Aripiprazole-induced alterations in axial skeletal ossification following in utero exposure in rats

Abstract

Aripiprazole, a third-generation atypical antipsychotic drug, crosses the placental barrier, yet its developmental skeletal effects remain unexplored. This study aimed to evaluate impact of prenatal aripiprazole exposure, administered at three different doses, on the ossification of axial skeleton in 20-day old rat fetuses. Forty pregnant Sprague–Dawley rats were assigned to four groups: control and three aripiprazole treated-groups receiving 3 mg/kg (low dose aripiprazole), 6 mg/kg (high dose aripiprazole), and 12 mg/kg (double high dose aripiprazole [DHDA]) daily from gestational days 6–19. Fetuses were delivered on gestation day 20, weighed, and processed for skeletal evaluation using Alizarin Red staining. Ossification of craniofacial bones, hyoid bone, vertebral centra and arches, sternum, and ribs were assessed and categorized as complete, delayed, or absent. A total of 151 fetuses were analyzed. Aripiprazole exposure induced a dose dependent reduction in fetal weight and in the number of completely ossified skeletal centers. Craniofacial bones, particularly parietal, interparietal, supraoccipital, and presphenoid, were the most affected. Significant impairments were observed in vertebral centra and arches across cervical, thoracic, lumbar, sacral, and coccygeal regions, with the DHDA group exhibiting the greatest deficits. Sternebrae ossification showed marked reduction, whereas rib ossification remained unaffected. A strong positive correlation was found between fetal weight and total number of complete ossification centers across all groups. Prenatal exposure to aripiprazole leads to dose dependent fetal growth restriction and delayed ossification of axial skeleton in rat fetuses. Future investigations should focus on the molecular mechanisms and consequences related to prenatal impacts of aripiprazole.

Introduction

Osteogenesis, the dynamic process of new bone formation, is meticulously orchestrated through the differentiation cascade of mesenchymal stem cells into osteoblasts. This critical developmental sequence proceeds via two distinct biological pathways: intramembranous or endochondral ossification [1]. Comprehensive skeletal assessments are routinely conducted in both developmental biology and toxicology research, employing diverse methodological approaches to analyze the integrity of the skeletal system [2, 3]. In developmental toxicology research, the time and the degree of prenatal ossification serves as a robust and reliable indicator for evaluating overall fetal maturity [3, 4]. Because the axial skeleton has demonstrated susceptibility to a broad spectrum of chemical agents, it has become the standard focus of examination in most developmental toxicity studies [3, 4].

The skull is composed entirely of craniofacial bones. The protective neurocranium is structurally divided into the cranial vault and base, with each component utilizing a separate osteogenic process; the vault forms directly via intramembranous ossification, while the base develops indirectly through endochondral ossification. The viscerocranium, comprising the entirety of the facial skeleton, is primarily derived from the first and second pharyngeal arches, with its ossification occurring predominantly by intramembranous ossification [5-7].

Developmentally, the vertebrae and ribs originate from the embryonic mesoderm, specifically from the sclerotome, which is the medial component of the somites. The initiation of this process is characterized by the genesis of hyaline cartilage models, that will be converted into bone through endochondral ossification. The developing ribs initially arise as integral extensions of the vertebral cartilage models before undergoing a process of separation and subsequent ventral elongation towards the sternum [8]. Variations and malformations in the development of the skull, vertebral column and the rib cage are evoked by exposure to a broad spectrum of xenobiotic agents, including, but not limited to ethanol, valproic acid, methanol and boric acid [9-11].

Extensive reports concerning in vitro, prenatal, and postnatal skeletogenesis collectively highlight that bone development is highly sensitive to the interplay of numerous environmental and intrinsic factors, including the migration and differentiation of cells, localized oxygen tension and angiogenesis. The observed effects of these factors on cartilage and bone development are primarily mediated by changes in the gene expression during mesenchymal differentiation, a process that is highly sensitive to the environmental signals [12-14]. Due to the foundational role of these regulatory elements in bone and cartilage development, any alteration—whether direct or indirect—caused by teratogenic agents during a critical period of mesenchymal differentiation can severely compromise these complex processes [15]. The regulation of bone growth and ongoing skeletal metabolism throughout the developmental period is critically mediated by several major endocrine players, notably thyroid hormones, calciotropic agents, growth hormone, and the essential sex hormones [16, 17].

