Evaluation of the reproductive and developmental risks of caffeine

A risk analysis of in utero caffeine exposure is presented utilizing epidemiological studies and animal studies dealing with congenital malformation, pregnancy loss, and weight reduction. These effects are of interest to teratologists, because animal studies are useful in their evaluation. Many of the epidemiology studies did not evaluate the impact of the "pregnancy signal," which identifies healthy pregnancies and permits investigators to identify subjects with low pregnancy risks. The spontaneous abortion epidemiology studies were inconsistent and the majority did not consider the confounding introduced by not considering the pregnancy signal. The animal studies do not support the concept that caffeine is an abortafacient for the wide range of human caffeine exposures. Almost all the congenital malformation epidemiology studies were negative. Animal pharmacokinetic studies indicate that the teratogenic plasma level of caffeine has to reach or exceed 60 μg/ml, which is not attainable from ingesting large amounts of caffeine in foods and beverages. No epidemiological study described the "caffeine teratogenic syndrome." Six of the 17 recent epidemiology studies dealing with the risk of caffeine and fetal weight reduction were negative. Seven of the positive studies had growth reductions that were clinically insignificant and none of the studies cited the animal literature. Analysis of caffeine's reproductive toxicity considers reproducibility and plausibility of clinical, epidemiological, and animal data. Moderate or even high amounts of beverages and foods containing caffeine do not increase the risks of congenital malformations, miscarriage or growth retardation. Pharmacokinetic studies markedly improve the ability to perform the risk analyses.     

Xenotransplantation Models to Study the Effects of Toxicants on Human Fetal Tissues

Many diseases that manifest throughout the lifetime are influenced by factors affecting fetal development. Fetal exposure to xenobiotics, in particular, may influence the development of adult diseases. Established animal models provide systems for characterizing both developmental biology and developmental toxicology. However, animal model systems do not allow researchers to assess the mechanistic effects of toxicants on developing human tissue. Human fetal tissue xenotransplantation models have recently been implemented to provide human‐relevant mechanistic data on the many tissue‐level functions that may be affected by fetal exposure to toxicants. This review describes the development of human fetal tissue xenotransplant models for testis, prostate, lung, liver, and adipose tissue, aimed at studying the effects of xenobiotics on tissue development, including implications for testicular dysgenesis, prostate disease, lung disease, and metabolic syndrome. The mechanistic data obtained from these models can complement data from epidemiology, traditional animal models, and in vitro studies to quantify the risks of toxicant exposures during human development     

Effects of pulsed ultrasound and temperature on the development of rat embryos in culture

Rat embryos in culture were exposed to pulsed ultrasound at SPTA intensity of 1.2 W/cm2 for 5, 15, and 30 min on day 9.5 of development. The whole embryo culture system allowed precise temperature control for directly examining the effects of ultrasound on the developing neural plate. After exposure, embryos were maintained in culture for a further 48 hr. No major morphological abnormalities were observed but a reduction in somite number occurred in the group insonated for 30 min, which was equivalent to a 2 hr delay in embryonic development. Similar delay in growth and "blistering" in the prosencephalon region of some embryos were observed after insonation for 15 min at 40.0 degrees C, an elevation of 1.5 degrees C over the temperature used for controls. Exposure to ultrasound for 15 min at 40 degrees C caused significant reduction in the growth of the head compared with that of control embryos. Heat shock genes for hsps 71/73 and 88 kD were induced after insonation for 30 min at 38.5 degrees C. Insonation did not cause any temperature changes in the culture medium. However, when the temperature of the culture medium was increased during insonation, defective development occurred. The results of these in vitro experiments suggest that ultrasound if resulting in significant hyperthermia could affect the development during early organogenesis of the neural plate and in particular they suggest that the embryo is at greater risk of damage during hyperthermic conditions. These results should provoke discussion of the concept that ultrasound in the febrile patient may present an increased embryonic risk which should be considered when deliberating on the use of diagnostic ultrasound procedures in the pregnant patient.     

Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta): A comparison by ultrasonography

The rhesus monkey has played an important role in the history of reproductive research. Because of limitations on the exportation and availability of this species, several other species of nonhuman primates have been considered as alternate models. Among them is the crab-eating, or cynomolgus, macaque (Macaca fascicularis), which displays similarities in developmental, reproductive, and physiological parameters. The use of both species of macaques for pregnancy-related studies necessitates the assessment of differences in growth and development through gestation. Observations during the embryonic and fetal periods in both species have been compared with diagnostic ultrasound. Results indicate no significant differences in size during the embryonic and early fetal periods, but a greater acceleration of growth in the rhesus begins at approximately 100–110 gestational days (GD). Analysis of embryonic and fetal heart rates indicate no differences between the two species. Normal predictive values for a variety of growth parameters including gestational sac, greatest length, biparietal diameter, and femur length have boon calculated by multiple regression analysis. These charts have proven useful for confirming the gestational age after timed matings and for predicting the age of animals for which the conception date is not known.     

The non-human primate oocyte and embryo as a model for women, or is it vice versa?

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.     

A novel approach to intrauterine viral inoculation of swine using PCV type 2 as a model

Experimentally intrauterine (IU) viral inoculation has been commonly used to circumvent maternal interference with transplacental infection of fetuses and to assess the effect of viral infection on fetal development or reproductive parameters. However, IU inoculation requires surgical procedures such as laparatomy and surgical incision of the uterus. Post-surgical complications, that frequently result in abortion or fetal death, have been a major disadvantage. An animal trial was conducted to evaluate the non-surgical procedure of ultrasound needle-guided transabdominal injection for IU inoculation of porcine circovirus type 2 (PCV2) since this virus has been reported to cause reproductive failure in pigs. Two groups of seven pregnant sows at mid- and late-gestation, respectively, were inoculated with PCV2 using an ultrasound needle-guided technique that delivered PCV2 directly into one of the fluid-filled fetal compartments. The effect of transabdominal in utero virus challenge on fetuses and sows was assessed until term. While five of six sham-inoculated control sows had no or minimal adverse affects from in utero injection, 10 of 14 virus-inoculated sows had dead and/or stillborn piglets and PCV2 infection was evident by polymerase chain reaction and/or immunohistochemistry. These results supported previous field and experimental observations that PCV2 may cause reproductive failure. In conclusion, ultrasound needle-guided transabdominal injection was a safe and efficient method for IU inoculation of virus in pigs.     

Effects of heat stress on mammalian reproduction

Heat stress can have large effects on most aspects of reproductive function in mammals. These include disruptions in spermatogenesis and oocyte development, oocyte maturation, early embryonic development, foetal and placental growth and lactation. These deleterious effects of heat stress are the result of either the hyperthermia associated with heat stress or the physiological adjustments made by the heat-stressed animal to regulate body temperature. Many effects of elevated temperature on gametes and the early embryo involve increased production of reactive oxygen species. Genetic adaptation to heat stress is possible both with respect to regulation of body temperature and cellular resistance to elevated temperature.     

Navigating the site for embryo implantation: Biomechanical and molecular regulation of intrauterine embryo distribution

The distribution of intrauterine embryo implantation site(s) in most mammalian species shows remarkably constant patterns: in monotocous species such as humans, an embryo tends to implant in the uterine fundus; in polytocous species such as rodents, embryos implant evenly along the uterine horns. These long-time evolved patterns bear great biological significance because disruption of these patterns can have adverse effects on pregnancies. However, lack of suitable models and in vivo monitoring techniques has impeded the progress in understanding the mechanisms of intrauterine embryo distribution. These obstacles are being overcome by genetically engineered mouse models and newly developed high-resolution ultrasound. It has been revealed that intrauterine embryo distribution involves multiple events including uterine sensing of an embryo, fine-tuned uterine peristaltic movements, time-controlled uterine fluid reabsorption and uterine luminal closure, as well as embryo orientation. Diverse molecular factors, such as steroid hormone signaling, lipid signaling, adrenergic signaling, developmental genes, ion/water channels, and potentially embryonic signaling are actively involved in intrauterine embryo distribution. This review covers the biomechanical and molecular aspects of intrauterine embryo distribution (embryo spacing at the longitudinal axis and embryo orientation at the vertical axis), as well as its pathophysiological roles in human reproductive medicine. Future progress requires multi-disciplinary research efforts that will integrate in vivo animal models, clinical cases, physiologically relevant in vitro models, and biomechanical/computational modeling. Understanding the mechanisms for intrauterine embryo distribution could potentially lead to development of therapeutics for treating related conditions in reproductive medicine.     

