Choose your own T-cell fate: creation of a narrative-based, decision-making activity to engage students in immunology.

Undergraduate courses in immunology are content-heavy and combined with a new, complex vocabulary, can be an overwhelming subject for students. In-class active learning approaches have been found to improve understanding of difficult concepts in science, technology, engineering and mathematics (STEM) disciplines; however, many undergraduate courses maintain a high dependence on lecture-style teaching because of time constraints, content demands and student resistance. We designed an online, out-of-class activity, the "Life and Death of a T cell", to complement a lecture on a complex immunological concept, T-cell development. Inspired by the "Choose Your Own Adventure" children's books, a fictional narrative was created in which students assume the role of a cell with a dream of becoming a helper T cell. Decision-making scenarios then prompt students to draw on their knowledge from the lecture to successfully navigate the steps of T-cell development. The activity was built on two platforms, Google Forms and H5P (HTML 5 Package), both of which are readily accessible and allow the inclusion of branching logic and the creation of a decision tree-based activity. An anonymous survey revealed that students found this interactive approach enjoyable, and their perceived understanding of the content significantly increased. Students appreciated the inclusion of a novel learning resource, with requests for similar activities to be developed for other immunological concepts. In conclusion, we developed a narrative-based, decision-making activity to complement a lecture on T-cell development. As an out-of-class activity, this style of learning approach can potentially capitalize on the benefits of active learning, while also overcoming barriers of student resistance.

Enhancing students' agency in learning anatomy vocabulary with a formative intervention design.

One of the major challenges for health science students is the rapid acquisition of a new vocabulary in anatomy comprising several hundred new words. Research has shown that vocabulary learning can be improved when students are directed to vocabulary strategies. This paper reported a study with a formative intervention design inspired by Vygotsky's method of double stimulation. In this design, the students were put in a structured situation that invited them to identify the challenges in learning anatomy and then provided them with active guidance and a range of anatomy vocabulary learning strategies that scaffolded them to work out a solution to the challenge and develop their individualized anatomy learning resources. The data were collected from surveys, pre and postquiz results, and group discussion transcripts. The results revealed students perceived one of the main challenges in learning anatomy was learning, memorizing, and remembering many new words. A key finding in our study was that the formative intervention enhanced students' agency in creating resources for learning anatomy vocabulary. In addition, the development of their understanding showed a recursive form: from concrete experiences to abstract concepts and then to concrete new practices.

Can drawing instruction help students with low visuospatial ability in learning anatomy?

Visuospatial skills are considered important attributes when learning anatomy and there is evidence suggesting that this ability can be improved with training techniques including drawing. The Mental Rotations Test (MRT) has been routinely used to assess visuospatial ability. This study aimed to introduce students to drawing as a learning strategy for anatomy. Undergraduate speech science anatomy students took part in a drawing tutorial (n = 92), completed an MRT test, pre- and post-tutorial tests, and surveys regarding their use and attitudes toward drawing as a study tool. The impact on their examination performance was then assessed. Regardless of MRT score or attitude to drawing, students who participated in the drawing tutorial demonstrated immediate improvement in post-tutorial test scores. Students in the drawing group performed better in most anatomy components of the examination, but the result did not reach statistical significance. There was only a positive correlation between MRT score and one type of anatomy question (non-image-based) and speech physics questions (r = 0.315, p = 0.002). The unexpected finding may relate to the MRT which assesses spatial rather than object visualization skills. Students who liked drawing also performed significantly better in word-based and speech physics questions. It is likely that the style of identification question did not require the mental manipulation ability assessed in the MRT. This study demonstrated that students with lower MRT scores are not outperformed in all aspects of anatomy assessment. The study highlights the importance of a more nuanced understanding of visuospatial skills required in anatomy.

A descriptive analysis of calls to the NSW Teratogen Information Service regarding use of anti-infectives during pregnancy.

