Int. J. Exp. Pathol. (2016), 97, 125–132
ORIGINAL ARTICLE
Effect of in utero exposure to the atypical anti-psychotic risperidone on histopathological features of the rat placenta K.P. Singh, Manoj K. Singh and Shrikant Gautam Neurobiology Lab., Department of Zoology, University of Allahabad, Allahabad, India
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY
SUMMARY
For clinical management of different forms of psychosis, both classical and atypical anti-psychotic drugs (APDs) are available. These drugs are widely prescribed, even during pregnancy considering their minimal extra-pyramidal side effects and teratogenic potential compared to classical APDs. Among AAPDs, risperidone (RIS) is a first-line drug of choice by physicians. The molecular weight of RIS is 410.49 g/mol; hence, it can easily cross the placental barrier and enter the foetal bloodstream. It is doi: 10.1111/iep.12176 not known whether or not AAPDs like RIS may affect the developing placenta and foetus adversely. Reports on this issue are limited and sketchy. Therefore, this study has evaluated the effects of maternal exposure to equivalent therapeutic doses of RIS on placental growth, histopathological and cytoarchitectural changes, and to establish a relationship between placental dysfunction and foetal outcomes. Pregnant rats (n = 24) were exposed to selected doses (0.8, 1.0 and 2.0 mg/kg) of RIS from gestation days 6–21. These dams were sacrificed; their placentas and foetuses were collected, morphometrically examined and further processed for histopathological Received for publication: 22 July 2015 examination. This study revealed that in utero exposure to equivalent therapeutic Accepted for publication: 31 January doses of RIS during organogenesis-induced placental dystrophy (size and weight), 2016 disturbed cytoarchitectural organization (thickness of different placental layers), Correspondence: histopathological lesions (necrosis in trophoblast with disruption of trophoblastic Krishna P. Singh septa and rupturing of maternal-foetal interface) and intrauterine growth restriction Neurobiology Lab of the foetuses. It may be concluded that multifactorial mechanisms might be Department of Zoology University of Allahabad involved in the dysregulation of structure and function of the placenta and of poor Allahabad foetal growth and development. India Tel./Fax: 91-0532-2640188 E-mails: [email protected]; [email protected]
Keywords atypical antipsychotics, histopathology, placenta, prenatal, risperidone
The placenta is an interface between dam and developing foetus. It also secures the embryo as a protective barrier against xenobiotic agents if administered during pre- and peri-conception. Although it is a temporary organ, its proper implantation on uterus and further growth plays an important role for development and growth of the embryo/foetus (Prouillac & Lecoeur 2010; Murphy et al. 2006). It is well documented that any kind of placental dysfunction, during early or late gestation, may adversely affect normal embryo-foetal development and growth (Furukawa et al. 2011a,b). Xenobiotics may not easily cross the placental barrier if the molecular weight of the agent is above 500 g/mol, but certain drugs/chemicals,
including drugs acting on the central nervous system (CNS) have the ability to cross the placental barrier easily as they are (on average) lower in molecular weight (400–600 g/mol) in humans (Newport et al. 2007). These drugs are also prevented from filtering through passive diffusion by the dense and complex network of blood capillaries. Hitherto it is not known whether or not molecules of psychotropic drugs, including anti-psychotics, affect the placental capillary network adversely at therapeutic dose range. Reports on this issue are limited and sketchy, and mostly cited at higher concentration of the drugs, causing developmental toxicity in the maternal environment as well as histopathological changes in the
© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology
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placenta. Searching both clinical and non-clinical literature revealed that the documentation about in utero exposure to atypical novel anti-psychotic drugs, and about their impact on histopathological impairments on the placenta, is negligible. For clinical management of different forms of psychosis, including schizophrenia, both classical (1st generation) and atypical/novel (2nd generation) anti-psychotic drugs are available on the world market. Atypical anti-psychotic drugs (AAPDs) are widely prescribed to adult populations (Galbaly et al. 2014; Stephenson et al. 2013; Toh et al. 2013), even to women of child-bearing age in view of their moderate side effects and lower teratogenic potential compared to classical APDs, which are associated with movement disorders (Costa et al. 2004). Among AAPDs, risperidone (RIS) is a first-line drug of choice by healthcare providers even during pregnancy (Paschetta et al. 2014; Sadowski et al. 2013). The molecular weight of this drug is 410.49 g/mol; hence, it can easily cross the placental barrier and enter the foetal circulation. The maternal transfer of the drug into foetuses takes place when both placenta and foetus are at developmental stage. Thus it is possible that prenatal exposure to RIS adversely affects normal development and growth of the placenta. Hence, placental impairment may be a bio-indicator of drug-induced effects on both the placenta and the developing foetus. Although several inducing mechanisms have been proposed by previous investigators (Prouillac & Lecoeur 2010; Newport et al. 2007), a mechanism to explain how a AAPDs such as RIS, induce placental dystrophy, either at a gross or a microscopic level. Therefore, the present study has been designed in order to evaluate how maternal exposure to equivalent therapeutic doses of RIS affects placental growth (size and weight) and histopathology and to establish a relationship between placental dysfunction and foetal outcomes under these conditions.
experimental doses were calibrated as 0.8 mg/kg (MHRD 9 4), 1.0 mg/kg (MHRD 9 6) and 2.0 mg/kg (MHRD 9 10) on the basis of per kg body weight per day, and their suitability to the rat animal model. The rationale for the selection of three doses of RIS was as per MRHD corrected for the higher metabolic rate of rats (i.e. 4–6 times faster than in humans) (Kapur et al. 2003). Four groups of pregnant female rats (n = 6) in each group were maintained. All the control and experimental rats were exposed from gestational day 6–21 (GD 6-21), either to the drug or to the vehicle alone. In this study, tablets of RIS (each tablet contains 2.0 mg of the drug molecule) were dissolved in 0.1N HCl solution. The selected doses of RIS (0.8, 1.0 and 2.0 mg) were gavaged (approximately 0.84–2.1 ml.) to sperm positive dams once daily (at 09.00 h) from GD 6-21 via a cannula. According to the protocol, RIS-treated rats of each experiment were sacrificed under phenobarbital anaesthesia on GD 21 (at 06.00 h), and foetuses along with their placenta were collected from both the uterine horns through uterectomy after transcardial perfusion with 10% formaldehyde. Placentas from all groups were cleaned, blotted dry, and then, size (longest diameter) was measured with the help of vernier callipers. The weight of each placenta was also recorded. One placenta from each uterine horn was randomly selected from each group and was cut into two equal parts and fixed in the same fixative for one week before processing for microscopic examination (paraffin microtomy). Transverse section(s) of each placenta were cut at 7l, and sections of highest diameter were selected for H&E staining to observe histopathological changes and to measure thickness of different layers with the help of capture pro software. All sections provided a transverse view of the medial region of the placenta, and eight sections of each placenta were examined, and selected measures recorded (Kosif et al. 2008). Foetal body size of each individual of four groups was measured from the anteriormost part of the snout to the tip of the tail using a prefixed length measuring board, and the weight was recorded.
