A retrospective review of factors associated with vasovasostomies in United States military members.

BACKGROUND: Men seeking a vasectomy should receive counseling prior to the procedure that includes discussion of later seeking a reversal. We sought to determine demographic factors that may predispose patients to possibly later seek a vasectomy reversal. METHODS: All U.S. Military electronic health records were searched between 2000 and 2009 for either a vasectomy or vasovasostomy procedure code. Aggregate demographic information was collected and statistical analysis performed. RESULT: A total of 82,945 patients had a vasectomy of which 4,485 had a vasovasostomy resulting in a vasovasostomy-to-vasectomy rate of 5.04%. The average age at vasovasostomy was 34.9±5.0, with an average interval of 4.1±2.2 years. Men undergoing a vasectomy at a younger age were more likely to have a vasovasostomy. Various religions did have statistically significant differences. Within ethnic groups, only Native Americans [OR=1.39 (95% CI 1.198-1.614)] and Asians [OR=0.501 (95% CI 0.364-0.690)] had statistically significant differences when compared to Caucasians. Men with more children at the time of vasectomy were more likely to have a vasovasostomy. CONCLUSION: Younger men, Native Americans, and men with more children at vasectomy were more likely to undergo a vasovasostomy. The reason for these differences is unknown, but this information may assist during pre-vasectomy counseling.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

BACKGROUND: In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Female reproductive disorders: the roles of endocrine-disrupting compounds and developmental timing.

OBJECTIVE: To evaluate the possible role of endocrine-disrupting compounds (EDCs) on female reproductive disorders emphasizing developmental plasticity and the complexity of endocrine-dependent ontogeny of reproductive organs. Declining conception rates and the high incidence of female reproductive disruptions warrant evaluation of the impact of EDCs on female reproductive health. DESIGN: Publications related to the contribution of EDCs to disorders of the ovary (aneuploidy, polycystic ovary syndrome, and altered cyclicity), uterus (endometriosis, uterine fibroids, fetal growth restriction, and pregnancy loss), breast (breast cancer, reduced duration of lactation), and pubertal timing were identified, reviewed, and summarized at a workshop. CONCLUSION(S): The data reviewed illustrate that EDCs contribute to numerous human female reproductive disorders and emphasize the sensitivity of early life-stage exposures. Many research gaps are identified that limit full understanding of the contribution of EDCs to female reproductive problems. Moreover, there is an urgent need to reduce the incidence of these reproductive disorders, which can be addressed by correlative studies on early life exposure and adult reproductive dysfunction together with tools to assess the specific exposures and methods to block their effects. This review of the EDC literature as it relates to female health provides an important platform on which women's health can be improved.

An ecological assessment of bisphenol-A: evidence from comparative biology.

This review assesses the effects of environmental concentrations of bisphenol-A (BPA) on wildlife. Water concentrations of BPA vary tremendously due to proximity to point and non-point sources, but reported concentrations in stream/river water samples are less than 21 microg/L, and concentrations in landfill leacheate are less than 17.2mg/L. Extensive evidence indicates that BPA induces feminization during gonadal ontogeny of fishes, reptiles, and birds, but in all cases the amount of BPA necessary to cause such ontogenetic disruption exceeds concentrations in the environment. Extensive evidence also exists that adult exposure to environmental concentrations of BPA is detrimental to spermatogenetic endpoints and stimulates vitellogenin synthesis in model species of fish. Most of the reported effects of BPA on vertebrate wildlife species can be attributed to BPA acting as an estrogen receptor agonist, but mechanisms of disruption in invertebrates are less certain. A comparison of measured BPA environmental concentrations with chronic values suggests that no significant margin of safety exists for the protection of aquatic communities against the toxicity of BPA. Further studies should examine the most vulnerable vertebrate and invertebrate species.

Seasonal variation in plasma sex steroid concentrations in juvenile American alligators.