BACKGROUND: MotherSafe is a free telephone-based counseling service for Australian consumers and health-care providers concerned about drug exposures during pregnancy and breastfeeding. Anti-infectives are the most commonly prescribed drugs for pregnant women. This study aims to provide a descriptive analysis of prospectively collected calls received by MotherSafe regarding anti-infective exposures during pregnancy between 2000 and 2020. Aggregate data were examined by type of caller, reason for call, pregnancy category and exposure type. Inductive thematic analysis of the comments recorded by MotherSafe counsellors at the time of call was undertaken. RESULTS: Over the study period, 25,890 calls related to exposure to anti-infectives during pregnancy (antibiotic, antiviral, and antifungal medications). Calls from patients were dominated by low-risk exposures (pregnancy category A) to drugs while calls from health care professionals related to drugs with limited human information (pregnancy category B3). Analysis of MotherSafe counsellor comments revealed over 200 instances of concerns relating to health care professional advice to the patient. Three themes emerged: incorrect or conflicting advice, poor counselling, and refusal to treat, prescribe or dispense. It is likely that these comments are biased to the negative as patients would not call MotherSafe if they were happy with HCP advice. However, the findings are concerning as they reveal an underlying lack of knowledge in some health care professionals which may have led to undertreatment of patients. This study reinforced the importance of Teratogen Information Services such as MotherSafe in providing counselling and clear communication of evidence-based information to guide decision-making, reducing potential emotional distress in pregnant women, and optimizing maternal, pregnancy and infant outcomes.

The effect of phenytoin on embryonic heart rate in Vivo.

Phenytoin is a known human teratogen with unknown etiology. Several mechanisms have been proposed including disturbances in folate metabolism, induction of embryonic hypoxia following phenytoin-induced bradycardia, free radical formation following re-oxygenation and phenytoin-induced maternal hyperglycemia. Using high frequency ultrasound, we demonstrated that phenytoin induced a dramatic decrease in the heart rate of embryos. This coincided with a moderate transient decrease in maternal heart rate and blood glucose levels. Embryonic heart rate had not fully recovered 24 h later in some embryos despite normal maternal physiological parameters. In a separate study, extent of hypoxia was measured using the marker pimonidazole. Phenytoin-exposed embryos did not demonstrate increased hypoxia compared to control embryos at 2, 4, 8 or 24 h dosing. Together our results show that phenytoin induces malformations as a result of a combination of insults: embryonic bradycardia, maternal bradycardia and maternal hyperglycemia. However, this does not appear to result in measurable embryonic hypoxia in our animal model.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

Calls to a Major Teratogen Information Service Regarding Exposures During Breastfeeding.

Background: MotherSafe is a free telephone-based counseling service for Australian consumers and health care providers concerned about drug exposures during pregnancy and breastfeeding. Calls relating to breastfeeding are relatively common and a source of significant distress to the breastfeeding mother, particularly if there is a lack of clarity regarding possible adverse effects of drug exposure on the infant. This study seeks to identify the medication exposures of concern for breastfeeding mothers and the information available to address these concerns. Aims: To review calls to MotherSafe about breastfeeding drug exposures during the 19-year period from 2000 to 2018 and to highlight drugs of concern and counseling issues. Materials and Methods: A retrospective descriptive assessment of a prospectively collected Access database was undertaken. Phone counseling records identified the medication (and other) exposures of concern regarding breastfeeding. The information about medication exposures via breastfeeding provided in consumer and product information (PI) was also reviewed. Results: Of a total of 315,158 calls received at MotherSafe between 2000 and 2018, 116,876 (37.1%) were regarding drug exposure via breastfeeding; 30% of these calls related to nonsteroidal anti-inflammatory drugs, antihistamines, antidepressants, simple analgesics, and antibiotics, and 5% were regarding an exposure specifically contraindicated when breastfeeding. Conclusions: Queries about medication exposures via breastfeeding represent a significant proportion of all the counseling calls to MotherSafe. This study demonstrates the inconsistent and often misleading information about breastfeeding exposures found in consumer and PI sheets and online and highlights the important role of Teratogen Information Services like MotherSafe in providing evidence-based information to both consumers and health care providers.

Fetal hypoxia and hyperglycemia in the formation of phenytoin-induced cleft lip and maxillary hypoplasia.

