A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part 2: Clinical perspectives and clinical methods in the evaluation of ageing skin

With the advancement of skin research, today's consumer has increased access to technological information about ageing skin and hair care products. As a result, there is a rapidly increasing demand for proof of efficacy of these products. Recognizing these demands has led to the development and validation of many clinical methods to measure and quantify ageing skin and the effects of anti-ageing treatments. Many of the current testing methods used to research and evaluate anti-ageing product claim to employ sophisticated instruments alongside more traditional clinical methods. Intelligent use of combined clinical methods has enabled the development of technologically advanced consumer products providing enhanced efficacy and performance. Of non-invasive methods for the assessment and quantification of ageing skin, there is a plethora of tools available to the clinical researcher as defined by key clinically observed ageing parameters: skin roughness and surface texture; fine lines and wrinkles; skin pigmentation; skin colour; firmness and elasticity; hair loss; and proliferative lesions. Furthermore, many clinical procedures for the evaluation of ageing skin treatments are combined with invasive procedures, which enable added-value to claims (such as identification and alteration of biochemical markers), particularly in those cases where perception of product effect needs additional support. As discussed herein, clinical methods used in the assessment of skin ageing are many and require a disciplined approach to their use in such investigations.     

An updated review of clinical methods in the assessment of ageing skin – New perspectives and evaluation for claims support

With the advancement of skin research, today's consumer has increased access to an informed understanding of ageing skin and its appendages, together with a plethora of targeted products to meet such needs. In recent years, increased legislative demands for quality evidential claims support have led not only to the development and validation of clinical methods to measure and quantify ageing skin, but also a clearer understanding of the skin ageing process–especially the impact of both its internal and external environments–as well as a tougher stance on clearly unjustifiable claims. Traditional testing methods used to research and evaluate anti‐ageing products claim to employ sophisticated instruments. Today, however, since the term anti‐ageing can be considered a misnomer, intelligent use of combined more advanced clinical methods has enabled the development of technologically improved consumer products providing enhanced efficacy and targeted performance. Non‐invasive methods for the assessment and quantification of the causes of ageing skin provide tools to the clinical researcher as defined by key clinically observed ageing parameters. Where evidence requires additional support, a number of clinical procedures evaluating ageing skin and hair products are combined with invasive procedures, thus enabling an added value to product claims. As discussed herein, given the enhanced understanding of ageing, we provide an update to our previous reviews of clinical methods used in the assessment of skin ageing, to include the wider aspects of environmental exposure; skin pigmentation; microbiome disturbance; surface topography; colour, radiance, and pH; and structural integrity–all requiring a disciplined approach to their use in dermatological investigations and product claims evidence.     

In vitro antioxidant activity and in vivo photoprotective effect of a red orange extract

Ultraviolet radiation causes damage to the skin, which may result in both precancerous and cancerous skinlesions and acceleration of skin ageing. Topical administration of enzymatic and non-enzymatic antioxidants is an effective strategy for protecting the skin against UV-mediated oxidative damage. Hence, a systematic study to evaluate the in vitro antioxidant activity and in vivo photoprotective effect of a standardized red orange extract (ROE) has been undertaken, where the main active ingredients are anthocyanins, hydroxycinnamic acids, flavanones and ascorbic acid. For the in vitro experiments, the ROE was tested in three models: (1) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test); (2) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) hydrochloride, of mixed dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles (LUVs) (LP-LUV test); and (3) UV-induced peroxidation of phospatidylcholine multilamellar vesicles (UV-IP test). The in vivo antioxidant/radical scavenger activity was assessed by determining the ability of topically applied ROE to reduce UVB-induced skin erythema in healthy human volunteers. The results obtained in the DPPH, LP-LUV and UV-IP tests demonstrated the strong antioxidant properties of ROE, with a clear relationship between ROE scavenger efficiency and its content in antioxidant compounds. In particular, the findings obtained in the UV-IP test provide a strong rationale for using this extract as a photoprotective agent. During in vivo experiments, ROE provided to efficiently protect against photooxidative skin damage when topically applied immediately after skin exposure to UVB radiations. Interestingly, the protective effect of ROE appears higher than that elicited by another natural antioxidant (tocopherol) commonly employed in cosmetic formulations. In conclusion, the present findings demonstrate that ROE affords excellent skin photoprotection, which is very likely a result of the antioxidant/radical scavenger activity of its active ingredients. Thus, ROE might have interesting applications in both anti-photoageing and after-sun cosmetic products.     