Antipsychotics are broadly categorized into two groups based on their core mechanism of action: typical antipsychotics, which function predominantly as antagonists of the dopamine receptor, and atypical antipsychotics, which utilize a wider spectrum of activity, by blocking both serotonin and dopamine receptors [18]. Because atypical antipsychotics are associated with a reduced incidence of adverse effects compared to typical antipsychotics, they offer a clinically advantageous option for prescribing to women in the reproductive age [19, 20].

Aripiprazole, classified as a third-generation atypical antipsychotic drug, holds a pivotal role in the pharmacological management of various mental health conditions, such as schizophrenia, major depressive disorder, autism and bipolar disorder [21]. The therapeutic action of this agent stems from its dual activity: exhibiting D2 dopaminergic partial agonism while simultaneously inducing full antagonism at the 5-HT2A serotoninergic receptor [22]. It is highly valued in clinical settings due to its robust therapeutic efficacy combined with notably low adverse effects, especially the extrapyramidal symptoms, when directly compared to conventional antipsychotics [21, 23]. Aripiprazole is currently classified under the U.S. FDA Pregnancy Category C, indicating that while animal reproduction studies have demonstrated potential adverse effects on fetal development, the available data are insufficient to confirm risks in humans. The drug’s necessity may still permit its cautious use in pregnant patients when significant maternal therapeutic benefit is prioritized over the associated theoretical fetal risks [24].

Pregnant women with serious psychiatric illnesses including schizophrenia or bipolar disorder often require maintaining the use of psychotropic drugs, such as antipsychotics, throughout the gestation and breastfeeding periods. The decision-making process regarding the initiation, continuation, or discontinuation of psychotropic medication throughout the gestational period mandates careful consideration of numerous, often complex, clinical and ethical variables [24].

Insufficient data exist to establish a clear conclusion on the reproductive safety profile of aripiprazole in pregnancy, such as its potential for structural malformations, neonatal adverse effects, or long-term behavioral/developmental teratogenicity [25]. However, previous research from our lab has confirmed that aripiprazole readily crosses the placental barrier, a critical factor which raises concerns regarding potential adverse effects on fetal development [26, 27]. Moreover, experimental evidence indicates that gestational exposure to aripiprazole significantly impairs the growth of rat fetuses, demonstrating a clear dose-dependent relationship. The observed adverse outcomes were primarily characterized by severe, symmetrical intrauterine growth restriction, manifested by a significant attenuation of fetal growth parameters. Secondary, but notable, effects included a pronounced increase in embryo-lethality and the limited occurrence of congenital anomalies [28, 29].

Due to the limited available literature evaluating aripiprazole’s influence on prenatal ossification, this study was designed to determine the effects of prenatal aripiprazole exposure in different doses on ossification of the axial skeleton in 20-day-old preterm rat fetuses.

Materials and Methods

Animals

In this study we utilized adult virgin female Sprague–Dawley rats weighing 150–250 g. Rats were housed individually in separate cages within a dedicated pathogen-free environment. Environmental conditions were rigorously adjusted to maintain room temperature 25°C and a standard 12-hours light/dark cycle. The animals were obtained from the Central Animal House Facility of the Arabian Gulf University. Prior to the initiation of the study and subsequent mating, all animals underwent a two-week period of environmental acclimation within their designated cages. To ensure standardization and adherence to animal welfare guidelines [30], all rats were provided with continuous access to food and water ad libitum and were handled by one dedicated investigator throughout the experiment. Following the acclimation period, mating was initiated by placing a fertile male Sprague–Dawley rat into a cage containing two females, remaining together overnight. Pregnancy was verified by microscopic evaluation of a vaginal smear. Examination of the smear on the next morning and observing spermatozoa is considered as the first day of gestation. This study was approved by the Research and Ethics Committee, College of Medicine and Health Sciences, Arabian Gulf University, Bahrain (Approval number E019-PI-11/20). All phases of the animal experimentation were conducted in strict compliance with globally accepted ethical guidelines and regulatory requirements. In the current study, all experimental procedures strictly adhered to the 3Rs principles (replacement, reduction, and refinement), as originally defined by Russell [31]. The rat model was chosen for this teratological assessment due to its cost-effectiveness and its capacity to produce multiple fetuses per litter significantly minimizes the total number of dams necessary for robust statistical analysis.