OBJECTIVE: Phenytoin exposure during the first trimester of pregnancy increases the risk of maxillary hypoplasia and cleft lip. The etiology of phenytoin embryopathy is unknown. Interestingly, phenytoin is also known to induce hyperglycemia in humans as well as rats. This study uses a rat model of fetal phenytoin syndrome to examine the role of hyperoxia, hyperglycemia, and arachidonic acid deficiency in the development of cleft lip and maxillary hypoplasia. METHODS: Pregnant rats were dosed with phenytoin during the critical period of lip development (day 11 of pregnancy) with or without supplemental oxygen, insulin, or arachidonic acid. The fetuses from all studies were examined at term. RESULTS: The frequency of cleft lip and maxillary hypoplasia was reduced by treating dams at the time of phenytoin exposure with either increased oxygen or insulin. However, in fetuses from phenytoin-treated dams dosed with arachidonic acid, the incidence of severe lip deformities remained the same although there was an increase in normal and mildly affected fetuses. Interestingly, this occurred in embryos from hyperglycemic dams. SIGNIFICANCE: Together, the results from these experiments suggest phenytoin-induced malformations may be a multifactorial process as malformations were not solely linked to a hyperglycemic state of the dam.

The effect of anti-emetic drugs on rat embryonic heart activity.

Nausea and vomiting of pregnancy (NVP) is the most common medical complaint during pregnancy affecting up to 70% of pregnant women worldwide. Some antiemetic medications (AEM) (droperidol, domperidone, granisetron, metoclopramide and trifluoperazine) used to treat NVP have the unwanted side effect of hERG blockade. The hERG potassium channel is essential for normal heart rhythm in both the adult human and the human and rat embryo. Animal studies show hERG blockade in the embryo causes bradycardia and arrhythmia leading to cardiovascular malformations and other birth defects. Whole rat embryo in vitro culture was used to determine the effect of the above listed AEM and meclizine on the heart rate of Gestational day 13 rat embryos. These embryos are similar in size and heart development to 5-6-week human embryo. The results showed that all of the AEMs caused a concentration-dependent bradycardia. Droperidol had the lowest margin of safety.

The effects of nifedipine and ivabradine on the functionality of the early rat embryonic heart. Are these drugs a risk in early human pregnancy?

BACKGROUND: When the human heart begins its earliest contractions from day 21, it lacks a functional autonomic nerve supply. Instead, contractions are generated by regular calcium transients later augmented by the funny current (If ) produced by sinoatrial-like cells. This study examined effects of blocking these currents in the early rat embryonic heart. METHODS: Rat embryos were incubated in vitro with either the calcium channel blocker nifedipine and/or the funny current (If ) blocker ivabradine for 1 hr to examine the effects of these drugs on the activity of the embryonic heart. RESULTS: On gestational day (GD) 10, nifedipine (0.45-1.8 µM) caused asystole at high concentrations (8/10 embryos at 1.8 µM and 3/10 embryos at 0.9 µM) and markedly increased embryonic heart rate (EHR) in all surviving embryos but likely reduced blood flow due to weak contractions. Ivabradine (1.5 µM) caused a 29% reduction in EHR in GD 10 embryos and a greater than 50% reduction in EHR for GD 11-14 embryos. Combined exposure to both nifedipine and ivabradine resulted in an additive effect. The increased EHR due to nifedipine was reduced by the ivabradine. CONCLUSION: The results suggest that exposure to nifedipine in human pregnancy 3-4 weeks postfertilization may cause a direct effect on the embryonic heart resulting in reduced blood flow leading to abnormal heart and/or blood vessel development and/or embryonic death. Accidental exposure to ivabradine in the organogenic period would be expected to cause embryonic bradycardia, hypoxia, malformations, and embryonic death. This drug is currently contraindicated in pregnancy.

Using the Jigsaw Method to Teach Abdominal Anatomy.

