A set of principles and practical suggestions for equitable fieldwork in biology.

Field biology is an area of research that involves working directly with living organisms in situ through a practice known as "fieldwork." Conducting fieldwork often requires complex logistical planning within multiregional or multinational teams, interacting with local communities at field sites, and collaborative research led by one or a few of the core team members. However, existing power imbalances stemming from geopolitical history, discrimination, and professional position, among other factors, perpetuate inequities when conducting these research endeavors. After reflecting on our own research programs, we propose four general principles to guide equitable, inclusive, ethical, and safe practices in field biology: be collaborative, be respectful, be legal, and be safe. Although many biologists already structure their field programs around these principles or similar values, executing equitable research practices can prove challenging and requires careful consideration, especially by those in positions with relatively greater privilege. Based on experiences and input from a diverse group of global collaborators, we provide suggestions for action-oriented approaches to make field biology more equitable, with particular attention to how those with greater privilege can contribute. While we acknowledge that not all suggestions will be applicable to every institution or program, we hope that they will generate discussions and provide a baseline for training in proactive, equitable fieldwork practices.

Anti-predator defences are linked with high levels of genetic differentiation in frogs.

Predator-prey interactions have been suggested as drivers of diversity in different lineages, and the presence of anti-predator defences in some clades is linked to higher rates of diversification. Warning signals are some of the most widespread defences in the animal world, and there is evidence of higher diversification rates in aposematic lineages. The mechanisms behind such species richness, however, are still unclear. Here, we test whether lineages that use aposematism as anti-predator defence exhibit higher levels of genetic differentiation between populations, leading to increased opportunities for divergence. We collated from the literature more than 3000 pairwise genetic differentiation values across more than 700 populations from over 60 amphibian species. We find evidence that over short geographical distances, populations of species of aposematic lineages exhibit greater genetic divergence relative to species that are not aposematic. Our results support a scenario where the use of warning signals could restrict gene flow, and suggest that anti-predator defences could impact divergence between populations and potentially have effects at a macro-evolutionary scale.

The effects of preformed vitamin A and provitamin A carotenoid supplementation on tadpoles of the poison frog Phyllobates vittatus.

Understanding the nutritional requirements of captive animals is necessary for proper animal husbandry, however, the specific dietary requirements for many amphibian species commonly kept in captivity are unknown. Like most vertebrates, frogs cannot synthesize carotenoids and must therefore obtain these essential nutrients through diet. It is unclear if amphibians can cleave provitamin A carotenoids to form vitamin A metabolically within the body, so common practice is to supplement their captive diets with both preformed vitamin A and provitamin A carotenoids. We carried out a feeding experiment in tadpoles of Phyllobates vittatus, a commonly kept poison frog species, to test the effects of supplementing a fish flake diet with a provitamin A carotenoid (2.5 mg/g ß-carotene) and vitamin A (0.033-0.066 µg/mL retinyl acetate), both individually and in combination. Contrary to our expectations, supplementation had either no effect or adverse effects on tadpole growth and survivorship. Tadpoles reared under supplemented diets with vitamin A showed higher mortality rates, coupled with symptoms of hypervitaminosis A. Survivors had a smaller body size and mass at metamorphosis. ß-carotene supplementation alone had no detectable effect. The vitamin A and ß-carotene levels in our supplemented diet have been shown to be harmless or benefit tadpoles of other species, yet our results indicate that adding these amounts to what is found in a generalist fish flake mix can have detrimental effects on P. vittatus tadpoles. More broadly, this study highlights the importance of creating husbandry guidelines based on the specific physiological needs of the species (or species groups) being kept in captivity, rather than general ones for all amphibians, as is often done.

Long-term sulforaphane-treatment restores redox homeostasis and prevents cognitive decline in middleaged female and male rats, but cannot revert previous damage in old animals.

