- Email: [email protected]
An in vivo murine model of rosiglitazone use in pregnancy
An in vivo murine model of rosiglitazone use in pregnancy Denise B. Klinkner, M.D.,a Hyun J. Lim, Ph.D.,b Estil Y. Strawn Jr., M.D.,c Keith T. Oldham, M.D.,a and Tara L. Sander, Ph.D.a a
Department of Surgery, Division of Pediatric Surgery, Medical College of Wisconsin and Children’s Research Institute; and Department of Biostatistics and c Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin b
Objective: To identify the effects of rosiglitazone use during murine pregnancy. Design: The effect of rosiglitazone on blastocyst development was determined by culturing two-cell mouse embryos with rosiglitazone for 72 hours. From January to June 2005, five independent groups of ICR/CD1 female mice were treated with rosiglitazone during pregnancy, from the time of identification of seminal plugs until delivery of pups. Setting: Controlled animal facility. Animal(s): Two-cell mouse embryos and an outbred line of mice, ICR/CD1. Intervention(s): Two-cell embryos were cocultured with rosiglitazone (10 M) for 72 hours and scored. Ten-week-old female ICR mice were mated. Females with seminal plugs then were randomized to rosiglitazone (10 or 0.1 mg/kg per day) or to carrier alone, by gavage, until delivery. Weekly weights were obtained, and pregnancy outcomes were documented. Main Outcome Measure(s): Blastocyst development, number of pups and pup weights, and morphological changes. Result(s): Embryos exposed to rosiglitazone progressed to the blastocyst stage within 72 hours. Pregnant animals demonstrated normal weight gain throughout pregnancy. Postnatal growth and litter size were not statistically different between groups. No changes in normal mouse neonate development were observed. Conclusion(s): Rosiglitazone did not impair murine blastocyst development in vitro or cause phenotypic harm to the mouse fetus when administered during pregnancy, suggesting potential safety for rosiglitazone use in pregnancy. (Fertil Steril威 2006;86(Suppl 3):1074 –9. ©2006 by American Society for Reproductive Medicine.) Key Words: Rosiglitazone, pregnancy, polycystic ovary syndrome, diabetes mellitus type 2
Thiazolidinedione drugs, such as troglitazone, pioglitazone, and rosiglitazone, are peroxisome proliferator–activated receptor gamma (PPAR␥) ligands. Rosiglitazone is the most potent known specific PPAR␥ ligand (1). It has potentially far-reaching effects on pathophysiologic processes, from cancer to atherosclerosis and diabetes. Rosiglitazone improves insulin sensitivity in peripheral tissue without stimulating the pancreatic beta cells, thereby also decreasing the possibility of hypoglycemic events. Thus, rosiglitazone has become a desirable agent for treating diabetes mellitus type 2, which currently is its primary clinical use. Because rosiglitazone effectively diminishes hyperinsulinemia, many have considered its use in treatment of women with polycystic ovary syndrome (PCOS). Polycystic ovary syndrome affects 4%–10% of premenopausal women (3.5–5 million women in the United States) Received December 21, 2005; revised and accepted March 17, 2006. Supported in part by an award from the Children’s Hospital of Wisconsin Research Foundation and Children’s Research Institute, Milwaukee, Wisconsin. Reprint requests: Tara L. Sander, Ph.D., Department of Surgery, Division of Pediatric Surgery, CVRC M4050, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226 (FAX: 414456-6473; E-mail: [email protected]).
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(2), causing hirsutism, oligomenorrhea, and anovulatory infertility. The disease involves a complex imbalance of androgens and insulin. Both obese and nonobese women with PCOS demonstrate insulin resistance and therefore develop hyperinsulinemia. In this circumstance, insulin stimulates androgen synthesis from the thecal cells (3) and decreases sex hormone– binding globulin synthesis in the liver (4), thus contributing to total free androgen in the plasma (5). The free androgen causes an imbalance in the hormonal regulation of ovulation. Therefore, these patients often seek treatment for infertility. Once the diagnosis of PCOS has been made, the current therapies initially begin with weight reduction for the obese and clomiphene citrate (CC) to induce ovulation. Treatment with CC carries the risk of multiple pregnancies and ovarian hyperstimulation, as well as the excessive cost associated with these complications. Furthermore, not all patients respond to the drug. Even those who ovulate and conceive have a 20% risk of spontaneous abortion and a 1% risk of stillbirth (6). Therefore, alternatives to CC have been sought. The use of insulin-sensitizing agents, that is, metformin, troglitazone, and rosiglitazone, has been reported in PCOS patients. Troglitazone improved ovulation rates (7) and re-
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sultant pregnancy (8), and rosiglitazone use resulted in normal pregnancy (9). Rosiglitazone treatment also restored menses in 96% of patients, with three resultant pregnancies (5). The ovulatory effects also were seen in nonobese patients (10). Overall results have been encouraging in CC-resistant PCOS patients. Ovulation is stimulated in women who are treated with metformin (11), troglitazone (12), or rosiglitazone (13) alone, with an additive effect in combination with CC. Women treated with thiazolidinediones are advised to discontinue the drug either at the time of ovulation or at confirmation of pregnancy to minimize exposure to the fetus (6). A small number of those who do ovulate become pregnant, and in one study, five of seven women had healthy infants, whereas two women miscarried (12). Because the rate of spontaneous abortion reaches 36%– 82% in women who are reported to have PCOS (14 –18), the cause of early pregnancy loss cannot be determined to be solely from the drug treatment. Conversely, PCOS patients treated with metformin throughout pregnancy have a lower rate of spontaneous abortion compared with untreated patients (8.8% vs. 41.9%; Jakubowicz et al. [15]). Thus, those taking metformin have been advised to continue administration of the drug throughout pregnancy (for a review of current use of insulin-sensitizing agents in pregnancy and PCOS, see Checa et al. [19]). This result suggests that treatment with an insulin-lowering agent may be desirable throughout pregnancy in PCOS patients. Rosiglitazone has become an attractive option for CCresistant PCOS patients who do not tolerate or respond to other oral agents (20). However, rosiglitazone is considered by the US Food and Drug Administration to be a pregnancy class C substance, because animal studies suggest that high doses of the drug cause mid- to late-gestational fetal death and growth retardation (6). Rats treated with rosiglitazone during gestation through lactation have reduced litter size, as well as decreased neonatal viability and postnatal growth (6). To the best of our knowledge, no published study has demonstrated specific effects of rosiglitazone use during pregnancy that lead to fetal death or growth retardation. More concerning, rosiglitazone has been identified in human fetal tissue during the first trimester, thus underscoring the importance of determining the potential effects of rosiglitazone on fetal development (21). Taken together, these reports have raised sufficient concerns that rosiglitazone use during pregnancy currently is considered to be contraindicated. As a result, physicians cautiously are considering the use of rosiglitazone as a PCOS treatment, because it is not clear whether rosiglitazone use during pregnancy is safe or harmful to the developing fetus. Because the drug has demonstrated potentially beneficial effects in a limited number of human case reports, but harmful effects have been observed in animal pregnancies, our study focused on investigating the effects of rosiglitazone treatment during murine pregnancy. Our initial experiments tested the in vitro effects of rosiglitazone on early Fertility and Sterility姞