This study evaluates a cooperative learning approach for teaching anatomy to health science students incorporating small group and peer instruction based on the jigsaw method first described in the 1970's. Fifty-three volunteers participated in abdominal anatomy workshops. Students were given time to become an "expert" in one of four segments of the topic (sub-topics) by allocating groups to work-stations with learning resources: axial computerized tomography (CT) of abdominal structures, axial CT of abdominal blood vessels, angiograms and venograms of abdominal blood vessels and structures located within abdominal quadrants. In the second part of workshop, students were redistributed into "jigsaw" learning groups with at least one "expert" at each workstation. The "jigsaw" learning groups then circulated between workstations learning all sub-topics with the "expert" teaching others in their group. To assess abdominal anatomy knowledge, students completed a quiz pre- and post- workshop. Students increased their knowledge with significant improvements in quiz scores irrespective of prior exposure to lectures or practical classes related to the workshop topic. The evidence for long-term retention of knowledge, assessed by comparing end-semester examination performance of workshop participants with workshop nonparticipants, was less convincing. Workshop participants rated the jigsaw workshop highly for both educational value and enjoyment and felt the teaching approach would improve their course performance. The jigsaw method improved anatomy knowledge in the short-term by engaging students in group work and peer-led learning, with minimal supervision required. Reported outcomes suggest that cooperative learning approaches can lead to gains in student performance and motivation to learn. Anat Sci Educ 00: 000-000. © 2018 American Association of Anatomists.

Ondansetron and teratogenicity in rats: Evidence for a mechanism mediated via embryonic hERG blockade.

The potent hERG channel blocking drug ondansetron is used off-label for treatment of nausea and vomiting in early pregnancy. Some human epidemiological studies have associated ondansetron with fetal cardiovascular defects and orofacial clefts. This study investigated the effects of ondanestron on embryonic heart rhythm of gestational day (GD) 13 rat embryos in vitro and then integrated the results with published animal teratology, and animal and human pharmacokinetic studies to perform a risk evaluation. Ondansetron caused concentration dependent bradycardia and arrhythmia. Cardiovascular malformations in rats occurred at exposures slightly higher than those in early human pregnancy. Together the results suggest that ondansetron can have teratogenic potential in rats and humans mediated via hERG block and severe heart rhythm disturbances in the embryo. The risk may be increased in human pregnancy if additional risk factors are present such as hypokalemia.

Medications and pregnancy: The role of community pharmacists - A descriptive study.

BACKGROUND: Safe use of medications during pregnancy requires a comprehensive understanding of risk-benefit profiles for individual treatments. Pharmacists are supported in this aspect by clinical information agencies (e.g. MotherSafe, a telephone-based teratogen information service) and reference texts. To what extent and for what reasons Australian pharmacists utilise these services/resources are yet unknown. Further, debate on replacement of conventionally defined medication safety in pregnancy categories (A, B1-3, C, D, X) by narratively stated safety evidence may affect pharmacists' routine practice. This study aimed to gauge pharmacists' experiences and resource needs in undertaking support roles regarding gestational drug use. METHODS: Semi-structured interviews (audio-recorded or documented using field notes) were performed with community pharmacists in Australia and transcribed verbatim. Inductive thematic analysis was conducted using the NVivo software (Version 11, QSR International). RESULTS: Data saturation was achieved with 24 interviews. Qualitative data yielded 5 emergent themes: barriers to effective counselling, patient trust, risk perception, role definition and practice support needs. Overall, participants relied on pregnancy categories, were risk averse and cautious in offering advice. Currently available data for unclassified and category B therapeutic agents (limited human data) were deemed inadequate. Reluctance to use the proposed narrative system was also expressed. DISCUSSION: This study highlights key barriers in the provision of maternal care by pharmacists and the potential tension present if the existing category system is replaced by a narrative one. These need to be addressed through training and development of practice support resources to enhance pharmacists' skills in evidence-based risk estimation and communication.

Long-term programming effects on blood pressure following gestational exposure to the IKr blocker Dofetilide.