Changes in surface configuration of the skin caused by ageing and application of cosmetics: three-dimensional analysis according to a new system based on image analysis and Fourier transformation

The surface configuration of the skin, as characterized by surface furrows and plateaux, is known to change with ageing and skin condition. There have been many attempts to analyse the surface configuration in order to evaluate the effect of skin care products. However, the systems proposed up to now are insufficient to obtain three-dimensional data.
A system based on image analysis and Fourier transformation has been developed. By using this system, we can make a quick and quantitative analysis of the three-dimensional surface configuration of the skin. This system was applied to measure changes in the surface configuration with ageing, and to clarify the effects of cosmetic cream.
As a result, it was found that the furrows become less clear, become parallel in one direction and the texture becomes coarse and irregular, as ageing continues. These changes are believed to correspond to deterioration of the metabolic and moisturizing functions of the skin.
It was also found that the furrows become clear and the texture becomes finer after the application of cream than before. This is believed to be the result of the improvement of the skin condition due to the moisturizing effect of the cream.     

Age‐related changes in pro‐opiomelanocortin (POMC) and related receptors in human epidermis

Much effort has been placed in cosmetic research for better understanding of the effects of ageing on skin’s appearance, structure, mechanical properties and function. It is now of common knowledge that UV radiations induce pre‐mature skin ageing notably in the epidermis where UV radiations induce keratinocyte differentiation. As UV radiations have also been shown to regulate the pro‐opiomelanocortin (POMC) peptide family in the skin and because no study has been conducted so far to investigate the age‐related changes in POMC and related receptors, we analysed POMC, MC‐1R, MC‐2R and MOR‐1 at mRNA level and MC‐1R, MC‐2R and MOR‐1 at protein level too in primary cultures of normal human keratinocytes obtained from female donors aged from 17 to 75 years old. Regarding the gene expressions, we observed that MC‐1R, MC‐2R and MOR‐1 suffered a dramatic decrease after 50 years of age, whereas POMC increased five‐fold. Western blot analysis confirmed these results except for MOR‐1 whose expression appeared to decrease at older age, around 70 years old. Immunostainings specific to MC‐1R, MC‐2R and MOR‐1 performed on full‐thickness skin biopsies also revealed an intense staining in the basal and spinous layers of a 30‐year‐old donor, whereas no reactivity could be observed in a 60‐year‐old one. We conclude that POMC and POMC‐related receptors suffer a dramatically disturbed balance with ageing and that this may be implicated in the general process of skin ageing.     

Screening tests of free radical scavengers for preventing sun-accelerated cutaneous ageing

Free radical formation has been shown to occur in UV-irradiated skin and a large body of evidence suggests that these reactive agents are responsible for sun-accelerated cutaneous ageing. The paper describes two tests for screening free radical scavengers potentially capable of inhibiting photo-induced skin alterations. They are based on analysis of thiobarbituric acid-reactive materials either in an acellular model in vitro or in the epidermis of rose bengal-sensitized mice after white light irradiation. The tests were illustrated with silymarin, a potent vegetable free radical scavenger.     

Zinc l-pyrrolidone carboxylate inhibits the UVA-induced production of matrix metalloproteinase-1 by in vitro cultured skin fibroblasts, whereas it enhances their collagen synthesis

Reduced collagen matrix in the dermis constitutes one of the characteristic features of chronologically aged skin, which is further enhanced on the sun-exposed portions of the body by chronic ultraviolet light (UV) irradiation, inducing the unique changes associated with skin photoageing. The zinc salt of l-pyrrolidone carboxylate (Zinc PCA) has long been used as a cosmetic ingredient, because of its astringent and anti-microbial properties. In the present study, by employing cultured normal human dermal fibroblasts, we found that Zinc PCA suppressed UVA-induced activation of activator protein-1 (AP-1) and reduced matrix metalloproteinase-1 production in these cells, which is thought to be involved in collagen degradation in photoaged skin. Moreover, Zinc PCA treatment of the cells increased the expression of an ascorbic acid transporter mRNA, SVCT2, but not SVCT1, resulting in the enhanced production of type I collagen. Based on these in vitro findings, we consider Zinc PCA to be a promising candidate for an anti-skin ageing agent.     