Identified and unidentified challenges for reproductive biotechnologies regarding infectious diseases in animal and public health.

The aim of the present paper is to review the known and theoretical risks for in vivo derived and in vitro produced embryos as well as for nuclear transferred or transgenic embryos in terms of animal diseases or diseases of public health consequence. For in vivo derived embryos, a considerable number of experiments and scientific investigations have resulted in recommended guidelines and procedures that ensure a high level of safety. The effectiveness of these measures has been validated by field experience with the safe transfer of several million embryos over the past three decades. In vitro produced embryos have several characteristics that differentiate them from the former, in particular a structure of the zona pellucida that results in a more frequent possible association of pathogens with the embryo. However, the guidelines prescribed by the IETS, the international standard setting body (OIE) and existing national regulatory frameworks are in place to minimize the risk of disease transmission. No specific public health risks have been identified to date with respect to in vivo or in vitro derived embryos. In regard to nuclear transferred and transgenic embryos, theoretical risks have been identified in relation to the potential effects on some intrinsic viruses such as endogenous retroviruses but very little targeted experimental work has been carried out on infectious diseases that could have adverse consequences on animal or human health. Although there has been no report of such adverse consequences associated with the limited number of animals produced to date by such reproductive technologies, a precautionary approach is warranted given the potential negative impacts and it would be prudent to restrict at this stage, the international movement of such "manipulated" embryos.     

Utilization of juvenile animal studies to determine the human effects and risks of environmental toxicants during postnatal developmental stages

BACKGROUND: Toxicology studies utilizing animals and in vitro cellular or tissue preparations have been used to study the toxic effects and mechanism of action of drugs and chemicals and to determine the effective and safe dose of drugs in humans and the risk of toxicity from chemical exposures. Testing in animals could be improved if animal dosing using the mg/kg basis was abandoned and drugs and chemicals were administered to compare the effects of pharmacokinetically and toxicokinetically equivalent serum levels in the animal model and human. Because alert physicians or epidemiology studies, not animal studies, have discovered most human teratogens and toxicities in children, animal studies play a minor role in discovering teratogens and agents that are deleterious to infants and children. In vitro studies play even a less important role, although they are helpful in describing the cellular or tissue effects of the drugs or chemicals and their mechanism of action. One cannot determine the magnitude of human risks from in vitro studies when they are the only source of toxicology data. METHODS: Toxicology studies on adult animals is carried out by pharmaceutical companies, chemical companies, the Food and Drug Administration (FDA), many laboratories at the National Institutes of Health, and scientific investigators in laboratories throughout the world. Although there is a vast amount of animal toxicology studies carried out on pregnant animals and adult animals, there is a paucity of animal studies utilizing newborn, infant, and juvenile animals. This deficiency is compounded by the fact that there are very few toxicology studies carried out in children. That is one reason why pregnant women and children are referred to as "therapeutic orphans." RESULTS: When animal studies are carried out with newborn and developing animals, the results demonstrate that generalizations are less applicable and less predictable than the toxicology studies in pregnant animals. Although many studies show that infants and developing animals may have difficulty in metabolizing drugs and are more vulnerable to the toxic effects of environmental chemicals, there are exceptions that indicate that infants and developing animals may be less vulnerable and more resilient to some drugs and chemicals. In other words, the generalization indicating that developing animals are always more sensitive to environmental toxicants is not valid. For animal toxicology studies to be useful, animal studies have to utilize modern concepts of pharmacokinetics and toxicokinetics, as well as "mechanism of action" (MOA) studies to determine whether animal data can be utilized for determining human risk. One example is the inability to determine carcinogenic risks in humans for some drugs and chemicals that produce tumors in rodents, When the oncogenesis is the result of peroxisome proliferation, a reaction that is of diminished importance in humans. CONCLUSIONS: Scientists can utilize animal studies to study the toxicokinetic and toxicodynamic aspects of drugs and environmental toxicants. But they have to be carried out with the most modern techniques and interpreted with the highest level of scholarship and objectivity. Threshold exposures, no-adverse-effect level (NOAEL) exposures, and toxic effects can be determined in animals, but have to be interpreted with caution when applying them to the human. Adult problems in growth, endocrine dysfunction, neurobehavioral abnormalities, and oncogenesis may be related to exposures to drugs, chemicals, and physical agents during development and may be fruitful areas for investigation. Maximum permissible exposures have to be based on data, not on generalizations that are applied to all drugs and chemicals. Epidemiology studies are still the best methodology for determining the human risk and the effects of environmental toxicants. Carrying out these focused studies in developing humans will be difficult. Animal studies may be our only alternative for answering many questions with regard to specific postnatal developmental vulnerabilities.     