Seasonal variation in plasma sex steroid concentrations is common in mature vertebrates, and is occasionally seen in juvenile animals. In this study, we examine the seasonal pattern of sex hormone concentration in juvenile American alligators (Alligator mississippiensis) and make a limited comparison of these seasonal patterns on two different lakes in Florida. Male juvenile alligators from a reference lake, Lake Woodruff, displayed temporal patterns in plasma testosterone (T) concentrations that appear to be seasonal. A similar pattern in plasma estradiol-17beta (E(2)) was observed in juvenile females from Lake Woodruff. Males had significantly elevated T concentrations during the spring and late summer, whereas females had elevated E(2) in the spring and late summer and significantly depressed E(2) concentrations during the winter. A limited 4-month survey of animals from contaminated Lake Apopka found a lack of such seasonality. These results suggest that: (1) healthy wild populations of juvenile alligators have a prolonged peripubescent period that is marked by seasonal hormonal cycles, (2) juvenile alligators exposed to environmental contaminants can lack such seasonal cyclicity, and (3) future studies of juvenile alligators should incorporate such seasonality into the experimental design.

Thyroid status in juvenile alligators (Alligator mississippiensis) from contaminated and reference sites on Lake Okeechobee, Florida, USA.

Exposure to environmental contaminants has been shown to alter normal thyroid function in various wildlife species, including the American alligator (Alligator mississippiensis). Abnormalities in circulating levels of the thyroid hormone thyroxine (T4) have been reported in juvenile alligators from several contaminated lakes in Florida. To further elucidate these functional thyroid abnormalities, this study examines the structure of thyroids and circulating T4 concentrations from juvenile alligators collected from three sites of varying contamination on Lake Okeechobee, Florida. The following variables were used to characterize thyroid morphology: epithelial cell height, width and area, percent colloid, and follicle area. These variables were compared among study sites and between genders. No difference was detected in epithelial cell height, epithelial cell area, or follicle area among the sites, whereas significant differences in epithelial cell width (p = 0.02) and percent colloid (p = 0.008) were found. Animals from the most contaminated site (Belle Glade) had significantly greater epithelial cell widths and significantly less colloid present in their follicles compared to animals from the reference site (West). Gender did not have a significant interaction with site for any variable measured. Thyroxine (T4) concentrations were elevated in the intermediately contaminated site (Conservation Area 3A) compared to the other sites (p < 0.0001). It is proposed that the disruptions seen in Lake Okeechobee alligators are due to disruptions at both the thyroid and extra-thyroidal tissues.

Temporal and spatial variation in plasma thyroxine (T4) concentrations in juvenile alligators collected from Lake Okeechobee and the northern Everglades, Florida, USA.

We examined variation in plasma thyroxine (T4) in juvenile American alligators (Alligator mississippiensis) collected from three sites within the Kissimmee River drainage basin (FL, USA). Based on historical sediment data, Moonshine Bay served as the low contaminant exposure site, Water Conservation Area 3A served as an intermediate contaminant exposure site, and Belle Glade served as the high contaminate exposure site. In May 1999, alligators (n = 22) from Water Conservation Area 3A exhibited higher T4 concentrations than animals from both Belle Glade (n = 22; p = 0.0003) and Moonshine Bay (n = 33; p = 0.001). In May 2000, alligators (n = 29) Water Conservation Area 3A again exhibited higher T4 concentrations than those from Belle Glade (n = 49; p = 0.02) but not those from Moonshine Bay (n = 40). No sexual dimorphism was observed among mean T4 concentrations within any of the sites during either year (p > 0.05). Animals within all sites exhibited higher T4 concentrations in May 2000 when compared to May 1999. When variance was examined, animals from Water Conservation Area 3A exhibited higher variance in plasma T4 concentrations than those from either Moonshine Bay or Belle Glade. We concluded that mean plasma T4 concentrations did not match the sediment contaminant mixture data presently available to us, whereas variance seems to be a more reliable indicator of contaminant exposure.

Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida lakes.