Materials and methods In this study, laboratory inbred female Wistar rats from our breeding colony, weighing 180 10 g, aged about 3 months, were used for the experimental procedures. Animals were housed in plastic cages with rice bran as bedding material at standard laboratory environment (24 2 °C, 12/12 h light/dark cycle and 60% RH). Standard rat food and tap water were made available ad libitum. Animals were maintained and used in accordance with the animal welfare act. After acclimatization, nulliparous female rats were allowed to mate with males overnight (ratio 2:1), and the next morning, presence of sperm in vaginal smears was designated gestation day zero (GD-0) for determining the onset of gestation. Such sperm positive rats were used further for the experimental procedures. RIS was purchased from the pharmaceutical market with trade name Risperdal (Torrent, India). The maximum recommended human dose (MRHD) of RIS is 04–12 mg/day. The
Statistical analysis All data are represented as mean and standard errors (meanSEM). Variables such as placental size and weight, thickness of placental layers, litter size, foetal body size and weight were statistically analysed using one-way ANOVA followed by post hoc Tukey’s multiple comparison test to determine difference amongst exposed groups and control. For all statistical values, minimum significance level was set at P < 0.05. All calculations were carried out with the help of Microsoft Excel (Microsoft Redmond Campus, Redmond, Washington, USA) and Statistica-10 software (Dell Statistica, Tulsa, OK, USA).
Ethical approval The present study was approved by the Institutional Animal Ethics Committee (IAEC), University of Allahabad, Allahabad, India. International Journal of Experimental Pathology, 2016, 97, 125–132
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Results Effect of prenatal exposure to RIS on placental size and weight One-way ANOVA followed by Tukey’s multiple comparison tests showed significant increase in placental size [F = (3, 56) 68.00, P < 0.001] and weight [F = (3, 56) 54.70, P < 0.001] in RIS-treated groups. In post hoc analysis, placental size was substantially (P < 0.001) increased (8.15% and 16.29%) at 0.8 mg and 1.0 mg dose groups, respectively, whereas it was significantly (P < 0.001) decreased (13.33%) at 2.0 mg/kg dose group as compared to control. Similarly, placental weight was significantly (P < 0.001) increased at 0.8 mg (15.80%) and 1.0 mg (64.36%) RIS dose groups, respectively, whereas the same was substantially (P < 0.001) decreased (54.02%) at 2.0 mg/kg drugexposed group in comparison with vehicle-treated group (Table 1, Figure 1). It appears that the drug effect was more intense for placental weight than size. Haemorrhages were found to be more severe on the ventral surface of each placenta in in utero exposed RIS in comparison with those of control placentas (Figure 1).
Effect of prenatal exposure to RIS on thickness of placental layers Statistical analysis (one-way ANOVA) displayed significant [F = (3, 28) 175.60, P < 0.001] alteration in total thickness of three layers (deciduas basalis, basal zone and labyrinthine zone) of RIS-treated placentas. The total thickness of three typical layers was increased (approximately 13% to 21.47%) substantially (P < 0.001) at 0.8 and 1.0 mg dose groups, respectively, while this measure was reduced (15.24%) substantially (P < 0.001) at 2.0 mg dose group in comparison with control (Figure 2). One-way ANOVA followed by post hoc Tukey’s test expressed that placental thickness of labyrinthine zone [F = (3, 28) 124.75, P < 0.001] was found to be significantly (P < 0.001) increased (approximately 24%) at 0.8 and 1.0 mg dose groups, and thickness of this zone was substantially (P < 0.001) reduced (approximately 18%) at 2.0 mg dose. In the basal zone, placental thickness was found to be significantly (P < 0.001) increased (approximately 50%) at 1.0 mg group only. In contrast to this, thickness of the decidual layer was substantially [F = (3, 28)124.46, P < 0.001] reduced (approximately 43% to 50%) in the 0.8
Figure 1 Effects of prenatal exposure to RIS on placental size and haemorrhages on ventral surface. Note that the intense haemorrhages on the surface of RIS-treated representative placenta of each groups.
Figure 2 Effect of prenatal exposure to RIS on placental thickness of different layers. All data represent Mean SEM. n = 8 per group. # indicates level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s test.
and 1.0 mg RIS-treated groups. In summary, the thickness of the basal and labyrinthine zone was substantially increased in the 0.8 and 1.0 mg dose groups, whereas thickness of these two layers was significantly decreased in the 2.0 mg dose group. The major impact of the drug, RIS, was seen in the labyrinthine zone.
Effect of prenatal exposure to RIS on histopathological changes and cytoarchitectural pattern of placentas In the deciduas basalis zone, no substantial changes were observed except increased fibrillation in RIS-treated groups as compared to controls (Figure 3). In the basal zone, the number of large giant cells and glycogen-rich cells were decreased in all the RIS-treated
Table 1 Effect of prenatal exposure to RIS on placental weight and diameter Indices
Control
0.8 mg RIS
1.0 mg RIS
2.0 mg RIS
Placental weight (g) Placental Diameter (cm)
0.35 0.02 1.35 0.02
0.40 0.02 ( 15.80%) 1.46# 0.02 ( 8.15%)
0.57#0.03 ( 64.36%) 1.57# 0.02 ( 16.29%)
0.16# 0.01 (54.02%) 1.17# 0.02 (13.33%)
All data represent Mean SEM, n = 15 per group. # Level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s multiple comparison test. Data in parenthesis represent percentage of reduction and increase with symbol ‘ ’ in RIS-treated groups in comparison with control.
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Figure 3 Thickness of deciduas basalis (DB), basal zone (BZ) and labyrinthine zone (LZ) in (a), control and RIS-exposed (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) at middle region of placenta. (10 9 magnification).
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Figure 4 Cellular organization of basal zone in control (a) and RIS treated placentas, (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) respectively. Large giant cells are marked as ( ) and glycogen cells are marked as ( ) and vacuoles are designated as star ( ). (40 9 magnification).
placentas. These cells were reduced substantially in the 2.0 mg dose group as compared to control and other drugtreated groups (Figure 4). The nuclei of the spongiotrophoblast and large giant cells were also found to be irregular in shape in the 1.0 and 2.0 mg dose groups when compared to control. In the labyrinthine zone, the trophoblastic septa of the placenta were drastically reduced and disrupted with loss of typical architecture in RIS-treated groups, while these trophoblastic septas were well developed and organized in control placentas (Figure 5). At some places, these septa were completely intermingled with the labyrinthine matrix in the 2.0 mg dose group. The trichorial membrane was
Figure 5 Cellular organization of labyrinthine zone (LZ) in control (a) and RIS-treated placentas, (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) respectively. Arrow ( ) represents rupturing sites of septas and star ( ) indicates vacuolization in placental matrix. Complete rupture of septas may be seen at 2.0 mg RIStreated placenta (d) (40 9 magnification).
disrupted at several places in a dose-dependent manner, thus facilitating the mixing of foetal and maternal blood in RIStreated placentas in comparison with control placentas. In the labyrinthine zone, the branching of blood vessels was decreased and maximum reduction was seen in the 2.0 mg dose group compared to controls (Figure 6). The maternal sinusoids were irregularly dilated with haemorrhage at several places. In this group, blood vessels were travelled a long distance into the labyrinthine zone without being branched and forming capillaries (exchange vessels). Thus, decreased and abnormal vessel formation in the labyrinthine zone of treated groups increase easy passage of the drug. Also, the blood vessels at the maternal interface in all the treated groups were not well branched, and haemorrhages were observed.