Aging is a complex and detrimental process, which disrupts most organs and systems within the organisms. The nervous system is morphologically and functionally affected during normal aging, and oxidative stress has been involved in age-related damage, leading to cognitive decline and neurodegenerative processes. Sulforaphane (SFN) is a hormetin that activates the antioxidant and anti-inflammatory responses. So, we aimed to evaluate if SFN long-term treatment was able to prevent age-associated cognitive decline in adult and old female and male rats. Memory was evaluated in adult (15-month-old), and old (21-month-old) female and male Wistar rats after three months of SFN treatment. Young rats (4-month-old) were used as age controls. The antioxidant response induction, the redox state (GSH/GSSG), and oxidative damage were determined in the brain cortex (Cx) and hippocampus (Hc). Our results showed that SFN restored redox homeostasis in the Cx and Hc of adult rats, thus preventing cognitive decline in both sexes; however, the redox responses were not the same in males and females. Old rats were not able to recover their redox state as adults did, but they had a mild improvement. These results suggest that SFN mainly prevents rather than reverts neural damage; though, there might also be a range of opportunities to use hormetins like SFN, to improve redox modulation in old animals.

Divergence, gene flow, and the origin of leapfrog geographic distributions: The history of colour pattern variation in Phyllobates poison-dart frogs.

The geographic distribution of phenotypic variation among closely related populations is a valuable source of information about the evolutionary processes that generate and maintain biodiversity. Leapfrog distributions, in which phenotypically similar populations are disjunctly distributed and separated by one or more phenotypically distinct populations, represent geographic replicates for the existence of a phenotype, and are therefore especially informative. These geographic patterns have mostly been studied from phylogenetic perspectives to understand how common ancestry and divergent evolution drive their formation. Other processes, such as gene flow between populations, have not received as much attention. Here, we investigate the roles of divergence and gene flow between populations in the origin and maintenance of a leapfrog distribution in Phyllobates poison frogs. We found evidence for high levels of gene flow between neighbouring populations but not over long distances, indicating that gene flow between populations exhibiting the central phenotype may have a homogenizing effect that maintains their similarity, and that introgression between 'leapfroging' taxa has not played a prominent role as a driver of phenotypic diversity in Phyllobates. Although phylogenetic analyses suggest that the leapfrog distribution was formed through independent evolution of the peripheral (i.e. leapfrogging) populations, the elevated levels of gene flow between geographically close populations poise alternative scenarios, such as the history of phenotypic change becoming decoupled from genome-averaged patterns of divergence, which we cannot rule out. These results highlight the importance of incorporating gene flow between populations into the study of geographic variation in phenotypes, both as a driver of phenotypic diversity and as a confounding factor of phylogeographic inferences.

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog.

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin.

A new species of Andean poison frog, Andinobates (Anura: Dendrobatidae), from the northwestern Andes of Colombia.

The poison frogs of the Colombian Andes, Pacific lowlands and Panama have been recently recognized as a new, monophyletic and well-supported genus: Andinobates. The species richness and distribution within Andinobates remain poorly understood due to the paucity of geographic, genetic and phenotypic data. Here we use a combination of molecular, bioacoustic and morphometric evidence to describe a new species of Andean poison frog: Andinobates cassidyhornae sp. nov. from the high elevation cloud forests of the Colombian Cordillera Occidental, in the northwestern Andes. The new species is associated to the bombetes group and characterized by a unique combination of ventral and dorsal color patterns. Data on 1119 bp from two mitochondrial markers allowed us to reject the null hypotheses that A. cassidyhornae sp. nov. is part of the phenotypically similar and geographically less distant species: A. opisthomelas, A. virolinensis or A. bombetes. The best available phylogenetic trees and the genetic distance to other Andinobates species further support this decision. Altogether, the advertisement call parameters unambiguously separated A. cassidyhornae sp. nov. calls from the calls of the three closest species. The new species adds to a poorly known and highly endangered genus of poison frogs that requires further studies and urgent conservation measures.

Two new species of Salamanders, Genus Bolitoglossa (Amphibia: Plethodontidae), from the Eastern Colombian Andes.