Soybean extract showed modulation of retinoic acid‐related gene expression of skin and photo‐protective effects in keratinocytes

Soy extracts are well known as medicinal and nutritional ingredients, and exhibit benefits towards human skin including depigmenting or anti‐ageing effects. Despite the wrinkle decreasing effects of retinoids on skin as an anti‐ageing ingredient, retinoid application can causes photo‐sensitive responses such as skin irritation. Thus, their daytime usage is not recommended. The aim of this study is the investigation into the activities of soybean extract as an anti‐ageing ingredient and their comparison to retinoids in this respect. Soybean extract decreased the relative ratio of MMP‐1/TIMP‐1 mRNA to the same degree as retinoic acid in normal human fibroblasts. It also affected mRNA levels of HAS2 and CRABP2 in normal human keratinocytes. Furthermore, we investigated its effect on mRNA expression of histidase, an enzyme that converts histidine into urocanic acid, the main UV light absorption factor of the stratum corneum. Unlike the complete inhibition of histidase exhibited by the mRNA expression of retinoic acid, the effect of soybean extract on histidase gene expression was weaker in normal human keratinocytes. Also, soybean extract pretreatment inhibited UVB‐induced cyclobutane pyrimidine dimer formation dose‐dependently in normal human keratinocytes. In this study, we found that soybean extract modulated retinoic acid‐related genes and showed photo‐protective effects. Our findings suggest that soybean extract could be an anti‐ageing ingredient that can be safely used under the sunlight.     

Intestinal uptake and immunological effects of carrageenan--current concepts

Carrageenans are a group of high molecular weight sulphated polygalactans which find extensive use in the food industry as thickening, gelling and protein-suspending agents. Although there is no evidence to suggest that the persorption of small amounts of carrageenans across the intestinal barrier poses an acute toxic hazard, they are known to be biologically active in a number of physiological systems and extended oral administration in laboratory animals has been shown to modify both in vivo and in vitro immune competence. Whereas this effect could be attributed to carrageenan having a selective toxic effect on antigen-processing macrophages, additional studies suggest that macrophages can also influence immune responses by the timed release of immunoregulatory mediators. Evidence in support of this comes from in vitro studies which demonstrate that carrageenan-treated macrophages can, depending on conditions and time of administration, release either stimulatory or inhibitory factors. The former is known to be the immunostimulatory agent interleukin 1 (IL-1). The inhibitory factor, which is produced at an early stage following exposure to non-toxic doses of carrageenans, has yet to be formally identified but it is believed to be a prostaglandin because of its specific mode of action and short biological half-life. At present it is impossible to relate these studies to the human situation. Although it is established that carrageenans can cross the intestinal barrier of experimental animals, there is no evidence to suggest that the limited uptake that may occur in man in any way interferes with normal immune competence. Nevertheless, increased exposure may occur in the neonate during weaning, and adults and children following allergic reactions and episodes of gastrointestinal disease. Further studies under such conditions now seem warranted in order to elucidate the possible immunological consequences which may be associated with enhanced uptake of carrageenans in vulnerable groups.     

Methods for assessment of cutaneous ageing

Quantitative assessment of skin ageing is necessary for the evaluation of age modifying treatments. The particular method used must depend on the target of the intended treatment. It should also clearly distinguish between the degree of chronic environmentally-induced trauma to the skin (predominantly photoageing) and the intrinsic ageing process. However, it is clear that no one measure will be adequate to define the extent of cutaneous ageing. Broadly speaking, the techniques available are either invasive or non-invasive. Both have their uses. For example, topically applied retinoic acid 0.05% has been shown to increase epidermal thickness and decrease stratum corneum replacement time, using an invasive technique to determine the value for the first and a non-invasive method to evaluate the second. Virtually all aspects of skin structure and function change with age. However, because of the importance with regard to appearance, some measure of solar elastotic degenerative change should be made. Profilometry techniques and ultrasound determination of skin thickness, as well as measurement of skin colour, may all have their use for this purpose.     