Effect of prenatal exposure to RIS on reproductive toxicity and congenital anomalies In this study, none of the foetuses were found dead or resorbed in the uteri of RIS-exposed dams. All foetuses were alive. The drug-induced effect on litter size was not significantly [F = (3, 20) 1.91, P > 0.05] reduced after one-way ANOVA. No external gross malformations were recorded in the drug-exposed groups.
Effect of prenatal exposure to RIS on foetal development and growth Table 2 exhibited significant dose-dependent reduction of foetal body size [F = (3, 36) 25.6, P < 0.001] and weight [F = (3, 36) 158.9, P < 0.001], respectively, in RIS-exposed foetuses, when one-way ANOVA was applied. Post hoc analysis of the data also displayed dose-dependent substantial International Journal of Experimental Pathology, 2016, 97, 125–132
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Figure 6 Typical organizations of blood capillaries at maternalfetal interface (a). Disruption of typical maternal-fetal branching and capillaries may be seen in RIS-treated placentas (b, c, and d respectively) (10X magnification).
Table 2 Effect of prenatal exposure to RIS on foetal body size and weight RIS doses
Body size (cm)
Control (vehicle) 0.8 mg/kg 1.0 mg/kg 2.0 mg/kg
3.62 3.43# 3.15# 2.99#
0.029 0.054 (5.24) 0.073 (12.98) 0.059 (17.40)
Body weight (g) 3.91 3.4 2.71# 2.62#
0.070 0.039 (13.04) 0.049 (30.76) 0.019 (32.82)
All data represent Mean SEM (n = 10 per group). # Level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s multiple comparison test. Data in parenthesis represent percentage of reduction (size and weight) in RIS-treated groups than control group.
(P < 0.001) deficit in foetal body size and weight at selected doses of RIS except foetal weight at 0.8 mg dose group. The percentage reduction of foetal size (5.24%, 12.98% and 17.40%) and weight (13.04%, 30.76% and 32.82%) was also found in dose-dependent manner at selected doses (0.8, 1.0 and 2 mg) of RIS. Thus, selected doses of RIS could induce foetal intrauterine growth restriction.
Discussion The current study revealed that in utero exposure to equivalent therapeutic doses of RIS during organogenesis-induced placental dystrophy (size and weight), disturbed cytoarchitectural organization (thickness of different placental layers), histopathological lesions (necrosis in trophoblast with disruption of trophoblastic septa and rupturing of maternalfoetal interface) and intrauterine growth restriction of foetuses (size and weight). No substantial alterations were recorded for foetal resorption, litter size, mortality and congenital anomalies. Our results are in agreement with those International Journal of Experimental Pathology, 2016, 97, 125–132
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investigators who have used different xenobiotics at different time points during pregnancy in animals and found placental toxicity and foetal toxicity, and established a correlation between placental development and growth of developing foetuses agents if they are (Ebaid 2013; Furukawa et al. 2013, 2011a,b; Raha et al. 2012; Kosif et al. 2008; Mishra & Singh 2008). The normal development of placenta starts rapidly after implantation into the uterus, through primary and secondary trophoblastic giant cells (Hafez & Tsutsumi 1966). These cells (progenitor cells) further divide and forms deciduas as the maternal part, and the basal and labyrinthine zones as foetal part respectively. Thus, the placenta is a susceptible target organ for xenobiotic agents if they are administered during early or preconception period of pregnancy. Maternal exposure to drugs or chemicals may induce placental injuries and subsequently lead to placental dystrophy, reproductive toxicity, impaired foetal growth and development as well as congenital malformations depending upon the drug doses and exposure period (Raha et al. 2012; Furukawa et al. 2011a,b). Although placental toxicity of some classical CNS acting drugs has been reported, literature, both clinical and preclinical, is almost silent on atypical anti-psychotic drugs (AAPDs) on this issue. The only report is one review article (Raha et al. 2012). Hence, this laboratory is a pioneer in reporting placental and foetal toxicity and their correlation when AAPDs like RIS was administered to pregnant rats. In our study, exposure to RIS at lower doses (0.8 and 1.0 mg/kg) induced placental hypertrophy as shown by increased placental weight which is a bio-indicator of altered foetal growth. This hypertrophy of placentas may occur due to the slightly unfavourable maternal environment such as hypoxia (Akay & Kockaya 2005; Eguchi et al. 1989; Lynch & Bruce 1989; Bruce 1976; Bruce & Cabral 1975) and poor transfer of glucose to developing foetuses through a maternal–placental–foetal axis (Lewis et al. 1997), decrease numbers of corpora lutea, implantation sites and numbers of implantation (generally less than six foetuses in the uterus) (Csapo & Wiest 1973) as well as hormonal imbalance such as oestrogen deficiency (Bartholomeusz et al. 1999; Csapo et al. 1974). Bruce and Cabral (1975) revealed that oestrogen and progesterone are essential for initiation and maintenance of pregnancy and closely associated to placental growth and development in rats. It is well documented that oestrogen is a known inhibitor of placental growth, and its mild deficiency induces placental hypertrophy (Ichikawa et al. 2006; Bartholomeusz et al. 1999; Csapo et al. 1974), whereas excess elevation of oestrogen inhibits the development of the placenta (Furukawa et al. 2013). Thus, placental hypertrophy may be a compensatory disposition against drug (RIS)-induced effects. In contrast to this, administration of the higher dose (2.0 mg) of RIS substantially decreased placental weight. This reverse macropathological observation of placentas may be associated with mitotic inhibition, massive apoptosis and/or necrosis of trophoblasts and excessive unfavourable
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maternal environment, resulted in the inhibition of placental development which leads to a small placenta (Akay & Kockaya 2005; Khera 1992; Graf et al. 1989; Padmanabhan et al. 1988; Singh & Padmanabhan 1980). Macroscopically, placental necrosis is associated with thinning, discoloration, haemorrhage, white spots, thick peripheral rim and white spots by maternal exposure to different xenobiotic agents (Akay & Kockaya 2005; Fuentes et al. 1996; Graf et al. 1989; Di Sant’Agnese et al. 1983) including some classical CNS acting drugs like valproic acid (Khera 1992) and chlorpromazine (Singh & Padmanabhan 1980). Topiramate, a newer anti-epileptic drug, could not induce small placentas when equivalent therapeutic doses were administered maternally from gestation day 9–12 (Mishra & Singh 2008). Although in this study the exposure period to the drug was limited to 4 days, this could be a reason for non-observable effects on placental weight. Overall, the results and potential inducing mechanisms for placental dystrophy are inconsistent, especially for psychotropic drugs in general and AAPDs (RIS) in particular. Histopathologically, total thickness and differential thickness of the three typical layers of placenta were increased substantially at lower doses of RIS (0.8 and 1.0 mg), respectively, whereas the same was decreased at higher dose (2.0 mg). This pattern is associated with placental weight gain and loss, respectively, with these doses. The drug impact was more discernible in the basal and labyrinthine zones. In the labyrinthine zone, trophoblastic septa, which act as a major component of the placental barrier, were drastically ruptured, thus facilitating increase in the porosity of bio-membrane due to loss of syncytiotrophoblasts (arranged in two layers) and cytotrophoblasts. Thus, maternal blood containing the drug molecules passes easily into foetal capillaries through dilated maternal sinusoids in the absence of a strong placental barrier. In this zone, capillary networking between maternal–foetal interfaces was also destroyed due to drug toxicity especially at higher dose (2.0 mg/kg). Hence, the trophoblast synthesis affects the placental growth and development adversely. Similarly, cellular organization of the basal zone (a junction zone) was also disrupted due to necrosis of spongiotrophoblasts, trophoblastic giant cells and glycogen-rich cells. The trophoblastic giant cells are one of the major endocrine cells of the placenta; they synthesize and secrete hormones/cytokines. It may be speculated that RIS could induced placental hormonal imbalance, irrespective of the drug doses. In our study, glycogen-rich cells were also found to be reduced in RIS-exposed groups. The degeneration of glycogen-rich cells is reflected in abnormal retention of extensive cytoplasmic vacuolation within glycogen-rich cells. In animal experiments, degeneration of glycogen-rich cells was also reported due to exposure of xenobiotic agents such as chlorpromazine (Singh & Padmanabhan 1980), streptozotocine (Padmanabhan et al. 1988) and 6-mercaptopurine (Furukawa et al. 2008). Placental apoptosis may be another potent inducing mechanism which operates through increased oxidative stress pathway, placental damage and