The salamander fauna of Colombia is very poorly known, probably because most research efforts have been devoted to anurans during the last two decades. Here, we describe two new species of the genus Bolitoglossa (Eladinea) from the eastern flank of the Eastern Colombian Andes (Cordillera Oriental), near the border with Venezuela. Bolitoglossa tamaense sp. nov. is distributed between 2000 to 2700 m.a.s.l. and Bolitoglossa leandrae sp. nov. is distributed in the low-lands at about 600 m. The new species are diagnosed by a combination of molecular (16S rRNA sequences), coloration, body size, and morphometric (number of maxillary and vomerine teeth and differences in foot webbing) characters. Both species face threats such as chytridiomycosis infections and habitat fragmentation that have already affected other sala-manders in the country. Thus, intensive field and museum work is needed to better document and perhaps protect the local salamander diversity.

Chronic consumption of a hypercaloric diet increases neuroinflammation and brain senescence, promoting cognitive decline in middle-aged female Wistar rats.

Being overweight and obesity are world health problems, with a higher prevalence in women, defined as abnormal or excessive fat accumulation that increases the risk of chronic diseases. Excess energy leads to adipose expansion, generating hypertrophic adipocytes that produce various pro-inflammatory molecules. These molecules cause chronic low-intensity inflammation, affecting the organism's functioning and the central nervous system (CNS), inducing neuroinflammation. The neuroinflammatory response during obesity occurs in different structures of the CNS involved in memory and learning, such as the cortex and the hippocampus. Here we analyzed how obesity-related peripheral inflammation can affect CNS physiology, generating neuroinflammation and promoting cellular senescence establishment. Since some studies have shown an increase in senescent cells during aging, obesity, and neurodegenerative diseases, we proposed that cellular senescence participation may contribute to the cognitive decline in an obesity model of middle-aged female Wistar rats. The inflammatory state of 6 and 13 months-old female Wistar rats fed with a hypercaloric diet was measured in serum and CNS (cortex and hippocampus). Memory was evaluated using the novel object recognition (NOR) test; the presence of senescent markers was also determined. Our data suggest that the systemic inflammation generated by obesity induces a neuroinflammatory state in regions involved in learning and memory, with an increase in senescent markers, thus proposing senescence as a potential participant in the negative consequences of obesity in cognition.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons.

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Sensory and memory processing in old female and male Wistar rat brain, and its relationship with the cortical and hippocampal redox state.

The brain is one of the most sensitive organs damaged during aging due to its susceptibility to the aging-related oxidative stress. Hence, in this study, the sensory nerve pathway integrity and the memory were evaluated and related to the redox state, the antioxidant enzymes function, and the protein oxidative damage in the brain cortex (Cx) and the hippocampus (Hc) of young (4-month-old) and old (24-month-old) male and female Wistar rats. Evoked potentials (EP) were performed for the auditory, visual, and somatosensory pathways. In both males and females, the old rat groups' latencies were larger in almost all waves when compared to the young same-sex animals. The novel object test was performed to evaluate memory. The superoxide dismutase and catalase antioxidant activity, as well as the protein oxidative damage, and the redox state were evaluated. Magnetic resonance (MR) imaging was used to obtain the diffusion tensor imaging, and the brain volume, while MR spectroscopy was used to obtain the brain metabolite concentrations (glutamine, glutamate, Myo-inositol, N-acetyl-aspartate, creatine) in the Cx and the Hc of young and old females. Our data suggest that, although there are limited variations regarding memory and nerve conduction velocity by sex, the differences concerning the redox status might be important to explain the dissimilar reactions during brain aging between males and females. Moreover, the increment in Myo-inositol levels in the Hc of old rats and the brain volume decrease suggest that redox state alterations might be correlated to neuroinflammation during brain aging.

[Prenatal diagnosis by amniocentesis. Clinical and cytogenetic experience in 1,500 cases]. / Diagnóstico prenatal por amniocentesis. Experiencia clínica y citogenética en 1,500 casos.