Neonatal and juvenile alligators from contaminated Lake Apopka in central Florida exhibit abnormal plasma sex steroid concentrations as well as morphological abnormalities of the gonad and phallus. This study addresses whether similar abnormalities occur in juvenile alligators inhabiting six other lakes in Florida. For analysis, animals were partitioned into two subsets, animals 40-79 cm total length (1-3 years old) and juveniles 80-130 cm total length (3-7 years old). Plasma testosterone (T) concentrations were lower in small males from lakes Apopka, Griffin, and Jessup than from Lake Woodruff National Wildlife Refuge (NWR). Similar differences were observed in the larger juveniles, with males from lakes Jessup, Apopka, and Okeechobee having lower plasma T concentrations than Lake Woodruff males. Plasma estradiol-17beta (E(2)) concentrations were significantly elevated in larger juvenile males from Lake Apopka compared to Lake Woodruff NWR. When compared to small juvenile females from Lake Woodruff NWR, females from lakes Griffin, Apopka, Orange, and Okeechobee had elevated plasma E(2) concentrations. Phallus size was significantly smaller in males from lakes Griffin and Apopka when compared to males from Lake Woodruff NWR. An association existed between body size and phallus size on all lakes except Lake Apopka and between phallus size and plasma T concentration on all lakes except lakes Apopka and Orange. Multiple regression analysis, with body size and plasma T concentration as independent covariables, explained the majority of the variation in phallus size on all lakes. These data suggest that the differences in sex steroids and phallus size observed in alligators from Lake Apopka are not limited to that lake, nor to one with a history of a major pesticide spill. Further work examining the relationship of sex steroids and phallus size with specific biotic and abiotic factors, such as antiandrogenic or estrogenic contaminants, is needed.

The functional and structural observations of the neonatal reproductive system of alligators exposed in ovo to atrazine, 2,4-D, or estradiol.

Wild alligators exposed to persistent organochlorine contaminants, municipal waste compounds, and contemporary-use herbicides exhibit reproductive alterations that are thought to be caused by endocrine disruption. This study tests the hypothesis that these alterations, at least in part, result from exposure of alligator embryos to contemporary-use herbicides. Alligator eggs were collected early in development, exposed to estradiol-17 beta, atrazine, or 2,4-D (at dosages of 0.14, 1.4, and 14 ppm, plus a dosage of 0.014 ppm for estradiol-17 beta only) before the period of gonadal differentiation, and incubated at a temperature that would produce either 100% males or 100% females. Analysis of histology was performed on the gonads and reproductive tracts of hatchlings. In females, epithelial cell height of the Müllerian duct and medullary regression of the ovary were assessed, whereas in males, sex-cord diameter was measured. Eggs incubated at the female-determining temperature produced all female hatchlings, whereas the estradiol-17 beta treatments caused the production of females at the male-determining temperature. Neither atrazine nor 2,4-D had this effect. Both Müllerian duct epithelial cell height and medullary regression were increased in estradiol-treated animals, but no differences were noted between herbicide-treated alligators and controls. A previous study found that male alligators exposed to 14 ppm atrazine had elevated gonadal aromatase activity, but there was no difference in sex-cord diameter in this or any other treatment group. Additionally, we observed that hepatic aromatase activity was not altered by in ovo exposure to any of the treatments. These results indicate that these herbicides alone are not responsible for the gonadal abnormalities previously reported for juvenile alligators from Lake Apopka and emphasize the importance of analyzing both the function (i.e., steroidogenic enzyme activity) and the structure (i.e., histological analysis) of the reproductive system. Structural assessment alone may be insufficient for detecting subtle endocrine alterations.

Reptiles as models of contaminant-induced endocrine disruption.