subsequently foetal growth restriction as evidenced in other AAPDs such as clozapine, olanzapine and aripiprazole (Baig et al. 2010; Martins et al. 2008; Zhang et al. 2006) in different regions of the brain. It was observed that aripiprazole (ARI) increased mitochondrial superoxide formation known to cause both mitochondrial and cytosolic oxidative damage (Raha et al., 2002 and Raha & Robinson 2000) followed by enhanced state of apoptosis (Fehsel et al. 2005). The majority of the evidence also suggests that AAPDs are associated with mitochondrial dysfunction mediating through electron transport chain preferably on complex I and V activity (Ji et al. 2009; Raha & Robinson 2003; Raha & Robinson 2000). Casanueva and Viteri (2003) proposed that some of the action of the AAPDs may occur via mitochondrial generated oxidative stress in the placenta especially, because the placenta is characterized by high levels of mitochondria. Further, increased oxidative stress along with mitochondrial dysfunction in the placenta could be linked to placental dystrophy, increased necrosis and apoptosis in trophoblastic cells, and foetal growth restriction and premature foetal loss (Gundogan et al. 2010). Myatt (2002) elaborated that oxidative stress may impact the ability of trophoblasts to transport nutrients between maternal and foetal circulation. Hence, dysregulation of placental hormones (synthesis and release) may adversely affect the placental growth and development. Similar to oxidative stress, a more convincing mechanism of action for AAPDs, olanzapine induced endoplasmic reticulum (ER) stress in in vitro studies (Lian et al. 2011; Kurosawa et al. 2007). Mechanistically, such combined cellular mitochondrial and endoplasmic reticular stress may influence the placental development and foetal growth in the in vitro model has not yet been established. The other possible and potential inducing mechanisms for AAPDs in placentas and foetuses may be associated with rate of placental passage of drug during gestation. Gentile (2010) reviewed the placental passage of both first- and second-generation anti-psychotics (AAPDs) and revealed that classical drugs like haloperidol have higher rate of placental passage (65%), hence are associated with enhanced teratogenicity and reproductive toxicity than quetiapine (AAPD) which has low placental passage (23.8%); therefore, low incidence of birth defects and less IUGR is reported. The placental passage of the other AAPDs like RIS ranged between them (49.20%), hence causes low incidence of complicated pregnancy outcomes and IUGR in both clinical and experimental trials (Newport et al. 2007). From this study, it may be concluded that multifactorial mechanisms could be involved in the dysregulation of structure and function of the placenta and of poor foetal growth and development in association with in utero exposure to AAPDs like RIS during critical period of organogenesis. Furthermore, experimental and clinical studies are urgently warranted to establish a relationship between other AAPDs in general and third-generation AAPDs like aripiprazole in particular with regard to placental development, in the interest of the disease affected population. Recently, FDA has approved some newer anti-psychotics such as paliperidone, International Journal of Experimental Pathology, 2016, 97, 125–132
Risperidone induced placental impairments iloperidone and aripiprazole which should also be evaluated in future studies.
Acknowledgement The authors would like to thank the Head, Department of Zoology, University of Allahabad for providing laboratory facilities. Financial supports from U.G.C., New Delhi, India, vide Project no. 37-394/2009 (SR) to KPS (PI), and MKS is thankfully acknowledged.
Conflict of interest There is no conflict of interest between authors and funding agency.
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Furukawa S., Hayashi S., Hayashi S. et al. (2011a) The relationship between fetal growth restriction and small placenta in 6-mercaptopurine exposed rat. Exp. Toxicol. Pathol. 63, 89–95. Furukawa S., Hayashi S., Usuda K. et al. (2011b) Toxicological pathology in the rat placenta. J. Toxicol. Pathol. 24, 95–111. Furukawa S., Hayashi S., Usuda K. et al. (2013) Effect of cisplatin on rat placenta development. Exp. Toxicol. Pathol. 65, 211–7. Galbaly M., Snellen M. & Power J. (2014) Antipsychotic drugs in pregnancy: a review of their maternal and fetal effects. Ther. Adv. Drug Saf. 5, 100–109. Graf R., Gossrau R. & Frank H.G. (1989) Placental toxicity in rats after administration of synthetic glucocorticoids. a morphological, histochemical and immunohistochemical investigation. Anat. Embryol. 180, 121–130. Gundogan F., Elwood G. & Mark P. (2010) Ethanol-induced oxidative stress and mitochondrial dysfunction in rat placenta: relevance to pregnancy loss. Alcohol. Clin. Exp. Res. 34, 415–423. Habermann F., Fritzsche J., Fuhlr€ uck F. et al. (2013) Atypical antipsychotic drugs and pregnancy outcome: a prospective, cohort study. J. Clin. Psychopharmacol. 33(4), 453–62. Hafez E.S.E. & Tsutsumi Y. (1966) Changes in endometrial vascularity during implantation and pregnancy in the rabbit. Am. J. Anat. 118, 249–282. Harding J. & Timko J. (2008) The use of psychotropic medications during pregnancy and lactation. Glob. Libr. Women’s Med. DOI 10.3843/GLOWM.10416 Ichikawa A., Seki N., Sugimoto T. et al. (2006) Effects of ketoconazole on placental weights in rats. Con Anom. 46, A23. Ji B., La Y. & Gao L. (2009) A comparative proteomic analysis of rat mitochondria from the cerebral cortex and hippocampus in response to antipsychotic medications. J. Proteome Res. 8, 3633–3641. Kapur S., VanderSpek S.C., Brownlee B.A. et al. (2003) Antipsychotic dosing in preclinical models is often unrepresentative of the clinical condition: a suggested solution based on in vivo occupancy. J. Pharmacol. Exp. Ther. 305, 625–631. Khera K.S. (1992) Valproic acid-induced placental and teratogenic effects in rats. Teratology. 45, 603–610. Kosif R., Akta G. & Oztekin A. (2008) Microscopic examination of placenta of rats prenatally exposed to Aloe barbadensis: a preliminary study. Int. J. Morphol. 26(2), 275–281. Kurosawa S., Hashimoto E., Ukai W. et al. (2007) Olanzapine potentiates neuronal survival and neural stem cell differentiation: regulation of endoplasmic reticulum stress response proteins. J. Neural. Transm. 114, 1121–1128. Lewis R.M., Batchelor D.C., Bassett N.S. et al. (1997) Perinatal growth disturbance in the spontaneously hypertensive rat. Pediatr. Res. 42, 758–764. Lian I.A., Loset M. & Mundal S.B. (2011) Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without preeclampsia. Placenta 32, 823–829. Lynch A.M. & Bruce N.W. (1989) Placental growth in rats exposed to carbon monoxide at selected stages of pregnancy. Biol. Neonate 56, 151–157. Martins M.R., Petronilho F.C., Gomes K.M. et al. (2008) Antipsychotic-induced oxidative stress in rat brain. Neurotox. Res. 13, 63–69. Mishra A. & Singh M. (2008) Topiramate induced histopathological changes in placenta of rats. Indian J. Exp. Biol. 46, 715–719. Murphy V.E., Smith R., Giles W.B. et al. (2006) Endocrine regulation of human fetal growth: the role of mother, placenta and fetus. Endocr. Rev. 27, 141–169.