Quantification of risk from fetal exposure to diagnostic ultrasound

Biomedical ultrasound may induce adverse effects in patients by either thermal or non-thermal means. Temperatures above normal can adversely affect biological systems, but effects also may be produced without significant heating. Thermally induced teratogenesis has been demonstrated in many animal species as well as in a few controlled studies in humans. Various maximum ‘safe’ temperature elevations have been proposed, although the suggested values range from 0.0 to 2.5° C. Factors relevant to thermal effects are considered, including the nature of the acoustic field in situ, the state of perfusion of the embryo/fetus, and the variation of sensitivity to thermal insult with gestational stage of development. Non-thermal mechanisms of action considered include acoustic cavitation, radiation force, and acoustic streaming. While cavitation can be quite destructive, it is extremely unlikely in the absence of stabilized gas bodies, and although the remaining mechanisms may occur in utero, they have not been shown to induce adverse effects. For example, pulsed, diagnostic ultrasound can increase fetal activity during exposure, apparently due to stimulation of auditory perception by radiation forces on the fetal head or auditory structures. In contrast, pulsed ultrasound also produces vascular damage near developing bone in the late-gestation mouse, but by a unknown mechanism and at levels above current US FDA output limits. It is concluded that: (1) thermal rather than nonthermal mechanisms are more likely to induce adverse effects in utero, and (2) while the probability of an adverse thermal event is usually small, under some conditions it can be disturbingly high.     

Estimation of gestational age in Egyptian native goats by ultrasonographic fetometry

The main aim of the present study was to estimate the gestational age of Egyptian goats by B-mode ultrasound measurement of embryonic or fetal parts throughout pregnancy. Trans-rectal (TR) ultrasonography (7 MHz) was carried out on 15 pregnant Egyptian does at Day 10 post mating on alternate days until Day 25 and then once at 3-5-day intervals until Day 50. Trans-abdominal (TA) ultrasonography (3.5-5 MHz) was carried out on the same animals from Days 25 to 130 at 3-5-day intervals. After imaging the embryo or the fetus, the following parameters were measured: length of the embryo or fetus (CRL), heart rate (FHR), biparital diameter (BPD), trunk diameter (TD), placentome size (PS), umbilical cord diameter (UCD) and femur length (FL). The average of days at which the embryonic vesicle was first determined by TR and TA ultrasonography was 16.98+/-1.97 and 27.87+/-3.48, respectively. The embryo proper with a beating heart was first determined by TR and TA ultrasonography at an average of 22.36+/-2.66 and 30.36+/-4.75 days, respectively. All the fetal measures were significantly (P<0.0001) correlated with the gestational age. With the exception of fetal heart rate (R(2)=0.551), all the measured fetal structures were highly correlated (R(2)> or =90) with the gestational age. In conclusion, the age of embryo or fetus in Egyptian does can be estimated by ultrasound measuring the crown rump length, biparital diameter, trunk diameter, placentome size, umbilical cord diameter and femur length.     

Molecular Reproduction & Development Volume 79, Issue 5, May 2012 cover image

Buffaloes (Bubalus bubalis) constitute an important animal in the dairy economy of India and South Asian countries. Implementation of embryo‐related artifi cial reproductive technologies will facilitate planned improvement strategies for this species. Yet more needs to be understood about its embryonic development. Verma et al. (in this issue) contribute to this understanding by identifying the stage of embryonic genome activation.     