A slow embryonic heart rate in early-mid gestation is associated with increased risk of embryonic death and malformation, however, the long-term consequences remain unknown. We administered Dofetilide (Dof, 2.5 mg/kg), a drug that produces embryo-specific bradycardia, to pregnant rats from gestational days 11-14. Embryonic heart rate and rhythm were determined using embryo culture. Cardiovascular function was assessed in surviving adult offspring at rest, during acute psychological stress (air jet stress, AJS), and after 7 days of repeated AJS. Dof reduced embryonic HR by 40% for ~8 h on each of the treatment days. On postnatal day 3, Dof offspring were ~10% smaller. Blood pressure was elevated in adult Dof rats (systolic blood pressure, night: 103.8 ± 3.9 vs. 111.2 ± 3.0 mmHg, P = 0.01). While the pressor response to AJS was similar in both groups (control 17.7 ± 3.4; Dof 18.9 ± 0.9 mmHg, P = 0.74), after 7 days repeated AJS, clear habituation was present in control (P = 0.0001) but not Dof offspring (P = 0.48). Only Dof offspring showed a small increase in resting blood pressure after 7 days repeated stress (+3.9 ± 1.7 mmHg, P = 0.05). The results indicate that embryonic bradycardia programs hypertension and impaired stress adaptation, and have implications for the maternal use of cardioactive drugs during pregnancy.

Editor's Highlight: Ethylene Glycol Teratogenicity: A Role for Embryonic Acidosis?

Ethylene glycol (EG) is a developmental toxicant in pregnant rats and mice. A suggested mechanism for this toxicity is that the EG metabolite, glycolic acid (GA), causes acidosis which may affect the embryonic heart rate (HR). This inhibition would cause periods of embryonic bradycardia and arrhythmia resulting in increased embryonic death and malformation in surviving embryos. This hypothesis was investigated using gestational day (GD) 11 and 13 rat embryos in vitro. Increasing concentrations of GA or lactic acid in the incubation medium caused a decrease in external pH (pHe) and a concentration-dependent decrease in embryonic HR. Increased concentrations of GA or lactic acid with pHe corrected to normal levels did not affect HR. Severely decreased pHe, caused by reduced NaHCO3 in the incubation medium, had little effect on the HR of GD 13 embryos but substantially reduced the HR of GD 11 embryos. These results suggest that increased monocarboxylate concentration (glycolate or lactate) needs to be in combination with increased H+ concentration (low pHe) to influence the embryonic HR. These results implicate the monocarboxylate transporter reported to be present in the early postnatal rat heart, the chick embryonic heart throughout development, and the chorioallantoic placenta. The results showed some evidence that the adverse effect of GA and reduced pHe on the embryonic HR increased with duration of exposure and hence lends support to the suggested mechanism of embryotoxicity for EG.

Early Gestational Hypoxia and Adverse Developmental Outcomes.

Hypoxia is a normal and essential part of embryonic development. However, this state may leave the embryo vulnerable to damage when oxygen supply is disturbed. Embryofetal response to hypoxia is dependent on duration and depth of hypoxia, as well as developmental stage. Early postimplantation rat embryos were resilient to hypoxia, with many surviving up to 1.5 hr of uterine clamping, while most mid-gestation embryos were dead after 1 hour of clamping. Survivors were small and many had a range of defects, principally terminal transverse limb reduction defects. Similar patterns of malformations occurred when embryonic hypoxia was induced by maternal hypoxia, interruption of uteroplacental flow, or perfusion and embryonic bradycardia. There is good evidence that high altitude pregnancies are associated with smaller babies and increased risk of some malformations, but these results are complicated by increased risk of pre-eclampsia. Early onset pre-eclampsia itself is associated with small for dates and increased risk of atrio-ventricular septal defects. Limb defects have clearly been associated with chorionic villus sampling, cocaine, and misoprostol use. Similar defects are also observed with increased frequency among fetuses who are homozygous for thalassemia. Drugs that block the potassium current, whether as the prime site of action or as a side effect, are highly teratogenic in experimental animals. They induce embryonic bradycardia, hypoxia, hemorrhage, and blisters, leading to transverse limb defects as well as craniofacial and cardiovascular defects. While evidence linking these drugs to birth defects in humans is not compelling, the reason may methodological rather than biological. Birth Defects Research 109:1358-1376, 2017.© 2017 Wiley Periodicals, Inc.

Abnormal pregnancy outcome associated with high-dose maternal tranylcypromine therapy: Case report and literature review.