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Myatt L. (2002) Role of placenta in preeclampsia. Endocrine 19, 103–111. Newport D.J., Calamaras M.R., DeVane C.L. et al. (2007) Atypical antipsychotic administration during late pregnancy: placental passage and obstetrical outcomes. Am. J. Psychiatry 164(8), 1214–1220. Padmanabhan R., Al-Zuhair A.G.H. & Aida Hussein A.L.I. (1988) Histopathological changes of the placenta in diabetes induced by maternal administration of streptozotocin during pregnancy in the rat. Con Anom. 28, 1–15. Paschetta E., Rosso G. & Maina G. (2014) Atypical antipsychotics, maternal and child outcomes: a critical review. Women’s Health. 2(1), 2. Prouillac C. & Lecoeur S. (2010) The role of placenta in fetal exposure to xenobiotics: importance of membrane transports and human models for transfer studies. Drug Metab. Dispos. 38, 1623–1635. Raha S., Myint A.T., Johnstone L. et al. (2002) Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase. Free Radical Biology and Medicine 32, 421–430. Raha S. & Robinson B.H. (2000) Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem. Sci. 25, 502–508.
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ORIGINAL ARTICLE
Effect of in utero exposure to the atypical anti-psychotic risperidone on histopathological features of the rat placenta K.P. Singh, Manoj K. Singh and Shrikant Gautam Neurobiology Lab., Department of Zoology, University of Allahabad, Allahabad, India
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY
SUMMARY
For clinical management of different forms of psychosis, both classical and atypical anti-psychotic drugs (APDs) are available. These drugs are widely prescribed, even during pregnancy considering their minimal extra-pyramidal side effects and teratogenic potential compared to classical APDs. Among AAPDs, risperidone (RIS) is a first-line drug of choice by physicians. The molecular weight of RIS is 410.49 g/mol; hence, it can easily cross the placental barrier and enter the foetal bloodstream. It is doi: 10.1111/iep.12176 not known whether or not AAPDs like RIS may affect the developing placenta and foetus adversely. Reports on this issue are limited and sketchy. Therefore, this study has evaluated the effects of maternal exposure to equivalent therapeutic doses of RIS on placental growth, histopathological and cytoarchitectural changes, and to establish a relationship between placental dysfunction and foetal outcomes. Pregnant rats (n = 24) were exposed to selected doses (0.8, 1.0 and 2.0 mg/kg) of RIS from gestation days 6–21. These dams were sacrificed; their placentas and foetuses were collected, morphometrically examined and further processed for histopathological Received for publication: 22 July 2015 examination. This study revealed that in utero exposure to equivalent therapeutic Accepted for publication: 31 January doses of RIS during organogenesis-induced placental dystrophy (size and weight), 2016 disturbed cytoarchitectural organization (thickness of different placental layers), Correspondence: histopathological lesions (necrosis in trophoblast with disruption of trophoblastic Krishna P. Singh septa and rupturing of maternal-foetal interface) and intrauterine growth restriction Neurobiology Lab of the foetuses. It may be concluded that multifactorial mechanisms might be Department of Zoology University of Allahabad involved in the dysregulation of structure and function of the placenta and of poor Allahabad foetal growth and development. India Tel./Fax: 91-0532-2640188 E-mails: [email protected]; [email protected]
Keywords atypical antipsychotics, histopathology, placenta, prenatal, risperidone
The placenta is an interface between dam and developing foetus. It also secures the embryo as a protective barrier against xenobiotic agents if administered during pre- and peri-conception. Although it is a temporary organ, its proper implantation on uterus and further growth plays an important role for development and growth of the embryo/foetus (Prouillac & Lecoeur 2010; Murphy et al. 2006). It is well documented that any kind of placental dysfunction, during early or late gestation, may adversely affect normal embryo-foetal development and growth (Furukawa et al. 2011a,b). Xenobiotics may not easily cross the placental barrier if the molecular weight of the agent is above 500 g/mol, but certain drugs/chemicals,
including drugs acting on the central nervous system (CNS) have the ability to cross the placental barrier easily as they are (on average) lower in molecular weight (400–600 g/mol) in humans (Newport et al. 2007). These drugs are also prevented from filtering through passive diffusion by the dense and complex network of blood capillaries. Hitherto it is not known whether or not molecules of psychotropic drugs, including anti-psychotics, affect the placental capillary network adversely at therapeutic dose range. Reports on this issue are limited and sketchy, and mostly cited at higher concentration of the drugs, causing developmental toxicity in the maternal environment as well as histopathological changes in the
© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology
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placenta. Searching both clinical and non-clinical literature revealed that the documentation about in utero exposure to atypical novel anti-psychotic drugs, and about their impact on histopathological impairments on the placenta, is negligible. For clinical management of different forms of psychosis, including schizophrenia, both classical (1st generation) and atypical/novel (2nd generation) anti-psychotic drugs are available on the world market. Atypical anti-psychotic drugs (AAPDs) are widely prescribed to adult populations (Galbaly et al. 2014; Stephenson et al. 2013; Toh et al. 2013), even to women of child-bearing age in view of their moderate side effects and lower teratogenic potential compared to classical APDs, which are associated with movement disorders (Costa et al. 2004). Among AAPDs, risperidone (RIS) is a first-line drug of choice by healthcare providers even during pregnancy (Paschetta et al. 2014; Sadowski et al. 2013). The molecular weight of this drug is 410.49 g/mol; hence, it can easily cross the placental barrier and enter the foetal circulation. The maternal transfer of the drug into foetuses takes place when both placenta and foetus are at developmental stage. Thus it is possible that prenatal exposure to RIS adversely affects normal development and growth of the placenta. Hence, placental impairment may be a bio-indicator of drug-induced effects on both the placenta and the developing foetus. Although several inducing mechanisms have been proposed by previous investigators (Prouillac & Lecoeur 2010; Newport et al. 2007), a mechanism to explain how a AAPDs such as RIS, induce placental dystrophy, either at a gross or a microscopic level. Therefore, the present study has been designed in order to evaluate how maternal exposure to equivalent therapeutic doses of RIS affects placental growth (size and weight) and histopathology and to establish a relationship between placental dysfunction and foetal outcomes under these conditions.