Study of the refirming effect of a plant complex

Loss of skin elasticity is one of the main problems of ageing. This is a mechanical property influenced by elastin, a protein in the dermis which, together with collagen and glycosaminoglycans, makes up the connective tissue. This tissue is affected by a large number of events (such as cutaneous ageing, pregnancy, slimming processes and cellulitis) which eventually cause it to change. At the same time, the metabolism of the proteins of the connective tissue decreases and there is an ever greater presence of enzymes, principally elastases and collagenases, which are responsible for breaking down the elastin and the collagen. One way to prevent such a loss of elasticity is to use active ingredients that are able to inhibit elastase enzymes. A plant complex was prepared using the following plants: lady's thistle (Silybum marianum GAERTN), alchemilla or yarrow (Alchemilla vulgaris L.), horsetail (Equisetum arvense L.) as well as germinated seeds (Glycine soja Siebold and Zucc., Triticum vulgare Vilars, Medicago sativa L., Raphanus sativus L.). The complex was standardized to give the corresponding active principles, silybin, tannins, silicon and peptides, respectively, and in vitro enzymatic tests were carried out to establish its ability to inhibit elastase. The study of enzymatic inhibition was carried out using two enzymes: (1) porcine pancreatic elastase (PPE), and (2) human leukocyte elastase (HLE). The results showed that the plant complex presents non-competitive inhibition in the order of 41.0% against PPE and 50.0% against HLE. An in vivo test was made alongside the in vitro test using an SEM 474 Cutometer (Courage & Khazaka) to study the elasticity of the skin, and positive effects were obtained when applying a cosmetic formulation containing 5% of the plant complex. Image analysis of duplicates of the cutaneous surface, before and after treatment began with a product containing 5% of plant complex and showed that wrinkles were decreased by 36.7%.     

The weak rate of sphingolipid biosynthesis shown by basal keratinocytes isolated from aged vs. young donors is fully rejuvenated after treatment with peptides of a potato hydrolysate

A new study was carried out to bring more information on the effect of the potato proteins ferment. Basal keratinocytes obtained from freshly excised skin samples of two groups of five donors, a young one (25–36‐year‐old) and an aged one (59–70‐year‐old) were established in culture. The results showed a downward trend in the content of all lipid fractions in untreated keratinocytes of aged donors when compared with young ones. We found major differences in the response of keratinocytes to potato proteins ferment treatment between young and old donors. Whereas the lipid content of cells from young donors increased either moderately or actually decreased in some cases in comparison with the untreated controls, the lipid biosynthesis was strongly stimulated in aged donors’ keratinocytes whose lipid contents globally became close to those found in young donors. However, the changes elicited by potato proteins ferment treatment were not seen at the same extent for all lipid classes. Cholesterol content increased up to three‐fold and alpha‐hydroxy fatty acids were augmented up to seven‐fold, whereas the increase in normal fatty acids was quite moderate. In sphingolipids labelled by incubation of keratinocytes in culture medium containing [¹⁴C]‐serine, ceramides and glucosylceramides in cells from aged donors showed the highest uptake of radioactivity, with somewhat less incorporation in sphingomyelin and gangliosides. Therefore, it seems that potato proteins ferment has a much more potent stimulatory activity on the lipid biosynthesis of basal keratinocytes of aged donors, thereby normalizing the cellular lipid content that obviously decreases along with ageing. Although our results were obtained only with basal keratinocytes in this study, potato proteins ferment could be beneficial to maintain an efficient skin barrier in ageing people, provided that the peptides can get through to the basal membrane upon topical application.     

Beyond UV radiation: A skin under challenge

Since ancient times, human beings have been trying to protect their skin against the adverse effects of the sun. From the first mineral sunscreens used by Egyptians, to the current more sophisticated ultraviolet (UVA/UVB) organic sunscreens, progress has been made in terms of sun protection and deeper knowledge of skin physiology has been acquired in the process. The solar spectrum is composed of radiations of various wavelengths having specific, as well as overlapping effects on skin. UVB is mainly responsible for sunburn and DNA dimer formation that can lead to mutation. UVA generates oxidative reactions affecting DNA, proteins and lipids, and is also immunosuppressive. Recently, visible light and infrared radiation (IR) have been associated with oxidative damage and IR has been additionally linked to adverse heat effects on skin. Numerous other extrinsic factors, related to environment and lifestyle, also affect the appearance of skin, precipitating ageing. New molecular mechanisms linking sun and environmental factors to skin ageing have been identified: IR affects mitochondrial integrity and specific heat receptors also mediate some of its effects, tryptophan is a chromophore for UVB, and the aryl hydrocarbon receptor (AhR) is activated by light and xenobiotics to alter skin physiology. Integrating all these new elements is changing the way we think about skin extrinsic ageing. Is UVA/UVB sunscreen protection still enough for our skin?     