Comparison of noninvasive methods for the evaluation of female reproductive condition in a large viviparous lizard,Tiliqua nigrolutea

We compared the diagnostic value of three noninvasive methods (radiography, ultrasound, and palpation) of evaluating reproductive status in females of a large viviparous skink, the blotched blue‐tongued lizard (Tiliqua nigrolutea). Radiography could be used to identify postovulatory females during the periovulatory period. However, in early to mid‐gestation, radiography did not allow differentiation of the relatively homogenous structures within the abdomen. In late gestation, once calcification of the embryonic skeleton had occurred, gestation could be diagnosed and embryos counted. Ultrasound allowed differentiation between preovulatory, postovulatory, and nonreproductive females during the periovulatory period. Ultrasound also allowed visualisation of follicles during the early and middle stages of gestation. However, the level of operator experience, and the misdiagnosis of artefacts limited the accuracy of diagnosis. In the later stages of gestation, ultrasound did allow the detection of identifiable features of the developing foetal unit (fluid‐filled foetal membranes, independent movement of the embryo, and visualisation of the heartbeat). Colour Doppler imaging could be used to visualise embryonic blood flow. Ultrasonography was not useful in detecting the number of embryos present. Palpation during the periovulatory period could be used to detect follicles and determine whether ovulation had occurred. However, during the remainder of the gestation period, palpation was unable to differentiate reproductive condition. This study provides a basis for the diagnosis of reproductive condition and problems in viviparous reptiles. Each of the diagnostic techniques is useful at different stages of gestation, and the use of a combined approach will give the most information throughout the reproductive cycle. Zoo Biol 21:253–268, 2002. © 2002 Wiley‐Liss, Inc.     

The Use of Epidemiology in Risk Assessment: Challenges and Opportunities

The assessment of risk from environmental and occupational exposures incorporates and synthesizes data from a variety of scientific disciplines including toxicology and epidemiology. Epidemiological data have offered valuable contributions to the identification of human health hazards, estimation of human exposures, quantification of the exposure–response relation, and characterization of risks to specific target populations including sensitive populations. As with any scientific discipline, there are some uncertainties inherent in these data; however, the best human health risk assessments utilize all available information, characterizing strengths and limitations as appropriate. Human health risk assessors evaluating environmental and occupational exposures have raised concerns about the validity of using epidemiological data for risk assessment due to actual or perceived study limitations. This article highlights three concerns commonly raised during the development of human health risk assessments of environmental and occupational exposures: (a) error in the measurement of exposure, (b) potential confounding, and (c) the interpretation of non-linear or non-monotonic exposure–response data. These issues are often the content of scientific disagreement and debate among the human health risk assessment community, and we explore how these concerns may be contextualized, addressed, and often ameliorated.     

The roles of cellular reactive oxygen species,oxidative stress and antioxidants in pregnancy outcomes

Reactive oxygen species (ROS) are generated as by-products of aerobic respiration and metabolism. Mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production, one of the central elements in signal transduction pathways involved in cell proliferation, differentiation and apoptosis. Antioxidants also ensure defenses against ROS-induced damage to lipids, proteins and DNA. ROS and antioxidants have been implicated in the regulation of reproductive processes in both animal and human, such as cyclic luteal and endometrial changes, follicular development, ovulation, fertilization, embryogenesis, embryonic implantation, and placental differentiation and growth. In contrast, imbalances between ROS production and antioxidant systems induce oxidative stress that negatively impacts reproductive processes. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Nutritional and environmental factors may contribute to such adverse pregnancy outcomes and increase the susceptibility of offspring to disease. This occurs, at least in part, via impairment of the antioxidant defense systems and enhancement of ROS generation which alters cellular signalling and/or damage cellular macromolecules. The links between oxidative stress, the female reproductive system and development of adverse pregnancy outcomes, constitute important issues in human and animal reproductive medicine. This review summarizes the role of ROS in female reproductive processes and the state of knowledge on the association between ROS, oxidative stress, antioxidants and pregnancy outcomes in different mammalian species.     

Mammalian prenatal development: the influence of maternally derived molecules

Normal fetal development is dependent upon an intricate exchange between mother and embryo. Several maternal and embryonic elements can influence this intimate interaction, including genetic, environmental or epigenetic factors, and have a significant impact on embryo development. The interaction of the genetic program of both mother and embryo, within the uterine environment, can shape the development of an individual. Accumulating data from animal models indicate that prenatal events may well initiate long‐term changes in the expression of the embryo genetic program, which persist, or may only become apparent, much later in the individual's life. Also, environmental conditions during prenatal development may prompt the adoption of different developmental pathways, leading to alternative life histories. This review focuses on environmental factors, specifically maternally derived molecules, to illustrate how they can influence in utero embryonic development and, by extension, adult life.