experimental doses were calibrated as 0.8 mg/kg (MHRD 9 4), 1.0 mg/kg (MHRD 9 6) and 2.0 mg/kg (MHRD 9 10) on the basis of per kg body weight per day, and their suitability to the rat animal model. The rationale for the selection of three doses of RIS was as per MRHD corrected for the higher metabolic rate of rats (i.e. 4–6 times faster than in humans) (Kapur et al. 2003). Four groups of pregnant female rats (n = 6) in each group were maintained. All the control and experimental rats were exposed from gestational day 6–21 (GD 6-21), either to the drug or to the vehicle alone. In this study, tablets of RIS (each tablet contains 2.0 mg of the drug molecule) were dissolved in 0.1N HCl solution. The selected doses of RIS (0.8, 1.0 and 2.0 mg) were gavaged (approximately 0.84–2.1 ml.) to sperm positive dams once daily (at 09.00 h) from GD 6-21 via a cannula. According to the protocol, RIS-treated rats of each experiment were sacrificed under phenobarbital anaesthesia on GD 21 (at 06.00 h), and foetuses along with their placenta were collected from both the uterine horns through uterectomy after transcardial perfusion with 10% formaldehyde. Placentas from all groups were cleaned, blotted dry, and then, size (longest diameter) was measured with the help of vernier callipers. The weight of each placenta was also recorded. One placenta from each uterine horn was randomly selected from each group and was cut into two equal parts and fixed in the same fixative for one week before processing for microscopic examination (paraffin microtomy). Transverse section(s) of each placenta were cut at 7l, and sections of highest diameter were selected for H&E staining to observe histopathological changes and to measure thickness of different layers with the help of capture pro software. All sections provided a transverse view of the medial region of the placenta, and eight sections of each placenta were examined, and selected measures recorded (Kosif et al. 2008). Foetal body size of each individual of four groups was measured from the anteriormost part of the snout to the tip of the tail using a prefixed length measuring board, and the weight was recorded.
Materials and methods In this study, laboratory inbred female Wistar rats from our breeding colony, weighing 180 10 g, aged about 3 months, were used for the experimental procedures. Animals were housed in plastic cages with rice bran as bedding material at standard laboratory environment (24 2 °C, 12/12 h light/dark cycle and 60% RH). Standard rat food and tap water were made available ad libitum. Animals were maintained and used in accordance with the animal welfare act. After acclimatization, nulliparous female rats were allowed to mate with males overnight (ratio 2:1), and the next morning, presence of sperm in vaginal smears was designated gestation day zero (GD-0) for determining the onset of gestation. Such sperm positive rats were used further for the experimental procedures. RIS was purchased from the pharmaceutical market with trade name Risperdal (Torrent, India). The maximum recommended human dose (MRHD) of RIS is 04–12 mg/day. The
Statistical analysis All data are represented as mean and standard errors (meanSEM). Variables such as placental size and weight, thickness of placental layers, litter size, foetal body size and weight were statistically analysed using one-way ANOVA followed by post hoc Tukey’s multiple comparison test to determine difference amongst exposed groups and control. For all statistical values, minimum significance level was set at P < 0.05. All calculations were carried out with the help of Microsoft Excel (Microsoft Redmond Campus, Redmond, Washington, USA) and Statistica-10 software (Dell Statistica, Tulsa, OK, USA).
Ethical approval The present study was approved by the Institutional Animal Ethics Committee (IAEC), University of Allahabad, Allahabad, India. International Journal of Experimental Pathology, 2016, 97, 125–132
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Results Effect of prenatal exposure to RIS on placental size and weight One-way ANOVA followed by Tukey’s multiple comparison tests showed significant increase in placental size [F = (3, 56) 68.00, P < 0.001] and weight [F = (3, 56) 54.70, P < 0.001] in RIS-treated groups. In post hoc analysis, placental size was substantially (P < 0.001) increased (8.15% and 16.29%) at 0.8 mg and 1.0 mg dose groups, respectively, whereas it was significantly (P < 0.001) decreased (13.33%) at 2.0 mg/kg dose group as compared to control. Similarly, placental weight was significantly (P < 0.001) increased at 0.8 mg (15.80%) and 1.0 mg (64.36%) RIS dose groups, respectively, whereas the same was substantially (P < 0.001) decreased (54.02%) at 2.0 mg/kg drugexposed group in comparison with vehicle-treated group (Table 1, Figure 1). It appears that the drug effect was more intense for placental weight than size. Haemorrhages were found to be more severe on the ventral surface of each placenta in in utero exposed RIS in comparison with those of control placentas (Figure 1).
Effect of prenatal exposure to RIS on thickness of placental layers Statistical analysis (one-way ANOVA) displayed significant [F = (3, 28) 175.60, P < 0.001] alteration in total thickness of three layers (deciduas basalis, basal zone and labyrinthine zone) of RIS-treated placentas. The total thickness of three typical layers was increased (approximately 13% to 21.47%) substantially (P < 0.001) at 0.8 and 1.0 mg dose groups, respectively, while this measure was reduced (15.24%) substantially (P < 0.001) at 2.0 mg dose group in comparison with control (Figure 2). One-way ANOVA followed by post hoc Tukey’s test expressed that placental thickness of labyrinthine zone [F = (3, 28) 124.75, P < 0.001] was found to be significantly (P < 0.001) increased (approximately 24%) at 0.8 and 1.0 mg dose groups, and thickness of this zone was substantially (P < 0.001) reduced (approximately 18%) at 2.0 mg dose. In the basal zone, placental thickness was found to be significantly (P < 0.001) increased (approximately 50%) at 1.0 mg group only. In contrast to this, thickness of the decidual layer was substantially [F = (3, 28)124.46, P < 0.001] reduced (approximately 43% to 50%) in the 0.8
Figure 1 Effects of prenatal exposure to RIS on placental size and haemorrhages on ventral surface. Note that the intense haemorrhages on the surface of RIS-treated representative placenta of each groups.
Figure 2 Effect of prenatal exposure to RIS on placental thickness of different layers. All data represent Mean SEM. n = 8 per group. # indicates level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s test.
and 1.0 mg RIS-treated groups. In summary, the thickness of the basal and labyrinthine zone was substantially increased in the 0.8 and 1.0 mg dose groups, whereas thickness of these two layers was significantly decreased in the 2.0 mg dose group. The major impact of the drug, RIS, was seen in the labyrinthine zone.
Effect of prenatal exposure to RIS on histopathological changes and cytoarchitectural pattern of placentas In the deciduas basalis zone, no substantial changes were observed except increased fibrillation in RIS-treated groups as compared to controls (Figure 3). In the basal zone, the number of large giant cells and glycogen-rich cells were decreased in all the RIS-treated
Table 1 Effect of prenatal exposure to RIS on placental weight and diameter Indices
Control
0.8 mg RIS
1.0 mg RIS
2.0 mg RIS
Placental weight (g) Placental Diameter (cm)
0.35 0.02 1.35 0.02
0.40 0.02 ( 15.80%) 1.46# 0.02 ( 8.15%)
0.57#0.03 ( 64.36%) 1.57# 0.02 ( 16.29%)
0.16# 0.01 (54.02%) 1.17# 0.02 (13.33%)
All data represent Mean SEM, n = 15 per group. # Level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s multiple comparison test. Data in parenthesis represent percentage of reduction and increase with symbol ‘ ’ in RIS-treated groups in comparison with control.