Material design using genetic algorithms and preparation of innovative UV-reflecting composite

The neck is a sun-exposed area. It seems to show the symptoms of photo-ageing as well as facial skin in the elderly. However, the physiological study of neck skin has hardly been reported. We examined the change of skin physiological properties at a neck site with ageing for 61 women (18—69 years old) compared with a cheek site. Water content in stratum corneum (SC) was higher, transepidermal water loss (TEWL) was lower and the turnover rate of SC judged from corneocyte area was slower at the neck site compared with the cheek site. Skin thickness was thinner, skin extensibility and elasticity were higher, skin grooves were deeper, and anisotropy of skin furrows was lower at the neck site than those at the cheek site. It was shown that the neck was also affected by sunlight but not so much as the cheek from the result of gelatinase activity detected in the tape stripped SC. Skin elasticity decreased with ageing at the neck site as well as the cheek site. Fine wrinkles were remarkably increased in the direction of Langer's line with ageing at the neck. Most skin physiological parameters at the neck showed the value between the cheek (heavily sun-exposed area) and back (not sun-exposed area). From these results, it was considered that not only intrinsic ageing but also photo-ageing affected the neck skin. We developed the prototype of cosmetics corresponding to neck skin physiology based on these results, and evaluated the effectiveness of the prototype product by a consumer test including skin measurement for 4 weeks. After treatment, water content increased, and it gave satisfaction in the skin colour brightness, skin elasticity and skin texture improvements for almost all volunteers. It was concluded that the prototype product was useful in neck skin treatment.     

Quantitative assessment of lactate and progerin production in normal human cutaneous cells during normal ageing: effect of an Alaria esculenta extract

Anti-ageing products are of a great importance in cosmetic fields. However, even if numerous strategies have been proposed to fight against skin ageing or to minimize its aesthetic impact since the beginning of the 'scientific cosmetology' era, the products basing their efficacy on the observation of pathological situations are rare. The most obvious pathology linked to the ageing of skin (notably) consists in the Hutchinson-Gilford Progeria Syndrome (HGPS), a rare disorder characterized by accelerated ageing and early death. In this disease the lamin A, a protein participating (with others lamins) in the formation of the nuclear lamina and implicated in nuclear stability, chromatin structure and gene expression, is present in a truncated version called progerin. In this study, we have examined the lactate and the progerin production of human normal cutaneous cells issued from subjects of different ages. Using a sensitive and specific progerin ELISA assay developed in house, we so provide the first quantitative demonstration of an increased progerin expression and lactate production in skin during ageing. Moreover, we have also demonstrated that in the selected experimental conditions, it was possible to down-regulate the progerin production of aged cells by using an algae extract. As this extract, an Alaria esculenta extract, could be used in cosmetic formulations, we suggest that a better understanding of the skin pathologies could be a useful tool in developing efficient active compounds, attractive for but not limited to cosmetic purposes.     

Natural skin surface pH is on average below 5, which is beneficial for its resident flora

Variable skin pH values are being reported in literature, all in the acidic range but with a broad range from pH 4.0 to 7.0. In a multicentre study (N = 330), we have assessed the skin surface pH of the volar forearm before and after refraining from showering and cosmetic product application for 24 h. The average pH dropped from 5.12 +/- 0.56 to 4.93 +/- 0.45. On the basis of this pH drop, it is estimated that the 'natural' skin surface pH is on average 4.7, i.e. below 5. This is in line with existing literature, where a relatively large number of reports (c. 50%) actually describes pH values below 5.0; this is in contrast to the general assumption, that skin surface pH is on average between 5.0 and 6.0. Not only prior use of cosmetic products, especially soaps, have profound influence on skin surface pH, but the use of plain tap water, in Europe with a pH value generally around 8.0, will increase skin pH up to 6 h after application before returning to its 'natural' value of on average below 5.0. It is demonstrated that skin with pH values below 5.0 is in a better condition than skin with pH values above 5.0, as shown by measuring the biophysical parameters of barrier function, moisturization and scaling. The effect of pH on adhesion of resident skin microflora was also assessed; an acid skin pH (4-4.5) keeps the resident bacterial flora attached to the skin, whereas an alkaline pH (8-9) promotes the dispersal from the skin.     