BACKGROUND: Tranylcypromine is a non-selective inhibitor of monamine oxidase which also inhibits the reuptake of norepinephrine. Spontaneous hypertensive reactions to the drug have been reported. In sheep tranylcypromine has been shown to cause a dose-dependent reduction in uterine blood flow. A similar effect in a pregnant woman might induce constriction of the uterine arteries and temporary fetal hypoxia. CASES: MotherSafe is a state-based Teratogen Information service and currently provides counselling to around 22,000 consumers and healthcare professionals annually regarding exposures during pregnancy and breastfeeding We report on the outcome of 2 pregnancies in a patient treated with high dose tranylcypromine as well as pimozide, diazepam and alprazolam. The first pregnancy resulted in fetal death and autopsy revealed facial dysmorphism with ocular hypertelorism, cardiac defect and placental infarcts. The second pregnancy continued to term but the baby had similar dysmorphic features as well as an atrio-ventricular septal defect and craniosynostosis. CONCLUSIONS: Due to their unpredictable interactions with many drugs and foods, MAO inhibitors such as tranylcypromine are not commonly used to treat depression and reports of use in pregnancy are rare. We report the outcome of 2 pregnancies with exposure to high doses of tranylcypromine resulting in children with a similar pattern of malformations. The aetiology is unknown but may relate to the vasoactive properties of the drug in above-therapeutic doses.

Control of the heart rate of rat embryos during the organogenic period.

The aim of this study was to gain insight into whether the first trimester embryo could control its own heart rate (HR) in response to hypoxia. The gestational day 13 rat embryo is a good model for the human embryo at 5-6 weeks gestation, as the heart is comparable in development and, like the human embryo, has no functional autonomic nerve supply at this stage. Utilizing a whole-embryo culture technique, we examined the effects of different pharmacological agents on HR under normoxic (95% oxygen) and hypoxic (20% oxygen) conditions. Oxygen concentrations ≤60% caused a concentration-dependent decrease in HR from normal levels of ~210 bpm. An adenosine agonist, AMP-activated protein kinase (AMPK) activator and KATP channel opener all caused bradycardia in normoxic conditions; however, putative antagonists for these systems failed to prevent or ameliorate hypoxia-induced bradycardia. This suggests that the activation of one or more of these systems is not the primary cause of the observed hypoxia-induced bradycardia. Inhibition of oxidative phosphorylation also decreased HR in normoxic conditions, highlighting the importance of ATP levels. The ß-blocker metoprolol caused a concentration-dependent reduction in HR supporting reports that ß1-adrenergic receptors are present in the early rat embryonic heart. The cAMP inducer colforsin induced a positive chronotropic effect in both normoxic and hypoxic conditions. Overall, the embryonic HR at this stage of development is responsive to the level of oxygenation, probably as a consequence of its influence on ATP production.

A comparison of drug-induced cardiotoxicity in rat embryos cultured in human serum or protein free media.

INTRODUCTION: Although much reproductive toxicology research is performed in live animals there is increasing use of in vitro techniques primarily to identify potential hazards with human exposure. As many in vitro studies are undertaken using protein free media, the standard protocol is to compare the effect concentration determined in vitro with the predicted therapeutic free plasma concentration in humans. The aim of the present study was to test this rationale by comparing the effect of a small number of therapeutic drugs on heart rate of rodent embryos cultured in human sera or protein free serum. METHODS: Whole rat embryos were cultured in protein-free media or human serum to which drugs (amiodarone, citalopram, dofetilide, haloperidol, paroxetine, quetiapine, or trazodone) known to induce embryonic bradycardia were added. Embryonic heart rate was observed before and after addition of drugs. RESULTS: Most of the tested drugs (5/7) caused a greater decrease in embryonic heart rate in human sera than predicted based on the protein binding of the drug. DISCUSSION: The results suggest that there is less unbound drug in the protein free media and/or more unbound drug in the human sera than predicted. Variables such as saturated protein binding and pH cannot fully explain our results. Since the results did not validate the original rationale, reproductive toxicity results obtained using protein free in vitro techniques may not have the large safety factors predicted on the basis of protein binding.