few consecutive warm years, higher temperatures for several consecutive years can have dramatic effects on reproductive success of orchids.

3D planning of ear prosthesis and navigated flapless surgery for craniofacial implants: A pilot study.

New 3D digital technologies can be applied to implant-supported ear prostheses to restore anatomical structures damaged by cancer, dysplasia, or trauma. However, several factors influence the accuracy of implant positioning using a cranial template. This pilot study describes an innovative navigated flapless surgery for craniofacial implants, prosthetically guided by 3D planning of the ear prosthesis. Laser surface scanning of the face allowed for mapping of the healthy ear onto the defect site, and projection of the volume and position of the final prosthesis. The projected ear volume was superimposed on the skull bone image obtained by cone-beam computed tomography (CBCT), performed with the navigation system marker plate positioned in the patient's mouth. The craniofacial implants were fitted optimally to the ear prosthesis. After system calibration, real-time navigated implant placement based on the virtual planning was performed with minimally invasive flapless surgery under local anesthesia. After 3 months of healing, digital impressions of the implants were made, and the digital manufacturing workflow was completed to manufacture the ear prosthesis anchored to the craniofacial implants. The proposed digital method facilitated implant positioning during flapless surgery, improving the ear prosthesis manufacturing process and reducing operation time, patient morbidity, and related costs. This protocol avoids the need for a reference tool fixed in the cranial bone, as is usually required for maxillofacial surgery, and confirmed that surgical navigation is useful for guiding the insertion of craniofacial implants during flapless surgery.

Optimal positioning of optodes on the scalp for personalized functional near-infrared spectroscopy investigations.

BACKGROUND: Application of functional Near InfraRed Spectroscopy (fNIRS) in neurology is still limited as a good optical coupling and optimized optode coverage of specific brain regions remains challenging, notably for prolonged monitoring. METHODS: We propose to evaluate a new procedure allowing accurate investigation of specific brain regions. The procedure consists in: (i) A priori maximization of spatial sensitivity of fNIRS measurements targeting specific brain regions, while reducing the number of applied optodes in order to decrease installation time and improve subject comfort. (ii) Utilization of a 3D neuronavigation device and usage of collodion to glue optodes on the scalp, ensuring good optical contact for prolonged investigations. (iii) Local reconstruction of the hemodynamic activity along the cortical surface using inverse modelling. RESULTS: Using realistic simulations, we demonstrated that maps derived from optimal montage acquisitions showed, after reconstruction, spatial resolution only slightly lower to that of ultra high density montages while significantly reducing the number of optodes. The optimal montages provided overall good quantitative accuracy especially at the peak of the spatially reconstructed map. We also evaluated real motor responses in two healthy subjects and obtained reproducible motor responses over different sessions. COMPARISON WITH EXISTING METHODS: We are among the first to propose a mathematical optimization strategy, allowing high sensitivity measurements. CONCLUSIONS: Our results support that using personalized optimal montages should allow to conduct accurate fNIRS studies in clinical settings and realistic lifestyle conditions.

Enhanced sensitivity in non-enzymatic glucose detection by improved growth kinetics of Ni-based nanostructures.

Ni-based nanostructures are attractive catalytic materials for many electrochemical applications, among which are non-enzymatic sensing, charge storage, and water splitting. In this work, we clarify the synthesis kinetics of Ni(OH)2/NiOOH nanowalls grown by chemical bath deposition at room temperature and at 50 °C. We applied the results to non-enzymatic glucose sensing, reaching a highest sensitivity of 31 mA cm-2mM-1. Using scanning electron microscopy, x-ray diffraction analysis and Rutherford backscattering spectrometry we found that the growth occurs through two regimes: first, a quick random growth leading to disordered sheets of Ni oxy-hydroxide, followed by a slower growth of well-aligned sheets of Ni hydroxide. A high growth temperature (50 °C), leading mainly to well-aligned sheets, offers superior electrochemical properties in terms of charge storage, charge carrier transport and catalytic action, as confirmed by cyclic voltammetry and electrochemical impedance spectroscopy analyses. The reported results on the optimization and application of low-cost synthesis of these Ni-based nanostructures have a large potential for application in catalysis, (bio)sensing, and supercapacitors areas.

Comparison of the spatial resolution of source imaging techniques in high-density EEG and MEG.