BACKGROUND: Genetic amniocentesis is performed in México 25 years ago but only few works have been published. OBJECTIVE: To analyze clinical and cytogenetic findings in consecutive patients submitted to genetic amniocentesis. MATERIAL AND METHOD: An analysis was made of the clinical features, amniocentesis results and pregnancy outcome in 1500 consecutive cases of genetic amniocentesis. RESULTS: Sixty-eight fetuses with chromosomopathy (4.5%) were detected and two, with an inborn error of metabolism. The most frequent abnormalities were trisomy 21 (32 cases), trisomy 18 (10 cases), trisomy 13(6 cases), 45,X (6 cases), 47,XXY (4 cases). Pregnancy outcome is known in 474 patients (32%). There were five fetal losses (1%). Of the 68 cases with chromosomopathy, the outcome is known in 45, of which, 29 (64%) decided to have an abortion while 16 (35%) continued the pregnancy, six had a spontaneous abortion or perinatal death and ten had an alive new born. Among fetuses with normal or balanced karyotype and normal ultrasound, 11 out of 419 (2.6%) had congenital anomalies. Two of them had a condition known to be related with epigenetic regulation, (Russell Silver and Angelman syndrome). CONCLUSIONS: Amniocentesis is a reliable and low risk method. Cytogenetic findings in this series are similar to those reported in the literature. Most patients with fetal disease decided to have an abortion. The finding of two patients with a condition related with abnormal epigenetic regulation suggests that the magnitude of this risk remains to be defined.

Speciation Associated with Shifts in Migratory Behavior in an Avian Radiation.

Migratory animals move up to thousands of kilometers every year [1]. Losses of migration (i.e., migratory drop-offs) occur when individuals of a migratory species stop migrating and establish founder sedentary populations, a phenomenon documented in birds [2-5] and butterflies [6]. In theory, losses-and also gains-of migration might promote speciation if sedentary and migratory populations become reproductively isolated [7-9]. Because migratory and sedentary strategies involve alternative physiological, behavioral, and morphological traits [10-13], divergence along multiple axes of organismal function is expected to accompany switches in migratory behavior, potentially accelerating speciation. We present evidence of speciation driven by a migratory drop-off in the fork-tailed flycatcher (Tyrannus savana) resulting in reproductive isolation likely driven by changes in breeding schedules (allochronic speciation [13-15]) and geographic isolation of breeding grounds (allopatric speciation [16]). Phylogenetic analyses across New World flycatchers (Tyrannidae) showed that an association between speciation and drop-offs is also observable at a macroevolutionary scale. Loss of migration was significantly more frequent than its gain, and speciation rates of migratory and partially migratory lineages (i.e., species having both migratory and sedentary populations) exceeded those of sedentary lineages. Models of trait evolution indicated that partial migration is an intermediate step between migratory and sedentary states in this family. Given that partial migration is widespread across migratory animals (e.g., of all migratory birds, ca. 51% are partially migratory [5]), speciation via switches in migratory behavior might be an important yet overlooked mechanism of animal diversification.

Does batrachotoxin autoresistance coevolve with toxicity in Phyllobates poison-dart frogs?

Toxicity is widespread among living organisms, and evolves as a multimodal phenotype. Part of this phenotype is the ability to avoid self-intoxication (autoresistance). Evolving toxin resistance can involve fitness tradeoffs, so autoresistance is often expected to evolve gradually and in tandem with toxicity, resulting in a correlation between the degrees of toxicity and autoresistance among toxic populations. We investigate this correlation in Phyllobates poison frogs, notorious for secreting batrachotoxin (BTX), a potent neurotoxin that targets sodium channels, using ancestral sequence reconstructions of BTX-sensing areas of the muscular voltage-gated sodium channel. Reconstructions suggest that BTX resistance arose at the root of Phyllobates, coinciding with the evolution of BTX secretion. After this event, little or no further evolution of autoresistance seems to have occurred, despite large increases in toxicity throughout the history of these frogs. Our results, therefore, provide no evidence in favor of an evolutionary correlation between toxicity and autoresistance, which conflicts with previous work. Future research on the functional costs and benefits of mutations putatively involved in BTX resistance, as well as their prevalence in natural populations, should shed light on the evolutionary mechanisms driving the relationship between toxicity and autoresistance in Phyllobates frogs.