embryogenesis, whereas later studies documented the in vivo effects of treatment throughout pregnancy. MATERIALS AND METHODS Murine Embryo Studies To test whether the PPAR␥ agonist rosiglitazone affects early embryogenesis, two-cell mouse embryos were treated with and without rosiglitazone, as described elsewhere for drug toxicity (22). Embryotech Laboratories, Inc. (Wilmington, MA) provided the embryos and expertise in culturing and collecting data. Fresh embryos were harvested from superovulated female B6C3F-1 ⫻ B6D2F-1 mice, the laboratory’s customary strain, at a standard predetermined hour, after hCG administration. The standard assay consisted of 21 embryos per test item and included a 21-embryo control. To the treatment group, rosiglitazone (Cayman Chemical, Ann Arbor, MI) was added fresh daily to the culture media in a 10 M concentration. Diethyl maleate (Sigma, St. Louis, MO), a well-recognized inhibitor of blastocyst development, served as a positive control (23). The carrier dimethyl sulfoxide (Sigma) was the negative control. Both the test and control embryos were set up in triplicate under oil and were cultured with 5% CO2 at 37°C until they were scored for development to expanded blastocysts. Development for the two-cell assay was scored at 72 hours. Greater than 80% blastocyst formation from the control group was required to validate the two-cell assay (http://www.embryotech.com/qc/mea.htm). Animals and Animal Care All studies were performed under a proposal approved in July 2004 by the Medical College of Wisconsin Institutional Animal Care and Use Committee, with approved addendums in October 2004. All animals were housed and cared for by the Medical College of Wisconsin Biomedical Research Center. A murine model was selected for a short gestation time of 19 –20 days, for litter sizes, and for ease of care. The ICR/CD1 mouse was chosen because of the ease of timed pregnancy (24) and large litter size of 10 –15 pups (personal communication, Charles River Laboratories). The animals were maintained in a pathogen-free environment. Groups were fed a high-fat (9%) chow diet to prevent cannibalism of pups at delivery (PicoLab Mouse Diet; PMI International LLC, Richmond, IN), with free access to both feed and water via nipple water bottles. All animals were obtained from Charles River Laboratories (Wilmington, MA). Upon arrival, mice were allowed 1 week to acclimate. Eighty 10-week-old CD1 female mice then were examined for signs of estrus, with those in estrus selected for 1:1 or 2:1 mating with ⱖ10-week-old males. The following morning, evidence of a seminal plug confirmed mating, and the females were separated from the males. Each cycle resulted in 9 to 15 plugs (11%–19% successful mating). The studies were repeated five times, and data were pooled for statistical analysis. Ear tags (National Band and Tag Co., Newport, 1075
KY) were placed to ease documentation, and weights (CS2000; Ohaus Corp., Pine Brook, NJ) were recorded at baseline and, subsequently, on a weekly basis. Drug Dosing and Administration Previous studies (25, 26) and a trial study with nonpregnant females confirmed the safety of a rosiglitazone dosage of 10 mg/kg per day in these animals without adverse side effects. Rosiglitazone is unstable once resuspended in solution and easily degrades within 4 hours at room temperature (27). Therefore, oral gavage was chosen to most closely approximate clinical use of the drug (absorption from the gastrointestinal tract) and to confirm timely treatment and equivalent doses between animals, as described elsewhere (25, 28). Dosing was based on a 25-g nonpregnant female, with no change in dose made throughout the study (24). The treatment group received 2.5 or 25 L of dimethyl sulfoxide with rosiglitazone–potassium (Cayman Chemical) diluted in sterile water to a final volume of 0.2 mL by oral gavage (concentration of 0.1 or 10 mg/mL, respectively). The control group received 2.5 or 25 L of dimethyl sulfoxide, diluted in room-temperature sterile water for an equal volume. The final drug dose was equivalent to 0.1 or 10 mg/kg per day, respectively. The 0.1 mg/kg per day dosing was approximately equivalent to the maximum recommended human dose of 8 mg per 70 kg, per day. Gavage needles (Popper and Sons, New Hyde Park, NY) were lubricated with nonspermicidal sterile lubricating jelly (First Priority, Inc., Elgin, IL) before each use. The gavage was performed by Biomedical Research Center–approved methods. The animals were observed after each administration. Any change in respiratory status or other clinical signs of deterioration mandated that the mouse be euthanized, which was performed by CO2 administration or by 2:1 ketamine-to-xylazine, injected intraperitoneally, followed by exsanguination. Effects From Treatment Gravid females were given dimethyl sulfoxide or rosiglitazone daily by oral gavage. Weekly weights were obtained and recorded. Before delivery, all females were separated to ensure proper assignment of pups. Litter size, individual pup weights and phenotypes, and viability were recorded, as described elsewhere (29, 30). Statistical Analysis Initial descriptive statistics were used to summarize the data. Distribution of continuous variables was examined for normality. The mean of the weight variable was compared using t test and analysis of variance with Bonferroni multiple comparisons adjustment (31). The generalized linear model was used, controlling for the number of pups. The graph of the mean weight at each measurement time also was used to overview the mean weight change over time. The growth curves analysis of the longitudinal data was performed to 1076
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investigate outcome measurement changes over time (32). For all analyses, the significance level used was .05. The analysis was performed using the SAS statistical package, version 9 (SAS Institute, Cary, NC). RESULTS Rosiglitazone Does Not Inhibit Blastocyst Formation Two-cell murine embryos were exposed to dimethyl sulfoxide control, rosiglitazone, or diethyl maleate for 72 hours. Consistent with work published elsewhere (23), all embryos exposed to diethyl maleate as a positive control failed to mature (data not shown). In Figure 1, no difference in blastocyst development was observed with rosiglitazone treatment. The embryos exposed to rosiglitazone appeared to progress in a similar fashion as the control embryos. These data suggest that rosiglitazone does not impact early, preimplantation embryonic development. Rosiglitazone Use During Murine Pregnancy The mouse pregnancy study included a control group, a group treated with 0.1 mg/kg per day of rosiglitazone throughout pregnancy, and a group treated with 10 mg/kg per day of rosiglitazone throughout pregnancy. Maternal weight gain did not differ across groups (data not shown). The number of pups and the weights of the pups were not different between treatment groups (Table 1). In addition, all pups reached developmental milestones of hair growth and eye opening similarly (Table 1). Postnatal weight gain also showed no differences between groups (Fig. 2). The highest recommended human equivalent dose (0.1 mg/kg per day)
FIGURE 1 Rosiglitazone effect on embryogenesis. Two-cell murine embryos were treated with 10 M rosiglitazone for 72 hours and then scored for stage of blastocyst development and compared with dimethyl sulfoxide carrier control. Of the control embryos, 100% reached blastocyst stage, and 100% of the rosiglitazone-treated embryos reached blastocyst stage. None of the diethyl maleate embryos reached blastocyst stage.