Historically, reptiles have been used as bioindicators of environmental contaminants and, currently, reptiles have the potential to elucidate the mechanisms of a newly described group of environmental contaminants--endocrine disrupters. Reptiles are particularly good models for studying endocrine altering compounds due to the fact that different species of reptiles have varying modes of gender determination (genotypic sex determination or temperature-dependent sex determination) and parity modes (oviparity or viviparity). This review focuses both on laboratory and field studies of contaminant-induced endocrine alterations in reptiles. Laboratory studies of oviparous reptiles with temperature-dependent sex determination reveal that embryonic exposure to natural hormones and many man-made chemicals (including the ubiquitous PCBs and common herbicides) can permanently alter the functioning of the reproductive system. It is hypothesized that similar permanent, organizational changes occur in wild reptiles exposed to endocrine-disrupting contaminants.

Cellular bioavailability of natural hormones and environmental contaminants as a function of serum and cytosolic binding factors.

Environmental contaminants have been reported to function as hormone mimics in various wildlife species. To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals. Hormone binding proteins from oviductal cytosol of the American alligator (Alligator mississippiensis) and yellow-bellied turtle (Trachemys scripta) were used in competitive binding assays with [3H] 17 beta-estradiol. Most of the environmental contaminants, and the potent, synthetic estrogen diethylstilbestrol (DES), did not interact with the cytosolic binding proteins. Among the compounds tested, o,p'-DDT and toxaphene exhibited the greatest affinity for the binding proteins. The functional consequence of the apparent lack of interaction of most contaminants with binding proteins was studied in a strain of yeast containing the human estrogen receptor (YES assay). The activation of YES with estradiol was reduced 30% in the presence of a physiological concentration (0.01 mg/mL) of human sex hormone binding globulin (SHBG), a hormone binding protein found in the blood. In contrast, the activity of DES was not inhibited by 0.01 mg/mL SHBG. Interestingly, ethinyl estradiol, a major component of contraceptives, did not appear to appreciably interact with SHBG in the YES system. Together, these data suggest that cytosolic and circulating binding proteins bind many environmental contaminants with much less affinity than native steroids. Therefore, such contaminants may be more hormonally active than previously hypothesized.

The reproductive cycle of the female American alligator (Alligator mississippiensis).

We examined ovarian and oviducal gross morphology and collected blood samples from wild female alligators in central Florida during most of the year. Females with vitellogenic follicles were observed throughout the year, although ovaries containing follicles larger than 15 mm were seen only during the spring and early summer (March-June). We detected a poor relationship between female body size and the number of vitellogenic follicles whereas body size was significantly correlated with clutch size. Plasma samples were analyzed for (1) estradiol-17beta (E2), testosterone (T), and corticosterone by specific radioimmunoassays; (2) vitellogenin by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and (3) total protein, phosphorus, and calcium by spectrophotometric assays. Reproductively active females showed elevated plasma concentrations of E2 during the fall (September-November) and spring (March-May) whereas non-reproductively active females exhibited basal levels with no apparent peaks. Vitellogenin was detected in the plasma during the same months that plasma E2 concentrations were elevated. Elevated plasma vitellogenin and E2 were not correlated with plasma total protein but were correlated with plasma calcium concentration. During late vitellogenesis, plasma T concentrations were elevated in reproductively active females coincident with a period of intense courtship and mating. Corticosterone plasma concentrations exhibit no significant monthly variation, nor apparent changes during various stages of reproductive activity although plasma concentrations were elevated during late gravidity. Our data suggest that female reproductive activity begins in the fall with an increase in plasma E2 concentration in September or October and vitellogenesis in October. Ovarian activity slows during winter and reactivates with the onset of spring.

Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants.