BACKGROUND: The present study aims at evaluating and comparing electrical and magnetic distributed source imaging methods applied to high-density Electroencephalography (hdEEG) and Magnetoencephalography (MEG) data. We used resolution matrices to characterize spatial resolution properties of Minimum Norm Estimate (MNE), dynamic Statistical Parametric Mapping (dSPM), standardized Low-Resolution Electromagnetic Tomography (sLORETA) and coherent Maximum Entropy on the Mean (cMEM, an entropy-based technique). The resolution matrix provides information of the Point Spread Functions (PSF) and of the Crosstalk functions (CT), this latter being also called source leakage, as it reflects the influence of a source on its neighbors. METHODS: The spatial resolution of the inverse operators was first evaluated theoretically and then with real data acquired using electrical median nerve stimulation on five healthy participants. We evaluated the Dipole Localization Error (DLE) and the Spatial Dispersion (SD) of each PSF and CT map. RESULTS: cMEM showed the smallest spatial spread (SD) for both PSF and CT maps, whereas localization errors (DLE) were similar for all methods. Whereas cMEM SD values were lower in MEG compared to hdEEG, the other methods slightly favored hdEEG over MEG. In real data, cMEM provided similar localization error and significantly less spatial spread than other methods for both MEG and hdEEG. Whereas both MEG and hdEEG provided very accurate localizations, all the source imaging methods actually performed better in MEG compared to hdEEG according to all evaluation metrics, probably due to the higher signal-to-noise ratio of the data in MEG. CONCLUSION: Our overall results show that all investigated methods provide similar localization errors, suggesting very accurate localization for both MEG and hdEEG when similar number of sensors are considered for both modalities. Intrinsic properties of source imaging methods as well as their behavior for well-controlled tasks, suggest an overall better performance of cMEM in regards to spatial resolution and spatial leakage for both hdEEG and MEG. This indicates that cMEM would be a good candidate for studying source localization of focal and extended generators as well as functional connectivity studies.

Quantum phase transition in an effective three-mode model of interacting bosons.

In this work we study an effective three-mode model describing interacting bosons. These bosons can be considered as exciton-polaritons in a semiconductor microcavity at the magic angle. This model exhibits quantum phase transition (QPT) when the parameters of the corresponding Hamiltonian are continuously varied. The properties of the Hamiltonian spectrum (e.g., the distance between two adjacent energy levels) and the phase space structure of the thermodynamic limit of the model are used to indicate QPT. The relation between spectral properties of the Hamiltonian and the corresponding classical frame of the thermodynamic limit of the model is established as indicative of QPT. The average number of bosons in a specific mode and the entanglement properties of the ground state as functions of the parameters are used to characterize the order of the transition and also to construct a phase diagram. Finally, we verify our results for experimental data obtained for a setting of exciton-polaritons in a semiconductor microcavity.

Fungemia due to Kodamaea ohmeri: First isolating in Italy. Case report and review of literature.

Kodamaea ohmeri, also known as Pichia ohmeri, is a yeast belonging to the Saccharomycetes family. In 2012, our hospital has recorded the first case of fungemia caused by K. ohmeri in an 80-year-old male, admitted to intensive care following an acute anterior-lateral myocardial infarction. K. ohmeri grew in blood cultures. Biochemical identification was confirmed using Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry and molecular sequencing. Antifungal susceptibility has been performed by broth dilution technique. This case confirms that K. ohmeri is an emergent pathogen even though rarely isolated in human disease. Permanent catheterization is a risk factor and may cause the persistence of a K. ohmeri infection, as well as support treatments (mechanical ventilation, tracheotomy, dialysis). Our therapeutic strategy has been empirical and based exclusively on tested antifungals MIC because EUCAST recommendations does not indicate breakpoints.

Wakefulness delta waves increase after cortical plasticity induction.

OBJECTIVE: Delta waves (DW) are present both during sleep and in wakefulness. In the first case, DW are considered effectors of synaptic plasticity, while in wakefulness, when they appear in the case of brain lesions, their functional meaning is not unanimously recognized. To throw light on the latter, we aimed to investigate the impact on DW exerted by the cortical plasticity-inducing protocol of intermittent theta burst stimulation (iTBS). METHODS: Twenty healthy subjects underwent iTBS (11 real iTBS and nine sham iTBS) on the left primary motor cortex with the aim of inducing long-term potentiation (LTP)-like phenomena. Five-minute resting open-eye 32-channel EEG, right opponens pollicis motor-evoked potentials (MEPs), and alertness behavioral scales were collected before and up to 30 min after the iTBS. Power spectral density (PSD), interhemispheric coherence between homologous sensorimotor regions, and intrahemispheric coherence were calculated for the frequency bands ranging from delta to beta. RESULTS: Real iTBS induced a significant increase of both MEP amplitude and DW PSD lasting up to 30 min after stimulation, while sham iTBS did not. The DW increase was evident over frontal areas ipsilateral and close to the stimulated cortex (electrode F3). Neither real nor sham iTBS induced significant modifications in the PSD of theta, alpha, and beta bands and in the interhemispheric coherence. Behavioral visuo-analogic scales score did not demonstrate changes in alertness after stimulations. No correlations were found between MEP amplitude and PSD changes in the delta band. CONCLUSIONS: Our data showed that LTP induction in the motor cortex during wakefulness, by means of iTBS, is accompanied by a large and enduring increase of DW over the ipsilateral frontal cortex. SIGNIFICANCE: The present results are strongly in favor of a prominent role of DW in the neural plasticity processes taking place during the awake state.

Pollen competition between two sympatric Orchis species (Orchidaceae): the overtaking of conspecific of heterospecific pollen as a reproductive barrier.

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post-pollination pre-zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.