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TABLE 1 Rosiglitazone (Rosi) treatment during murine pregnancy. Parameter No. of plugs No. pregnant No. of pups per litter Birth weight (g) Male (%) Hair (PND 8) Eyes open (PND 15)
Control
Rosi (0.1)a
Rosi (10)b
23 18 10.67 ⫾ 1.94 1.61 ⫾ 0.16 46 18/18 18/18
15 15 11.07 ⫾ 2.81 1.60 ⫾ 0.17 51 15/15 10/15
20 15 10.06 ⫾ 1.27 1.62 ⫾ 0.19 48 15/15 12/15
Note: Paired t test, P⬎.05 for each experimental group vs. control. PND ⫽ postnatal day. a 0.1 mg/kg per day. b 10 mg/kg per day. Klinkner. Rosiglitazone use in murine pregnancy. Fertil Steril 2006.
and a two log– higher dose of 10 mg/kg per day do not appear to interfere with normal murine fetal development. Adverse Outcomes Throughout the course of the study, two maternal deaths occurred at 2 weeks postpartum. Autopsy identified small bowel obstruction as the cause of death, which has been reported in lactating mice (33). Thus, two litters were dispersed among the remaining mothers of the same treatment group. Pup loss from those control litters was significantly greater, apparently as a result of higher pup to mother ratios. Thirty-nine pups were excluded from postnatal weight gain analysis because of death during the period of observation. Cause of death was cannibalism for the majority, with one pup born with ascites.
FIGURE 2 Mean weekly pup weights. Weights were obtained weekly on control (C), rosiglitazone (R), and human-dosaged–rosiglitazone (HR) pups. No differences were observed between groups. (Analysis of variance with Bonferroni adjustment, P⬎.05).
Klinkner. Rosiglitazone use in murine pregnancy. Fertil Steril 2006.
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DISCUSSION Peroxisome proliferator–activated receptor gamma is a ligand-activated nuclear hormone receptor that is found in adipocytes, colonocytes, endothelial cells, and cells of the monocyte lineage (34). During embryonic development, PPAR␥ is expressed in bovine oocytes at all preimplantation stages, from oocyte to hatched blastocyst (35). Furthermore, PPAR␥ has been identified in both the inner cell mass and the trophectoderm. Jain et al. (36) demonstrated PPAR␥ expression in the urogenital sinus and in brown adipose tissue at E15.5 of murine embryogenesis. Interestingly, PPAR␥ has been demonstrated in endothelial cells of the mouse uterus as early as day 1 of pregnancy (37). In the rat uterus, PPAR␥ was expressed at day 11 of pregnancy, increased by day 13, and subsequently decreased (38). Troglitazone administration between day 9 and day 11 then increased PPAR␥ expression in the placenta and reduced the mortality of the fetuses. A deficiency of PPAR␥ in the developing embryo leads to improper development of the placental vasculature and also is linked to myocardial thinning and death by gestational day 10 in mice (39, 40), suggesting that PPAR␥ plays an important role in fetal development. Activation of PPAR␥ and retinoid X receptor, a heterodimeric partner of PPAR␥, has been shown to play a role in fatty acid uptake in human placental trophoblasts, yet another important aspect of fetal development (41). Others have demonstrated that rosiglitazone prevents human trophoblast invasion from first-trimester placenta, a key step in implantation (42). In vitro use of rosiglitazone has been demonstrated to interfere with pathways for normal cardiac valvular development (43). Taken together, these observations demonstrate that PPAR␥ clearly plays a role in placental and cardiac development in in vivo rodent models. Thus, inappropriate activation at key portions of development may result in adverse developmental outcomes. 1077
Natural ligands for PPAR␥ appear to be polyunsaturated fatty acids and eicosanoids, such as 15-deoxy-delta 12, 14prostaglandin J2 (15d-PGJ2) (34). The relationship between eicosanoids and PPAR␥ was studied in early pregnancy in mice. Blocking the 12/15-lipoxygenase– derived eicosanoid resulted in failure of endometrial implantation, whereas treatment with rosiglitazone rescued the embryos and led to proper implantation (37). This observation suggested that PPAR␥ was involved in the maintenance of pregnancy. Given that PPAR␥-deficient mice have abnormal placental development and that rosiglitazone promotes implantation in these mice, PPAR␥ ligands may be beneficial in preventing spontaneous abortions in women with PCOS. Rosiglitazone may be of therapeutic value at other specific points during pregnancy. Berry et al. (44) demonstrated that PPAR isoforms change with onset of labor, compared with women undergoing Cesarean sections, suggesting that PPAR expression and function may have a role in maintenance of pregnancy or initiation of labor. Lappas et al. (45) have suggested that rosiglitazone may be useful in preventing or stopping preterm labor. They studied the effects of 15d-PGJ2 and rosiglitazone on placental cytokine release from tissue obtained from full-term pregnancies delivered via Caesarean section for breech or previous C-section. Both PPAR␥ ligands dramatically decreased the release of IL-6, IL-8, and TNF-␣. Theoretically, modulating the inflammatory response by inhibiting release of IL-6, IL-8, and TNF-␣ would halt preterm labor. No study has demonstrated this hypothesis in vivo, nor has safety for this clinical application in late pregnancy been determined. On the basis of information provided in the Physicians’ Desk Reference (6) regarding rosiglitazone use during pregnancy, we expected a decrease in the number of pups born and a delay in weight gain. We confirmed the lack of toxicity to the embryo up to 72 hours. Our in vivo findings in pregnant mice were not consistent with the manufacturer’s data in rats and rabbits. Combined with the limited case reports of rosiglitazone use in human pregnancy (5, 9, 13, 46 – 48), these findings suggest that rosiglitazone use may be acceptable for use in pregnancy for women with diabetes mellitus type 2 or with PCOS. Because rosiglitazone has been documented to cross the placenta at 10 weeks’ gestational age (21), the safety and efficacy of use in human pregnancy should be demonstrated before widespread use. Since much of murine development has been delineated, we conducted our work in mice. The added benefits of short gestation and ease of care supported our animal selection. However, no commercially available genetic mouse model of PCOS or diabetes mellitus type 2 exists, pointing to the genetic variability in these diseases and subsequent difficulty in recreating human diseases in mice. The ICR mouse, an outbred line, may reflect the genetic variability in the human population. However, the effects seen in this line cannot be applied broadly. As noted by other investigators, there can be difficulty with mouse models: genetically different mice responded to rosiglitazone in a variable fashion (49). 1078