Sulforaphane - role in aging and neurodegeneration.

In the last several years, numerous molecules derived from plants and vegetables have been tested for their antioxidant, anti-inflammatory, and anti-aging properties. One of them is sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables. SFN activates the antioxidant and anti-inflammatory responses by inducing Nrf2 pathway and inhibiting NF-κB. It also has an epigenetic effect by inhibiting HDAC and DNA methyltransferases and modifies mitochondrial dynamics. Moreover, SFN preserves proteome homeostasis (proteostasis) by activating the proteasome, which has been shown to lead to increased cellular lifespan and prevent neurodegeneration. In this review, we describe some of the molecular and physical characteristics of SFN, its mechanisms of action, and the effects that SFN treatment induces in order to discuss its relevance as a "miraculous" drug to prevent aging and neurodegeneration.

Long-Term Moderate Exercise Combined with Metformin Treatment Induces an Hormetic Response That Prevents Strength and Muscle Mass Loss in Old Female Wistar Rats.

Sarcopenia is a syndrome characterized by a progressive and generalized skeletal muscle mass and strength loss, as well as a poor physical performance, which as strongly been associated with aging. Sedentary lifestyle in the elderly contributes to this condition; however, physical activity improves health, reducing morbidity and mortality. Recent studies have shown that metformin (MTF) can also prevent muscle damage promoting muscular performance. To date, there is great controversy if MTF treatment combined with exercise training improves or nullifies the benefits provided by physical activity. This study is aimed at evaluating the effect of long-term moderate exercise combined with MTF treatment on body composition, strength, redox state, and survival rate during the life of female Wistar rats. In this study, rats performed moderate exercise during 20 of their 24 months of life and were treated with MTF for one year or for 6 months, i.e., from 12 to 24 months old and 18 to 24 months old. The body composition (percentage of fat, bone, and lean mass) was determined using a dual-energy X-ray absorption scanner (DXA), and grip strength was determined using a dynamometer. Likewise, medial and tibial nerve somatosensory evoked potentials were evaluated and the redox state was measured by HPLC, calculating the GSH/GSSG ratio in the gastrocnemius muscle. Our results suggest- that the MTF administration, both in the sedentary and the exercise groups, might activate a mechanism that is directly related to the induction of the hormetic response through the redox state modulation. MTF treatment does not eliminate the beneficial effects of exercise throughout life, and although MTF does not increase muscle mass, it increases longevity.

Genome Sequences Reveal Cryptic Speciation in the Human Pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus that causes life-threatening lung infections. About 500,000 people are exposed to H. capsulatum each year in the United States, and over 60% of the U.S. population has been exposed to the fungus at some point in their life. We performed genome-wide population genetics and phylogenetic analyses with 30 Histoplasma isolates representing four recognized areas where histoplasmosis is endemic and show that the Histoplasma genus is composed of at least four species that are genetically isolated and rarely interbreed. Therefore, we propose a taxonomic rearrangement of the genus.IMPORTANCE The evolutionary processes that give rise to new pathogen lineages are critical to our understanding of how they adapt to new environments and how frequently they exchange genes with each other. The fungal pathogen Histoplasma capsulatum provides opportunities to precisely test hypotheses about the origin of new genetic variation. We find that H. capsulatum is composed of at least four different cryptic species that differ genetically and also in virulence. These results have implications for the epidemiology of histoplasmosis because not all Histoplasma species are equivalent in their geographic range and ability to cause disease.