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Figure 3 Thickness of deciduas basalis (DB), basal zone (BZ) and labyrinthine zone (LZ) in (a), control and RIS-exposed (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) at middle region of placenta. (10 9 magnification).
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Figure 4 Cellular organization of basal zone in control (a) and RIS treated placentas, (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) respectively. Large giant cells are marked as ( ) and glycogen cells are marked as ( ) and vacuoles are designated as star ( ). (40 9 magnification).
placentas. These cells were reduced substantially in the 2.0 mg dose group as compared to control and other drugtreated groups (Figure 4). The nuclei of the spongiotrophoblast and large giant cells were also found to be irregular in shape in the 1.0 and 2.0 mg dose groups when compared to control. In the labyrinthine zone, the trophoblastic septa of the placenta were drastically reduced and disrupted with loss of typical architecture in RIS-treated groups, while these trophoblastic septas were well developed and organized in control placentas (Figure 5). At some places, these septa were completely intermingled with the labyrinthine matrix in the 2.0 mg dose group. The trichorial membrane was
Figure 5 Cellular organization of labyrinthine zone (LZ) in control (a) and RIS-treated placentas, (b) (0.8 mg), (c) (1.0 mg) and (d) (2.0 mg) respectively. Arrow ( ) represents rupturing sites of septas and star ( ) indicates vacuolization in placental matrix. Complete rupture of septas may be seen at 2.0 mg RIStreated placenta (d) (40 9 magnification).
disrupted at several places in a dose-dependent manner, thus facilitating the mixing of foetal and maternal blood in RIStreated placentas in comparison with control placentas. In the labyrinthine zone, the branching of blood vessels was decreased and maximum reduction was seen in the 2.0 mg dose group compared to controls (Figure 6). The maternal sinusoids were irregularly dilated with haemorrhage at several places. In this group, blood vessels were travelled a long distance into the labyrinthine zone without being branched and forming capillaries (exchange vessels). Thus, decreased and abnormal vessel formation in the labyrinthine zone of treated groups increase easy passage of the drug. Also, the blood vessels at the maternal interface in all the treated groups were not well branched, and haemorrhages were observed.
Effect of prenatal exposure to RIS on reproductive toxicity and congenital anomalies In this study, none of the foetuses were found dead or resorbed in the uteri of RIS-exposed dams. All foetuses were alive. The drug-induced effect on litter size was not significantly [F = (3, 20) 1.91, P > 0.05] reduced after one-way ANOVA. No external gross malformations were recorded in the drug-exposed groups.
Effect of prenatal exposure to RIS on foetal development and growth Table 2 exhibited significant dose-dependent reduction of foetal body size [F = (3, 36) 25.6, P < 0.001] and weight [F = (3, 36) 158.9, P < 0.001], respectively, in RIS-exposed foetuses, when one-way ANOVA was applied. Post hoc analysis of the data also displayed dose-dependent substantial International Journal of Experimental Pathology, 2016, 97, 125–132
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Figure 6 Typical organizations of blood capillaries at maternalfetal interface (a). Disruption of typical maternal-fetal branching and capillaries may be seen in RIS-treated placentas (b, c, and d respectively) (10X magnification).
Table 2 Effect of prenatal exposure to RIS on foetal body size and weight RIS doses
Body size (cm)
Control (vehicle) 0.8 mg/kg 1.0 mg/kg 2.0 mg/kg
3.62 3.43# 3.15# 2.99#
0.029 0.054 (5.24) 0.073 (12.98) 0.059 (17.40)
Body weight (g) 3.91 3.4 2.71# 2.62#
0.070 0.039 (13.04) 0.049 (30.76) 0.019 (32.82)
All data represent Mean SEM (n = 10 per group). # Level of significance at P < 0.001 between control and exposed groups for one-way ANOVA followed by Tukey’s multiple comparison test. Data in parenthesis represent percentage of reduction (size and weight) in RIS-treated groups than control group.
(P < 0.001) deficit in foetal body size and weight at selected doses of RIS except foetal weight at 0.8 mg dose group. The percentage reduction of foetal size (5.24%, 12.98% and 17.40%) and weight (13.04%, 30.76% and 32.82%) was also found in dose-dependent manner at selected doses (0.8, 1.0 and 2 mg) of RIS. Thus, selected doses of RIS could induce foetal intrauterine growth restriction.
Discussion The current study revealed that in utero exposure to equivalent therapeutic doses of RIS during organogenesis-induced placental dystrophy (size and weight), disturbed cytoarchitectural organization (thickness of different placental layers), histopathological lesions (necrosis in trophoblast with disruption of trophoblastic septa and rupturing of maternalfoetal interface) and intrauterine growth restriction of foetuses (size and weight). No substantial alterations were recorded for foetal resorption, litter size, mortality and congenital anomalies. Our results are in agreement with those International Journal of Experimental Pathology, 2016, 97, 125–132
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investigators who have used different xenobiotics at different time points during pregnancy in animals and found placental toxicity and foetal toxicity, and established a correlation between placental development and growth of developing foetuses agents if they are (Ebaid 2013; Furukawa et al. 2013, 2011a,b; Raha et al. 2012; Kosif et al. 2008; Mishra & Singh 2008). The normal development of placenta starts rapidly after implantation into the uterus, through primary and secondary trophoblastic giant cells (Hafez & Tsutsumi 1966). These cells (progenitor cells) further divide and forms deciduas as the maternal part, and the basal and labyrinthine zones as foetal part respectively. Thus, the placenta is a susceptible target organ for xenobiotic agents if they are administered during early or preconception period of pregnancy. Maternal exposure to drugs or chemicals may induce placental injuries and subsequently lead to placental dystrophy, reproductive toxicity, impaired foetal growth and development as well as congenital malformations depending upon the drug doses and exposure period (Raha et al. 2012; Furukawa et al. 2011a,b). Although placental toxicity of some classical CNS acting drugs has been reported, literature, both clinical and preclinical, is almost silent on atypical anti-psychotic drugs (AAPDs) on this issue. The only report is one review article (Raha et al. 2012). Hence, this laboratory is a pioneer in reporting placental and foetal toxicity and their correlation when AAPDs like RIS was administered to pregnant rats. In our study, exposure to RIS at lower doses (0.8 and 1.0 mg/kg) induced placental hypertrophy as shown by increased placental weight which is a bio-indicator of altered foetal growth. This hypertrophy of placentas may occur due to the slightly unfavourable maternal environment such as hypoxia (Akay & Kockaya 2005; Eguchi et al. 1989; Lynch & Bruce 1989; Bruce 1976; Bruce & Cabral 1975) and poor transfer of glucose to developing foetuses through a maternal–placental–foetal axis (Lewis et al. 1997), decrease numbers of corpora lutea, implantation sites and numbers of implantation (generally less than six foetuses in the uterus) (Csapo & Wiest 1973) as well as hormonal imbalance such as oestrogen deficiency (Bartholomeusz et al. 1999; Csapo et al. 1974). Bruce and Cabral (1975) revealed that oestrogen and progesterone are essential for initiation and maintenance of pregnancy and closely associated to placental growth and development in rats. It is well documented that oestrogen is a known inhibitor of placental growth, and its mild deficiency induces placental hypertrophy (Ichikawa et al. 2006; Bartholomeusz et al. 1999; Csapo et al. 1974), whereas excess elevation of oestrogen inhibits the development of the placenta (Furukawa et al. 2013). Thus, placental hypertrophy may be a compensatory disposition against drug (RIS)-induced effects. In contrast to this, administration of the higher dose (2.0 mg) of RIS substantially decreased placental weight. This reverse macropathological observation of placentas may be associated with mitotic inhibition, massive apoptosis and/or necrosis of trophoblasts and excessive unfavourable