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One potential reason that we did not observe an inhibition of postnatal growth with rosiglitazone administration is that treatment was discontinued upon delivery. Peroxisome proliferator–activated receptor␥ has been identified in murine breast tissue of lactating mice (36), and activation of PPAR␥ may affect milk production. We chose not to treat throughout lactation, so as to more closely mimic human treatment models. For example, infants of diabetic mothers often are fed formula if the mother continues rosiglitazone treatment after delivery. With the data that we present here, additional studies may be warranted in human beings. This study provides one of the first reports of the effects of rosiglitazone use in pregnancy. Although this work demonstrates no phenotypic evidence of altered fetal development, additional studies must be undertaken before human studies. Echocardiography will allow assessment of cardiac development and function without requiring that the animals be sacrificed, and magnetic resonance imaging likewise will provide insight into other potential structural changes. Although few drugs are considered entirely safe in pregnancy, rosiglitazone may be a drug with benefits to consider once the risks have been fully evaluated. Acknowledgments: The authors gratefully acknowledge Alexandra LerchGaggl, Ph.D., and Mary Holtz, Ph.D., at the Medical College of Wisconsin for assistance with mouse husbandry and mating. The authors thank Kirkwood Pritchard Jr., Ph.D., and John Densmore, M.D., at the Medical College of Wisconsin and Children’s Research Institute for intellectual contributions to the study.
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9. Cataldo NA, Abbasi F, McLaughlin TL, Lamendola C, Reaven GM. Improvement in insulin sensitivity followed by ovulation and pregnancy in a woman with polycystic ovary syndrome who was treated with rosiglitazone. Fertil Steril 2001;76:1057–9. 10. Baillargeon JP, Jakubowicz DJ, Iuorno MJ, Jakubowicz S, Nestler JE. Effects of metformin and rosiglitazone, alone and in combination, in nonobese women with polycystic ovary syndrome and normal indices of insulin sensitivity. Fertil Steril 2004;82:893–902. 11. Vandermolen DT, Ratts VS, Evans WS, Stovall DW, Kauma SW, Nestler JE. Metformin increases the ovulatory rate and pregnancy rate from clomiphene citrate in patients with polycystic ovary syndrome who are resistant to clomiphene citrate alone. Fertil Steril 2001;75: 310 –5. 12. Mitwally MF, Kuscu NK, Yalcinkaya TM. High ovulatory rates with use of troglitazone in clomiphene-resistant women with polycystic ovary syndrome. Hum Reprod 1999;14:2700 –3. 13. Ghazeeri G, Kutteh WH, Bryer-Ash M, Haas D, Ke RW. Effect of rosiglitazone on spontaneous and clomiphene citrate-induced ovulation in women with polycystic ovary syndrome. Fertil Steril 2003;79:562– 6. 14. Clifford K, Rai R, Watson H, Regan L. An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Hum Reprod 1994;9:1328 –32. 15. Jakubowicz DJ, Iuorno MJ, Jakubowicz S, Roberts KA, Nestler JE. Effects of metformin on early pregnancy loss in the polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:524 –9. 16. Liddell HS, Sowden K, Farquhar CM. Recurrent miscarriage: screening for polycystic ovaries and subsequent pregnancy outcome. Aust NZ J Obstet Gynaecol 1997;37:402– 6. 17. Regan L, Braude PR, Trembath PL. Influence of past reproductive performance on risk of spontaneous abortion. BMJ 1989;299:541–5. 18. Sagle M, Bishop K, Ridley N, Alexander FM, Michel M, Bonney RC, et al. Recurrent early miscarriage and polycystic ovaries. BMJ 1988; 297:1027– 8. 19. Checa MA, Requena A, Salvador C, Tur R, Callejo J, Espinos JJ, et al. Insulin-sensitizing agents: use in pregnancy and as therapy in polycystic ovary syndrome. Hum Reprod Update 2005;11:375–90. 20. De Leo V, la Marca A, Petraglia F. Insulin-lowering agents in the management of polycystic ovary syndrome. Endocr Rev 2003;24: 633– 67. 21. Chan LY, Yeung JH, Lau TK. Placental transfer of rosiglitazone in the first trimester of human pregnancy. Fertil Steril 2005;83:955– 8. 22. Bedaiwy MA, Miller KF, Goldberg JM, Nelson D, Falcone T. Effect of metformin on mouse embryo development. Fertil Steril 2001;76: 1078 –9. 23. Gardiner CS, Reed DJ. Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo. Biol Reprod 1994;51:1307–14. 24. Rayburn WF, Sienko AE, Gonzalez CL, Christensen HD, Kupiec TC, Paulsen AL, et al. Impact of antenatal exposure of mice to fenfluramine on cardiac development and long-term growth of the offspring. Drug Chem Toxicol 2000;23:419 –31. 25. Ryan MJ, Didion SP, Mathur S, Faraci FM, Sigmund CD. PPAR(gamma) agonist rosiglitazone improves vascular function and lowers blood pressure in hypertensive transgenic mice. Hypertension 2004;43:661– 6. 26. Phillips JW, Barringhaus KG, Sanders JM, Yang Z, Chen M, Hesselbacher S, et al. Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes. Circulation 2003;108:1994 –9. 27. Cox PJ, Ryan DA, Hollis FJ, Harris AM, Miller AK, Vousden M, et al. Absorption, disposition, and metabolism of rosiglitazone, a potent thiazolidinedione insulin sensitizer, in humans. Drug Metab Dispos 2000;28:772– 80. 28. Bernard BK, Hoberman AM, Brown WR, Ranpuria AK, Christian MS. Oral (gavage) two-generation (one litter per generation) reproduction study of pentachlorophenol (penta) in rats. Int J Toxicol 2002;21:301–18. 29. Jagetia GC, Baliga MS. Induction of developmental toxicity in mice treated with Alstonia scholaris (Sapthaparna) in utero. Birth Defects Res B Dev Reprod Toxicol 2003;68:472– 8.