Improvement in skin color achieved by smoking cessation

It has been well known that habitual smoking accelerates premature skin ageing recognized as ‘smoker's face’. However, the effect of smoking cessation on the appearance of skin has not been elucidated. The aim of this study was to evaluate objectively the effect of smoking cessation on the skin's appearance. The stratum corneum carbonyl protein level and skin colour of the cheek and the hand were measured. The change before and during the smoking cessation treatment (0, 2, 4, 8 and 12 weeks), and the success or failure in smoking cessation, was compared and examined. Eighty‐four cases who had smoking cessation treatment were examined. The level of the stratum corneum carbonyl protein did not show any difference comparing before and after treatment for the smoking cessation success group and the failure group. The lightness of skin colour showed an upward tendency 4–12 weeks after starting the treatment in the success group and increased significantly compared with the failure group. The redness showed a significant decrease in comparison with before the treatment, and it also showed a significant decrease compared with the failure group. The yellowness did not show any clear tendency. Also, the haemoglobin showed a decreased tendency. Furthermore, multivariate statistical analysis showed a possibility that the lightness and haemoglobin could be changed by smoking cessation treatment. In conclusion, our study showed that an upward tendency of skin lightness was seen to correspond with a haemoglobin decrease accompanied by smoking cessation. If we can easily measure skin improvement as an effect of smoking cessation, it is thought to be a useful aid for smoking cessation support.     

Intrinsic and extrinsic factors in skin ageing: a review

As the proportion of the ageing population in industrialized countries continues to increase, the dermatological concerns of the aged grow in medical importance. Intrinsic structural changes occur as a natural consequence of ageing and are genetically determined. The rate of ageing is significantly different among different populations, as well as among different anatomical sites even within a single individual. The intrinsic rate of skin ageing in any individual can also be dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light. Photodamage, which considerably accelerates the visible ageing of skin, also greatly increases the risk of cutaneous neoplasms. As the population ages, dermatological focus must shift from ameliorating the cosmetic consequences of skin ageing to decreasing the genuine morbidity associated with problems of the ageing skin. A better understanding of both the intrinsic and extrinsic influences on the ageing of the skin, as well as distinguishing the retractable aspects of cutaneous ageing (primarily hormonal and lifestyle influences) from the irretractable (primarily intrinsic ageing), is crucial to this endeavour.     

Direct stimulatory effect of ghrelin on pituitary release of LH through a nitric oxide-dependent mechanism that is modulated by estrogen

Ghrelin, a gut peptide with key actions on food intake and GH secretion, has been recently recognized as potential regulator of reproductive function. Thus, in adult female rats, ghrelin has been proven to modulate GnRH/LH secretion, with predominant inhibitory effects in vivo. We analyze herein potential direct pituitary effects of ghrelin on basal and GnRH-stimulated gonadotropin secretion in prepubertal female rats, and its interplay with ovarian inputs, nitric oxide (NO), and hypothalamic differentiation. In the experimental setting, pituitaries from intact and ovariectomized prepubertal female rats were challenged with ghrelin in vitro and LH secretion was monitored. Our results demonstrate that 1) ghrelin consistently stimulated in vitro pituitary LH secretion under different experimental conditions; 2) the sensitivity to ghrelin, expressed either as the minimal effective dose or the amplitude of the LH response, was modulated by ovarian inputs; 3) the blockade of estrogen action significantly augmented the stimulatory effect of ghrelin; 4) the stimulatory effect of ghrelin on LH secretion required proper NO synthesis; and 5) the ability of ghrelin to elicit LH secretion in vitro was preserved after alteration (masculinization) of brain sexual differentiation. Overall, our present data reinforce the concept that ghrelin participates in the control of LH secretion, with potential stimulatory actions at the pituitary level that require the presence of NO and are modulated by ovarian signals.