Many environmental contaminants alter the reproduction of animals by altering the development and function of the endocrine system. The ability of environmental contaminants to alter the endocrine system of alligators was studied both in a descriptive study in which juvenile alligators from a historically contaminated lake were compared to animals from a control lake and in an experimental study in which hatchling control alligators were exposed in ovo to several endocrine-disrupting standards and two modern-use herbicides. Endocrine status was assessed by examining plasma hormone concentrations, gonadal-adrenal mesonephros (GAM) aromatase activity, and gonadal histopathology. In the descriptive study, juvenile alligators from the contaminated lake had significantly lower plasma testosterone concentrations (29.2 pg/ml compared to 51.3 pg/ml), whereas plasma 17 beta-estradiol concentrations did not vary when compared to controls. GAM aromatase activity was significantly decreased n the alligators from the contaminated lake (7.6 pmol/g/hr compared to 11.4 pmol/g/hr). In the experimental study, the endocrine-disrupting standards had the expected effects. 17 beta-Estradiol and tamoxifen caused sex reversal from male to female, with a corresponding increase in aromatase activity. Vinclozolin had no apparent effect on male or female alligators. Among the herbicides tested, atrazine induced GAM aromatase activity in male hatchling alligators that was neither characteristic of males nor females, although testicular differentiation was not altered. Exposure to 2,4-dichlorophenoxyacetic acid had no effect on the endocrine parameters that were measured. Together, these studies show that exposure to some environmental chemicals (such as atrazine) can alter steroidogenesis in alligators, but the endocrine alterations previously noted for Lake Apopka, Florida, alligators can not be fully explained by this mechanism.

Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator.

Reports of reproductive abnormalities in the American alligator from Lake Apopka, Florida, have been linked to a spill of DDT and other pesticides suspected of having hormonelike activity. To determine whether environmental chemicals had the potential to function as exogenous hormones in the American alligator, we examined the ability of chemicals to bind the estrogen receptor (aER) and progesterone receptor (aPR) in a protein extract prepared from the oviduct of the alligator. In competition binding assays with [3H]17 beta-estradiol, some DDT metabolites showed inhibition of [3H]17 beta-estradiol binding to aER. A combination of DDTs demonstrated an additive decrease in [3H]17 beta-estradiol binding to aER. Modern-use chemicals such as alachlor, trans-nonachlor, endosulfan, and atrazine also competed with [3H]17 beta-estradiol for binding to the aER. To test the effect of chemicals identified in alligator eggs from Lake Apopka on [3H]17 beta-estradiol binding, we mixed these chemicals at concentrations measured in eggs in the competition binding assay. 2,2-bis(4-chlorophenyl)-N-(methoxymethyl)acetamide (p,p'-DDD) and trans-nonachlor, both found in Lake Apopka, interacted with aER, whereas others such as chlordane and toxaphene did not. Surprisingly, combinations of these chemicals decreased [3H]17 beta-estradiol binding in a greater than additive manner. To assess the ability of chemicals to interact with aPR, we performed commpetition binding assays with the synthetic progestin [3H]R5020. Most of the chemicals tested did not reduce [3H]R5020 binding to aPR, whereas endosulfan, alachlor, and kepone inhibited binding. These results provide the first evidence that environmental chemicals bind the aER and aPR from the American alligator, supporting the hypothesis that the reported reproductive abnormalities may be related to the modulation of endocrine-related responses. The findings that combinations of chemicals demonstrated a greater than additive interaction with the aER and some chemicals bind to the aPR in the competition binding assay are novel. This suggests that interactions of these chemicals with the endocrine system are complex.

Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant.

Endocrine disrupting chemicals can potentially alter the reproductive physiology of fishes. To test this hypothesis, serum was collected from common carp (Cyprinus carpio) at five riverine locations in Minnesota. Male fish collected from an effluent channel below the St. Paul metropolitan sewage treatment plant had significantly elevated serum egg protein (vitellogenin) concentrations and significantly decreased serum testosterone concentrations compared to male carp collected from the St. Croix River, classified as a National Wild and Scenic River. Carp collected from the Minnesota River, which receives significant agricultural runoff, also exhibited depressed serum testosterone concentrations, but no serum vitellogenin was apparent. These data suggest that North American rivers are receiving estrogenic chemicals that are biologically active, as has been reported in Great Britain.

Plasma insulin-like growth factor-I concentration during the reproductive cycle of the American alligator (Alligator mississippiensis).