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30. Christensen HD, Rayburn WF, Parker KM, Gonzalez CL, Gold KP. Chronic prenatal exposure to carbamazepine and perinatal outcomes of C3H/He mice. Am J Obstet Gynecol 2004;190:259 – 63. 31. Altman D. Practical statistics for medical research. London: Chapman and Hall/CRC, 1997. 32. Diggle PJ, Heagerty P, Liang KY, Zeger SL. Analysis of longitudinal data. New York: Oxford University Press, Wiley & Sons, Inc., 2002. 33. Kunstyr I. Paresis of peristalsis and ileus lead to death in lactating mice. Lab Anim 1986;20:32–5. 34. Chen YE, Fu M, Zhang J, Zhu X, Lin Y, Akinbami MA, et al. Peroxisome proliferator-activated receptors and the cardiovascular system. Vitam Horm 2003;66:157– 88. 35. Mohan M, Malayer JR, Geisert RD, Morgan GL. Expression patterns of retinoid X receptors, retinaldehyde dehydrogenase, and peroxisome proliferator activated receptor gamma in bovine preattachment embryos. Biol Reprod 2002;66:692–700. 36. Jain S, Pulikuri S, Zhu Y, Qi C, Kanwar YS, Yeldandi AV, et al. Differential expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivators steroid receptor coactivator-1 and PPAR-binding protein PBP in the brown fat, urinary bladder, colon, and breast of the mouse. Am J Pathol 1998;153:349 –54. 37. Li Q, Cheon YP, Kannan A, Shanker S, Bagchi IC, Bagchi MK. A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice. J Biol Chem 2004;279:11570 – 81. 38. Asami-Miyagishi R, Iseki S, Usui M, Uchida K, Kubo H, Morita I. Expression and function of PPARgamma in rat placental development. Biochem Biophys Res Commun 2004;315:497–501. 39. Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, et al. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell 1999;4:597– 609. 40. Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, et al. PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol Cell 1999;4:585–95. 41. Schaiff WT, Bildirici I, Cheong M, Chern PL, Nelson DM, Sadovsky Y. Peroxisome proliferator-activated receptor-gamma and retinoid X receptor signaling regulate fatty acid uptake by primary human placental trophoblasts. J Clin Endocrinol Metab 2005;90:4267–75. 42. Fournier T, Pavan L, Tarrade A, Schoonjans K, Auwerx J, RochetteEgly C, et al. The role of PPAR-gamma/RXR-alpha heterodimers in the regulation of human trophoblast invasion. Ann NY Acad Sci 2002;973: 26 –30. 43. Sandor TL, Noll L, Klinkner DB, Weihrauch D, He BJ, Kaul S, et al. Rosiglitazone antagonizes VEGF signalling and NFATc1 activation in cardiac valve endothelium. Endothelium 2006;13:181–90. 44. Berry EB, Eykholt R, Helliwell RJ, Gilmour RS, Mitchell MD, Marvin KW. Peroxisome proliferator-activated receptor isoform expression changes in human gestational tissues with labor at term. Mol Pharmacol 2003;64:1586 –90. 45. Lappas M, Permezel M, Georgiou HM, Rice GE. Regulation of proinflammatory cytokines in human gestational tissues by peroxisome proliferator-activated receptor-gamma: effect of 15-deoxy-Delta (12,14)-PGJ(2) and troglitazone. J Clin Endocrinol Metab 2002;87: 4667–72. 46. Kalyoncu NI, Yaris F, Ulku C, Kadioglu M, Kesim M, Unsal M, et al. A case of rosiglitazone exposure in the second trimester of pregnancy. Reprod Toxicol 2005;19:563– 4. 47. Vaughan TB, Bell DS. Stockpiling of ovarian follicles and the response to rosiglitazone. Diabetes Care 2005;28:2333– 4. 48. Yaris F, Yaris E, Kadioglu M, Ulku C, Kesim M, Kalyoncu NI. Normal pregnancy outcome following inadvertent exposure to rosiglitazone, gliclazide, and atorvastatin in a diabetic and hypertensive woman. Reprod Toxicol 2004;18:619 –21. 49. Leiter EH, Reifsnyder PC, Zhang W, Pan HJ, Xiao Q, Mistry J. Differential endocrine responses to rosiglitazone therapy in new mouse models of type 2 diabetes. Endocrinology 2006;147:919 –26.
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Department of Surgery, Division of Pediatric Surgery, Medical College of Wisconsin and Children’s Research Institute; and Department of Biostatistics and c Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin b
Objective: To identify the effects of rosiglitazone use during murine pregnancy. Design: The effect of rosiglitazone on blastocyst development was determined by culturing two-cell mouse embryos with rosiglitazone for 72 hours. From January to June 2005, five independent groups of ICR/CD1 female mice were treated with rosiglitazone during pregnancy, from the time of identification of seminal plugs until delivery of pups. Setting: Controlled animal facility. Animal(s): Two-cell mouse embryos and an outbred line of mice, ICR/CD1. Intervention(s): Two-cell embryos were cocultured with rosiglitazone (10 M) for 72 hours and scored. Ten-week-old female ICR mice were mated. Females with seminal plugs then were randomized to rosiglitazone (10 or 0.1 mg/kg per day) or to carrier alone, by gavage, until delivery. Weekly weights were obtained, and pregnancy outcomes were documented. Main Outcome Measure(s): Blastocyst development, number of pups and pup weights, and morphological changes. Result(s): Embryos exposed to rosiglitazone progressed to the blastocyst stage within 72 hours. Pregnant animals demonstrated normal weight gain throughout pregnancy. Postnatal growth and litter size were not statistically different between groups. No changes in normal mouse neonate development were observed. Conclusion(s): Rosiglitazone did not impair murine blastocyst development in vitro or cause phenotypic harm to the mouse fetus when administered during pregnancy, suggesting potential safety for rosiglitazone use in pregnancy. (Fertil Steril威 2006;86(Suppl 3):1074 –9. ©2006 by American Society for Reproductive Medicine.) Key Words: Rosiglitazone, pregnancy, polycystic ovary syndrome, diabetes mellitus type 2
Thiazolidinedione drugs, such as troglitazone, pioglitazone, and rosiglitazone, are peroxisome proliferator–activated receptor gamma (PPAR␥) ligands. Rosiglitazone is the most potent known specific PPAR␥ ligand (1). It has potentially far-reaching effects on pathophysiologic processes, from cancer to atherosclerosis and diabetes. Rosiglitazone improves insulin sensitivity in peripheral tissue without stimulating the pancreatic beta cells, thereby also decreasing the possibility of hypoglycemic events. Thus, rosiglitazone has become a desirable agent for treating diabetes mellitus type 2, which currently is its primary clinical use. Because rosiglitazone effectively diminishes hyperinsulinemia, many have considered its use in treatment of women with polycystic ovary syndrome (PCOS). Polycystic ovary syndrome affects 4%–10% of premenopausal women (3.5–5 million women in the United States) Received December 21, 2005; revised and accepted March 17, 2006. Supported in part by an award from the Children’s Hospital of Wisconsin Research Foundation and Children’s Research Institute, Milwaukee, Wisconsin. Reprint requests: Tara L. Sander, Ph.D., Department of Surgery, Division of Pediatric Surgery, CVRC M4050, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226 (FAX: 414456-6473; E-mail: [email protected]).