The impact of a monophasic continuous estro-progestogenic treatment on Latin American menopausal women.

OBJECTIVE: The present study was undertaken to assess the impact, effectiveness and safety of a monophasic hormone replacement treatment (HRT) for continuous use with regards to the clinical effects, bleeding patterns and lipid profile of menopausal women in four Latin American countries. DESIGN: Three hundred and six postmenopausal women with natural menopause and uterus present were recruited. This was a multicentre prospective, clinical trial; the participating countries were Brazil (BR), Colombia (CO), Mexico (MX) and Argentina (AR). The study period was 12 months. The HRT regime was formulated in tablets containing 2 mg estradiol E2 and 1mg norethisterone acetate (NETA); one visit every 3 months was solicited. METHODS: HRT was given as one tablet every day without interruption for 1 year. Climacteric complaints, side-effects, reason for discontinuation, bleeding patterns, lipid profile at baseline and 12 months of treatment were documented. RESULTS: There were no significant differences between the four populations on clinical measurements. Thirty-four women discontinued, 13 for bleeding problems. The five most common side-effects were mastalgia, bleeding problems, headache, pelvic pain and nausea. 44.8% of women experienced scanty vaginal bleeding during the first 3 months of therapy. Ninety seven percent of women had amenorrhea at the end of the study in MX, BR and AR, and 100% in CO. Body weight was constant during the study, and no correlation was found between body weight and total days with bleeding. The Kupperman index score was used to evaluate the climacteric complaints, and the score decreased from a mean of 25.4 to 5.1 at 12-months visit. Total cholesterol levels were significantly reduced in BR and CO (P < 0.05) between baseline and the final sample; serum triglycerides remained unchanged, HDL-cholesterol was significantly increased in MX (P < 0.05), and LDL-cholesterol was significantly reduced in CO (P < 0.05). The results of this 1-year study emphasize that a continuous combined HRT regimen with 2 mg E2/1 mg NETA is an attractive alternative for postmenopausal women who are at least 1 year after their menopause and optimally 2 years after their menopause. Although the combination of 2 mg E2 with 1 mg NETA in a continuous combined therapy scheme has been in use in the Nordic countries for over a decade and in Latin America for the last 6 years, there have been no previous published reports on its effectivity in Latin American women. This publication reports the experience in a group of 306 Latin American women, and it is the first Latin American publication with this formulation.

A 13-month multicenter clinical experience of a low-dose monophasic oral contraceptive containing 20 microg ethinylestradiol and 75 microg gestodene in Latin American women.

This prospective, multicenter study was conducted to evaluate the contraceptive reliability, cycle control and tolerability of a 21-day oral contraceptive regimen containing 20 microg ethinylestradiol and 75 microg gestodene in four Latin American countries (Mexico, Argentina, Brazil and Colombia). Participants took trial medication daily for 21 days. Contraceptive efficacy, cycle control and tolerability were evaluated over a period of 13 cycles. Efficacy data gathered from 5,109 treatment cycles were obtained from 393 participants. The trial medication proved to be an effective contraceptive and provided good cycle control. One pregnancy because of poor compliance was recorded. This resulted in a study Pearl index of 0.25. Forty-six percent of Latin American women reported one intracyclic spotting bleeding episode and 37.6% reported one intracyclic breakthrough bleeding (medium/excessive bleeding) episode during cycles 2-4 (primary target). Overall, intracyclic bleeding was reported in 41%. Overall, there was a trend towards a lower incidence of spotting in all the countries and this difference had statistical significance between Argentina and the others three countries (p < 0.05) during cycles 2-4. This trend was also apparent with respect to breakthrough bleeding, but again the difference did not achieve statistical significance. The discontinuation rate because of adverse events was low (3%); no serious adverse events were reported. More than 78% of the women in the four countries maintained constant body weight or lost weight (2 kg) during the study. The treatment effect on blood pressure was negligible. There were no appreciable changes in mean laboratory values over the course of the study.