Insulin-like growth factor I (IGF-I) is involved in the growth, reproduction, and development of vertebrates. In this study, we report plasma IGF-I concentrations throughout the reproductive cycle of the American alligator (Alligator mississippiensis) and consider these concentrations with respect to several reproductive steroid hormones and ions involved in reptilian reproduction. Mean plasma IGF-I concentrations were significantly different among reproductive stages. Maximal plasma concentrations of IGF-I were observed throughout gravidity. Plasma IGF-I concentration was not correlated with plasma concentrations of estradiol-17 beta, calcium, phosphorus, albumin, total protein, or body size, but was correlated with plasma progesterone concentration. We hypothesize that the elevated concentrations of IGF-I in gravid female alligators are due to the synthesis of this hormone for incorporation into eggs as well as residual IGF-I that could function in the stimulation of uterine secretory products.

Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment.

The development of the male reproductive ducts and external genitalia in vertebrates is dependent on elevated androgen concentrations during embryonic development and the period of postnatal growth. We have observed that a population of juvenile alligators living on Lake Apopka exhibit significantly smaller penis size (24% average decrease) and lower plasma concentrations of testosterone (70% lower concentrations) when compared to animals of similar size on Lake Woodruff. In addition to smaller phalli, no relationship exists between plasma testosterone concentrations and penile size in males from Lake Apopka, whereas a positive relationship exists for males from Lake Woodruff. The alligators on Lake Apopka are known to have elevated concentrations of the antiandrogenic DDT breakdown product p.p'-DDE stored in their fat. We suggest a number of hypotheses that could explain the modification in the phenotype of the juvenile male living in Lake Apopka. These modifications in phenotype include a smaller penis size, lower plasma androgen concentrations, and lack of responsiveness of the penis to the plasma androgens present.

Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife.

Many environmental contaminants disrupt the vertebrate endocrine system. Although they may be no more sensitive to endocrine-disrupting contaminants (EDCs) than other vertebrates, reptiles are good sentinels of exposure to EDCs due to the lability in their sex determination. This is exemplified by a study of alligators at Lake Apopka, Florida, showing that EDCs have altered the balance of reproductive hormones resulting in reproductive dysfunction. Such alterations may be activationally or organizationally induced. Much research emphasizes the former, but a complete understanding of the influence of EDCs in nature can be generated only after consideration of both activational and organizational alterations. The organizational model suggests that a small quantity of an EDC, administered during a specific period of embryonic development, can permanently modify the organization of the reproductive, immune, and nervous systems. Additionally, this model helps explain evolutionary adaptations to naturally occurring estrogenic compounds, such as phytoestrogens.

Identification of sex in hatchling loggerhead turtles (Caretta caretta) by analysis of steroid concentrations in chorioallantoic/amniotic fluid.

A major difficulty in sea turtle conservation is the inability to nonlethally and noninvasively identify the sex of hatching sea turtles. Traditional sexing techniques such as plasma sex steroid quantification cannot be applied to hatchlings without sacrificing the hatchlings or utilizing invasive procedure. This paper presents a technique for sexing hatchling sea turtles by analysis of sex steroid concentrations in egg chorioallantoic/amniotic fluid (CAF). Metabolites of estradiol-17 beta (E) and testosterone (T) in CAF are best expressed as an index or E:T ratio. Chorioallantoic/amniotic fluid E:T ratios for males (0.5 +/- 0.1) were significantly lower than those for females (2.2 +/- 0.3). When separated by utilizing an E:T ratio of 1.25 as the determinant index value, 27 of 28 hatchlings were designated correctly as males (E:T < 1.25) or females (E:T > or = 1.25). Sex was verified for all hatchlings by gonadal histology. This study shows significant concentrations of T and E metabolites in CAF and plasma of hatchling loggerhead turtles and illustrates the use of a nonlethal, noninvasive method for determining sex, which could be potentially utilized for other endangered reptile and avian species.