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(2), causing hirsutism, oligomenorrhea, and anovulatory infertility. The disease involves a complex imbalance of androgens and insulin. Both obese and nonobese women with PCOS demonstrate insulin resistance and therefore develop hyperinsulinemia. In this circumstance, insulin stimulates androgen synthesis from the thecal cells (3) and decreases sex hormone– binding globulin synthesis in the liver (4), thus contributing to total free androgen in the plasma (5). The free androgen causes an imbalance in the hormonal regulation of ovulation. Therefore, these patients often seek treatment for infertility. Once the diagnosis of PCOS has been made, the current therapies initially begin with weight reduction for the obese and clomiphene citrate (CC) to induce ovulation. Treatment with CC carries the risk of multiple pregnancies and ovarian hyperstimulation, as well as the excessive cost associated with these complications. Furthermore, not all patients respond to the drug. Even those who ovulate and conceive have a 20% risk of spontaneous abortion and a 1% risk of stillbirth (6). Therefore, alternatives to CC have been sought. The use of insulin-sensitizing agents, that is, metformin, troglitazone, and rosiglitazone, has been reported in PCOS patients. Troglitazone improved ovulation rates (7) and re-
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sultant pregnancy (8), and rosiglitazone use resulted in normal pregnancy (9). Rosiglitazone treatment also restored menses in 96% of patients, with three resultant pregnancies (5). The ovulatory effects also were seen in nonobese patients (10). Overall results have been encouraging in CC-resistant PCOS patients. Ovulation is stimulated in women who are treated with metformin (11), troglitazone (12), or rosiglitazone (13) alone, with an additive effect in combination with CC. Women treated with thiazolidinediones are advised to discontinue the drug either at the time of ovulation or at confirmation of pregnancy to minimize exposure to the fetus (6). A small number of those who do ovulate become pregnant, and in one study, five of seven women had healthy infants, whereas two women miscarried (12). Because the rate of spontaneous abortion reaches 36%– 82% in women who are reported to have PCOS (14 –18), the cause of early pregnancy loss cannot be determined to be solely from the drug treatment. Conversely, PCOS patients treated with metformin throughout pregnancy have a lower rate of spontaneous abortion compared with untreated patients (8.8% vs. 41.9%; Jakubowicz et al. [15]). Thus, those taking metformin have been advised to continue administration of the drug throughout pregnancy (for a review of current use of insulin-sensitizing agents in pregnancy and PCOS, see Checa et al. [19]). This result suggests that treatment with an insulin-lowering agent may be desirable throughout pregnancy in PCOS patients. Rosiglitazone has become an attractive option for CCresistant PCOS patients who do not tolerate or respond to other oral agents (20). However, rosiglitazone is considered by the US Food and Drug Administration to be a pregnancy class C substance, because animal studies suggest that high doses of the drug cause mid- to late-gestational fetal death and growth retardation (6). Rats treated with rosiglitazone during gestation through lactation have reduced litter size, as well as decreased neonatal viability and postnatal growth (6). To the best of our knowledge, no published study has demonstrated specific effects of rosiglitazone use during pregnancy that lead to fetal death or growth retardation. More concerning, rosiglitazone has been identified in human fetal tissue during the first trimester, thus underscoring the importance of determining the potential effects of rosiglitazone on fetal development (21). Taken together, these reports have raised sufficient concerns that rosiglitazone use during pregnancy currently is considered to be contraindicated. As a result, physicians cautiously are considering the use of rosiglitazone as a PCOS treatment, because it is not clear whether rosiglitazone use during pregnancy is safe or harmful to the developing fetus. Because the drug has demonstrated potentially beneficial effects in a limited number of human case reports, but harmful effects have been observed in animal pregnancies, our study focused on investigating the effects of rosiglitazone treatment during murine pregnancy. Our initial experiments tested the in vitro effects of rosiglitazone on early Fertility and Sterility姞
embryogenesis, whereas later studies documented the in vivo effects of treatment throughout pregnancy. MATERIALS AND METHODS Murine Embryo Studies To test whether the PPAR␥ agonist rosiglitazone affects early embryogenesis, two-cell mouse embryos were treated with and without rosiglitazone, as described elsewhere for drug toxicity (22). Embryotech Laboratories, Inc. (Wilmington, MA) provided the embryos and expertise in culturing and collecting data. Fresh embryos were harvested from superovulated female B6C3F-1 ⫻ B6D2F-1 mice, the laboratory’s customary strain, at a standard predetermined hour, after hCG administration. The standard assay consisted of 21 embryos per test item and included a 21-embryo control. To the treatment group, rosiglitazone (Cayman Chemical, Ann Arbor, MI) was added fresh daily to the culture media in a 10 M concentration. Diethyl maleate (Sigma, St. Louis, MO), a well-recognized inhibitor of blastocyst development, served as a positive control (23). The carrier dimethyl sulfoxide (Sigma) was the negative control. Both the test and control embryos were set up in triplicate under oil and were cultured with 5% CO2 at 37°C until they were scored for development to expanded blastocysts. Development for the two-cell assay was scored at 72 hours. Greater than 80% blastocyst formation from the control group was required to validate the two-cell assay (http://www.embryotech.com/qc/mea.htm). Animals and Animal Care All studies were performed under a proposal approved in July 2004 by the Medical College of Wisconsin Institutional Animal Care and Use Committee, with approved addendums in October 2004. All animals were housed and cared for by the Medical College of Wisconsin Biomedical Research Center. A murine model was selected for a short gestation time of 19 –20 days, for litter sizes, and for ease of care. The ICR/CD1 mouse was chosen because of the ease of timed pregnancy (24) and large litter size of 10 –15 pups (personal communication, Charles River Laboratories). The animals were maintained in a pathogen-free environment. Groups were fed a high-fat (9%) chow diet to prevent cannibalism of pups at delivery (PicoLab Mouse Diet; PMI International LLC, Richmond, IN), with free access to both feed and water via nipple water bottles. All animals were obtained from Charles River Laboratories (Wilmington, MA). Upon arrival, mice were allowed 1 week to acclimate. Eighty 10-week-old CD1 female mice then were examined for signs of estrus, with those in estrus selected for 1:1 or 2:1 mating with ⱖ10-week-old males. The following morning, evidence of a seminal plug confirmed mating, and the females were separated from the males. Each cycle resulted in 9 to 15 plugs (11%–19% successful mating). The studies were repeated five times, and data were pooled for statistical analysis. Ear tags (National Band and Tag Co., Newport, 1075