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maternal environment, resulted in the inhibition of placental development which leads to a small placenta (Akay & Kockaya 2005; Khera 1992; Graf et al. 1989; Padmanabhan et al. 1988; Singh & Padmanabhan 1980). Macroscopically, placental necrosis is associated with thinning, discoloration, haemorrhage, white spots, thick peripheral rim and white spots by maternal exposure to different xenobiotic agents (Akay & Kockaya 2005; Fuentes et al. 1996; Graf et al. 1989; Di Sant’Agnese et al. 1983) including some classical CNS acting drugs like valproic acid (Khera 1992) and chlorpromazine (Singh & Padmanabhan 1980). Topiramate, a newer anti-epileptic drug, could not induce small placentas when equivalent therapeutic doses were administered maternally from gestation day 9–12 (Mishra & Singh 2008). Although in this study the exposure period to the drug was limited to 4 days, this could be a reason for non-observable effects on placental weight. Overall, the results and potential inducing mechanisms for placental dystrophy are inconsistent, especially for psychotropic drugs in general and AAPDs (RIS) in particular. Histopathologically, total thickness and differential thickness of the three typical layers of placenta were increased substantially at lower doses of RIS (0.8 and 1.0 mg), respectively, whereas the same was decreased at higher dose (2.0 mg). This pattern is associated with placental weight gain and loss, respectively, with these doses. The drug impact was more discernible in the basal and labyrinthine zones. In the labyrinthine zone, trophoblastic septa, which act as a major component of the placental barrier, were drastically ruptured, thus facilitating increase in the porosity of bio-membrane due to loss of syncytiotrophoblasts (arranged in two layers) and cytotrophoblasts. Thus, maternal blood containing the drug molecules passes easily into foetal capillaries through dilated maternal sinusoids in the absence of a strong placental barrier. In this zone, capillary networking between maternal–foetal interfaces was also destroyed due to drug toxicity especially at higher dose (2.0 mg/kg). Hence, the trophoblast synthesis affects the placental growth and development adversely. Similarly, cellular organization of the basal zone (a junction zone) was also disrupted due to necrosis of spongiotrophoblasts, trophoblastic giant cells and glycogen-rich cells. The trophoblastic giant cells are one of the major endocrine cells of the placenta; they synthesize and secrete hormones/cytokines. It may be speculated that RIS could induced placental hormonal imbalance, irrespective of the drug doses. In our study, glycogen-rich cells were also found to be reduced in RIS-exposed groups. The degeneration of glycogen-rich cells is reflected in abnormal retention of extensive cytoplasmic vacuolation within glycogen-rich cells. In animal experiments, degeneration of glycogen-rich cells was also reported due to exposure of xenobiotic agents such as chlorpromazine (Singh & Padmanabhan 1980), streptozotocine (Padmanabhan et al. 1988) and 6-mercaptopurine (Furukawa et al. 2008). Placental apoptosis may be another potent inducing mechanism which operates through increased oxidative stress pathway, placental damage and
subsequently foetal growth restriction as evidenced in other AAPDs such as clozapine, olanzapine and aripiprazole (Baig et al. 2010; Martins et al. 2008; Zhang et al. 2006) in different regions of the brain. It was observed that aripiprazole (ARI) increased mitochondrial superoxide formation known to cause both mitochondrial and cytosolic oxidative damage (Raha et al., 2002 and Raha & Robinson 2000) followed by enhanced state of apoptosis (Fehsel et al. 2005). The majority of the evidence also suggests that AAPDs are associated with mitochondrial dysfunction mediating through electron transport chain preferably on complex I and V activity (Ji et al. 2009; Raha & Robinson 2003; Raha & Robinson 2000). Casanueva and Viteri (2003) proposed that some of the action of the AAPDs may occur via mitochondrial generated oxidative stress in the placenta especially, because the placenta is characterized by high levels of mitochondria. Further, increased oxidative stress along with mitochondrial dysfunction in the placenta could be linked to placental dystrophy, increased necrosis and apoptosis in trophoblastic cells, and foetal growth restriction and premature foetal loss (Gundogan et al. 2010). Myatt (2002) elaborated that oxidative stress may impact the ability of trophoblasts to transport nutrients between maternal and foetal circulation. Hence, dysregulation of placental hormones (synthesis and release) may adversely affect the placental growth and development. Similar to oxidative stress, a more convincing mechanism of action for AAPDs, olanzapine induced endoplasmic reticulum (ER) stress in in vitro studies (Lian et al. 2011; Kurosawa et al. 2007). Mechanistically, such combined cellular mitochondrial and endoplasmic reticular stress may influence the placental development and foetal growth in the in vitro model has not yet been established. The other possible and potential inducing mechanisms for AAPDs in placentas and foetuses may be associated with rate of placental passage of drug during gestation. Gentile (2010) reviewed the placental passage of both first- and second-generation anti-psychotics (AAPDs) and revealed that classical drugs like haloperidol have higher rate of placental passage (65%), hence are associated with enhanced teratogenicity and reproductive toxicity than quetiapine (AAPD) which has low placental passage (23.8%); therefore, low incidence of birth defects and less IUGR is reported. The placental passage of the other AAPDs like RIS ranged between them (49.20%), hence causes low incidence of complicated pregnancy outcomes and IUGR in both clinical and experimental trials (Newport et al. 2007). From this study, it may be concluded that multifactorial mechanisms could be involved in the dysregulation of structure and function of the placenta and of poor foetal growth and development in association with in utero exposure to AAPDs like RIS during critical period of organogenesis. Furthermore, experimental and clinical studies are urgently warranted to establish a relationship between other AAPDs in general and third-generation AAPDs like aripiprazole in particular with regard to placental development, in the interest of the disease affected population. Recently, FDA has approved some newer anti-psychotics such as paliperidone, International Journal of Experimental Pathology, 2016, 97, 125–132
Risperidone induced placental impairments iloperidone and aripiprazole which should also be evaluated in future studies.
Acknowledgement The authors would like to thank the Head, Department of Zoology, University of Allahabad for providing laboratory facilities. Financial supports from U.G.C., New Delhi, India, vide Project no. 37-394/2009 (SR) to KPS (PI), and MKS is thankfully acknowledged.
Conflict of interest There is no conflict of interest between authors and funding agency.
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