KY) were placed to ease documentation, and weights (CS2000; Ohaus Corp., Pine Brook, NJ) were recorded at baseline and, subsequently, on a weekly basis. Drug Dosing and Administration Previous studies (25, 26) and a trial study with nonpregnant females confirmed the safety of a rosiglitazone dosage of 10 mg/kg per day in these animals without adverse side effects. Rosiglitazone is unstable once resuspended in solution and easily degrades within 4 hours at room temperature (27). Therefore, oral gavage was chosen to most closely approximate clinical use of the drug (absorption from the gastrointestinal tract) and to confirm timely treatment and equivalent doses between animals, as described elsewhere (25, 28). Dosing was based on a 25-g nonpregnant female, with no change in dose made throughout the study (24). The treatment group received 2.5 or 25 L of dimethyl sulfoxide with rosiglitazone–potassium (Cayman Chemical) diluted in sterile water to a final volume of 0.2 mL by oral gavage (concentration of 0.1 or 10 mg/mL, respectively). The control group received 2.5 or 25 L of dimethyl sulfoxide, diluted in room-temperature sterile water for an equal volume. The final drug dose was equivalent to 0.1 or 10 mg/kg per day, respectively. The 0.1 mg/kg per day dosing was approximately equivalent to the maximum recommended human dose of 8 mg per 70 kg, per day. Gavage needles (Popper and Sons, New Hyde Park, NY) were lubricated with nonspermicidal sterile lubricating jelly (First Priority, Inc., Elgin, IL) before each use. The gavage was performed by Biomedical Research Center–approved methods. The animals were observed after each administration. Any change in respiratory status or other clinical signs of deterioration mandated that the mouse be euthanized, which was performed by CO2 administration or by 2:1 ketamine-to-xylazine, injected intraperitoneally, followed by exsanguination. Effects From Treatment Gravid females were given dimethyl sulfoxide or rosiglitazone daily by oral gavage. Weekly weights were obtained and recorded. Before delivery, all females were separated to ensure proper assignment of pups. Litter size, individual pup weights and phenotypes, and viability were recorded, as described elsewhere (29, 30). Statistical Analysis Initial descriptive statistics were used to summarize the data. Distribution of continuous variables was examined for normality. The mean of the weight variable was compared using t test and analysis of variance with Bonferroni multiple comparisons adjustment (31). The generalized linear model was used, controlling for the number of pups. The graph of the mean weight at each measurement time also was used to overview the mean weight change over time. The growth curves analysis of the longitudinal data was performed to 1076
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investigate outcome measurement changes over time (32). For all analyses, the significance level used was .05. The analysis was performed using the SAS statistical package, version 9 (SAS Institute, Cary, NC). RESULTS Rosiglitazone Does Not Inhibit Blastocyst Formation Two-cell murine embryos were exposed to dimethyl sulfoxide control, rosiglitazone, or diethyl maleate for 72 hours. Consistent with work published elsewhere (23), all embryos exposed to diethyl maleate as a positive control failed to mature (data not shown). In Figure 1, no difference in blastocyst development was observed with rosiglitazone treatment. The embryos exposed to rosiglitazone appeared to progress in a similar fashion as the control embryos. These data suggest that rosiglitazone does not impact early, preimplantation embryonic development. Rosiglitazone Use During Murine Pregnancy The mouse pregnancy study included a control group, a group treated with 0.1 mg/kg per day of rosiglitazone throughout pregnancy, and a group treated with 10 mg/kg per day of rosiglitazone throughout pregnancy. Maternal weight gain did not differ across groups (data not shown). The number of pups and the weights of the pups were not different between treatment groups (Table 1). In addition, all pups reached developmental milestones of hair growth and eye opening similarly (Table 1). Postnatal weight gain also showed no differences between groups (Fig. 2). The highest recommended human equivalent dose (0.1 mg/kg per day)
FIGURE 1 Rosiglitazone effect on embryogenesis. Two-cell murine embryos were treated with 10 M rosiglitazone for 72 hours and then scored for stage of blastocyst development and compared with dimethyl sulfoxide carrier control. Of the control embryos, 100% reached blastocyst stage, and 100% of the rosiglitazone-treated embryos reached blastocyst stage. None of the diethyl maleate embryos reached blastocyst stage.
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TABLE 1 Rosiglitazone (Rosi) treatment during murine pregnancy. Parameter No. of plugs No. pregnant No. of pups per litter Birth weight (g) Male (%) Hair (PND 8) Eyes open (PND 15)
Control
Rosi (0.1)a
Rosi (10)b
23 18 10.67 ⫾ 1.94 1.61 ⫾ 0.16 46 18/18 18/18
15 15 11.07 ⫾ 2.81 1.60 ⫾ 0.17 51 15/15 10/15
20 15 10.06 ⫾ 1.27 1.62 ⫾ 0.19 48 15/15 12/15
Note: Paired t test, P⬎.05 for each experimental group vs. control. PND ⫽ postnatal day. a 0.1 mg/kg per day. b 10 mg/kg per day. Klinkner. Rosiglitazone use in murine pregnancy. Fertil Steril 2006.
and a two log– higher dose of 10 mg/kg per day do not appear to interfere with normal murine fetal development. Adverse Outcomes Throughout the course of the study, two maternal deaths occurred at 2 weeks postpartum. Autopsy identified small bowel obstruction as the cause of death, which has been reported in lactating mice (33). Thus, two litters were dispersed among the remaining mothers of the same treatment group. Pup loss from those control litters was significantly greater, apparently as a result of higher pup to mother ratios. Thirty-nine pups were excluded from postnatal weight gain analysis because of death during the period of observation. Cause of death was cannibalism for the majority, with one pup born with ascites.
FIGURE 2 Mean weekly pup weights. Weights were obtained weekly on control (C), rosiglitazone (R), and human-dosaged–rosiglitazone (HR) pups. No differences were observed between groups. (Analysis of variance with Bonferroni adjustment, P⬎.05).
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DISCUSSION Peroxisome proliferator–activated receptor gamma is a ligand-activated nuclear hormone receptor that is found in adipocytes, colonocytes, endothelial cells, and cells of the monocyte lineage (34). During embryonic development, PPAR␥ is expressed in bovine oocytes at all preimplantation stages, from oocyte to hatched blastocyst (35). Furthermore, PPAR␥ has been identified in both the inner cell mass and the trophectoderm. Jain et al. (36) demonstrated PPAR␥ expression in the urogenital sinus and in brown adipose tissue at E15.5 of murine embryogenesis. Interestingly, PPAR␥ has been demonstrated in endothelial cells of the mouse uterus as early as day 1 of pregnancy (37). In the rat uterus, PPAR␥ was expressed at day 11 of pregnancy, increased by day 13, and subsequently decreased (38). Troglitazone administration between day 9 and day 11 then increased PPAR␥ expression in the placenta and reduced the mortality of the fetuses. A deficiency of PPAR␥ in the developing embryo leads to improper development of the placental vasculature and also is linked to myocardial thinning and death by gestational day 10 in mice (39, 40), suggesting that PPAR␥ plays an important role in fetal development. Activation of PPAR␥ and retinoid X receptor, a heterodimeric partner of PPAR␥, has been shown to play a role in fatty acid uptake in human placental trophoblasts, yet another important aspect of fetal development (41). Others have demonstrated that rosiglitazone prevents human trophoblast invasion from first-trimester placenta, a key step in implantation (42). In vitro use of rosiglitazone has been demonstrated to interfere with pathways for normal cardiac valvular development (43). Taken together, these observations demonstrate that PPAR␥ clearly plays a role in placental and cardiac development in in vivo rodent models. Thus, inappropriate activation at key portions of development may result in adverse developmental outcomes. 1077
Natural ligands for PPAR␥ appear to be polyunsaturated fatty acids and eicosanoids, such as 15-deoxy-delta 12, 14prostaglandin J2 (15d-PGJ2) (34). The relationship between eicosanoids and PPAR␥ was studied in early pregnancy in mice. Blocking the 12/15-lipoxygenase– derived eicosanoid resulted in failure of endometrial implantation, whereas treatment with rosiglitazone rescued the embryos and led to proper implantation (37). This observation suggested that PPAR␥ was involved in the maintenance of pregnancy. Given that PPAR␥-deficient mice have abnormal placental development and that rosiglitazone promotes implantation in these mice, PPAR␥ ligands may be beneficial in preventing spontaneous abortions in women with PCOS. Rosiglitazone may be of therapeutic value at other specific points during pregnancy. Berry et al. (44) demonstrated that PPAR isoforms change with onset of labor, compared with women undergoing Cesarean sections, suggesting that PPAR expression and function may have a role in maintenance of pregnancy or initiation of labor. Lappas et al. (45) have suggested that rosiglitazone may be useful in preventing or stopping preterm labor. They studied the effects of 15d-PGJ2 and rosiglitazone on placental cytokine release from tissue obtained from full-term pregnancies delivered via Caesarean section for breech or previous C-section. Both PPAR␥ ligands dramatically decreased the release of IL-6, IL-8, and TNF-␣. Theoretically, modulating the inflammatory response by inhibiting release of IL-6, IL-8, and TNF-␣ would halt preterm labor. No study has demonstrated this hypothesis in vivo, nor has safety for this clinical application in late pregnancy been determined. On the basis of information provided in the Physicians’ Desk Reference (6) regarding rosiglitazone use during pregnancy, we expected a decrease in the number of pups born and a delay in weight gain. We confirmed the lack of toxicity to the embryo up to 72 hours. Our in vivo findings in pregnant mice were not consistent with the manufacturer’s data in rats and rabbits. Combined with the limited case reports of rosiglitazone use in human pregnancy (5, 9, 13, 46 – 48), these findings suggest that rosiglitazone use may be acceptable for use in pregnancy for women with diabetes mellitus type 2 or with PCOS. Because rosiglitazone has been documented to cross the placenta at 10 weeks’ gestational age (21), the safety and efficacy of use in human pregnancy should be demonstrated before widespread use. Since much of murine development has been delineated, we conducted our work in mice. The added benefits of short gestation and ease of care supported our animal selection. However, no commercially available genetic mouse model of PCOS or diabetes mellitus type 2 exists, pointing to the genetic variability in these diseases and subsequent difficulty in recreating human diseases in mice. The ICR mouse, an outbred line, may reflect the genetic variability in the human population. However, the effects seen in this line cannot be applied broadly. As noted by other investigators, there can be difficulty with mouse models: genetically different mice responded to rosiglitazone in a variable fashion (49). 1078
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Rosiglitazone use in murine pregnancy
One potential reason that we did not observe an inhibition of postnatal growth with rosiglitazone administration is that treatment was discontinued upon delivery. Peroxisome proliferator–activated receptor␥ has been identified in murine breast tissue of lactating mice (36), and activation of PPAR␥ may affect milk production. We chose not to treat throughout lactation, so as to more closely mimic human treatment models. For example, infants of diabetic mothers often are fed formula if the mother continues rosiglitazone treatment after delivery. With the data that we present here, additional studies may be warranted in human beings. This study provides one of the first reports of the effects of rosiglitazone use in pregnancy. Although this work demonstrates no phenotypic evidence of altered fetal development, additional studies must be undertaken before human studies. Echocardiography will allow assessment of cardiac development and function without requiring that the animals be sacrificed, and magnetic resonance imaging likewise will provide insight into other potential structural changes. Although few drugs are considered entirely safe in pregnancy, rosiglitazone may be a drug with benefits to consider once the risks have been fully evaluated. Acknowledgments: The authors gratefully acknowledge Alexandra LerchGaggl, Ph.D., and Mary Holtz, Ph.D., at the Medical College of Wisconsin for assistance with mouse husbandry and mating. The authors thank Kirkwood Pritchard Jr., Ph.D., and John Densmore, M.D., at the Medical College of Wisconsin and Children’s Research Institute for intellectual contributions to the study.
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