Experimental design

A total of forty pregnant dams were randomly allocated into four experimental groups: a control group and three treated groups (n=10 per group). Treatment administration in the treated groups was performed via gastric intubation using a specialized rubber feeding tube (14–16 gauge) daily from gestational day 6 through 19. A solution of Aripiprazole (15 mg tablets; Apotex Inc.) was prepared daily by dissolving the tablets in distilled water to achieve a concentration of (10 mg/ml) then was administered to the pregnant dams once daily at a consistent time (9 am ±30 minutes) after fresh preparation. Aripiprazole doses were adjusted daily according to the weight of the dams. Group A (control group) received the vehicle, an equivalent volume of distilled water, administered daily via gastric intubation. Group B received (low dose) 3 mg/kg, Group C received (high dose) 6 mg/kg and Group D received (double high dose) 12 mg/kg aripiprazole daily. To maintain exposure consistency, all aripiprazole doses were adjusted daily based on individual maternal body weight.

The calculation of the precise dose administered to the rats adhered to the following equation:

Rat dose (mg/kg)=human equivalent dose (mg/kg)×Km ratio of rat

Km ratio conversion factor=6.2 [32, 33].

In the current study, we converted the maximum recommended human therapeutic dose (MRTD): 30 mg/day, to a rat dose and utilized as the low dose of aripiprazole (LDA).

To investigate the upper limits of risk, we included two higher-dose groups that exceed the MRTD after conversion to a rat dose. The high dose of aripiprazole (HDA) was twice the LDA, and the double high dose of aripiprazole (DHDA) was quadruple the LDA. Based on an assumed average woman weight of 65 kg for calculating the human equivalent dose, the three experimental aripiprazole doses utilized in the rat model were: 3 mg/kg/body weight (LDA), 6 mg/kg/body weight (HDA), and 12 mg/kg/body weight (DHDA).

On gestation day 20, at 9 am ±30 minutes dams were deeply anesthetized by ether and the fetuses were surgically delivered via cesarean section. Dams were then euthanized by CO2 inhalation.

All fetuses were cleared of their membranes and separated from placentae before their individual fetal weights (FWs) were precisely documented.

Staining process

From each individual dam, half of the number of fetuses were randomly allocated for skeletal analysis via Alizarin Red S staining, while the remaining half underwent fixation in Bouin’s solution, and were already used in a previously published study [29]. A total of 151 fetuses were collected for skeletal analysis via Alizarin Red S staining, comprising 39 fetuses from the control group, 38 from LDA group, 45 fetuses from HDA group and 29 fetuses from DHDA group. For skeletal preparation, the fetuses were sacrificed then they were eviscerated and underwent standardized clearing preparation. Each fetus was immersed in a water bath of 70°C for 30 seconds to facilitate skin removal. The staining procedure strictly followed the standard methodology developed by Dawson [34]. Briefly, the skeletal staining protocol commenced with the immersion of each fetus in 95% ethanol for a 48-hour dehydration period, followed by transfer into a 1% potassium hydroxide solution for 1–2 days to achieve adequate soft tissue clearing prior to staining. The fetus was subsequently immersed into a newly prepared 1% potassium hydroxide mixed with few drops of prepared Alizarin Red S solution and the fetus was maintained in this staining solution for a period of 48 to 72 hours. Overstraining was reformed by storing the samples in the Mall’s Solution (79% distilled water, 20% glycerin, and 1% KOH). After staining, the skeletal specimens were subjected to a gradual clearing process, involving sequential transfer through ascending concentrations of glycerin (30%, 50%, and 70%) over a 2–3 days period between each change. The specimens were then permanently stored in pure 100% glycerin, to which thymol was added specifically to prevent fungal contamination.

Examination of the ossification

For the assessment of ossification centers, a specific skeletal scoring chart was adopted, based on the methodology previously published by Nash and Persaud [35]. This chart specifically delineates the expected ossification centers present in the Sprague–Dawley rat fetus at gestational day 20. Assessment of the ossification centers of axial skeleton in each fetus was performed using a Zeiss Stemi 2000-C dissecting microscope. We focused on scoring the ossification centers of the craniofacial bones, the hyoid bone, the vertebral centra and arches, sternum, and ribs, with all findings meticulously recorded. Craniofacial skeleton examined in this stage covered 28 ossification centers per fetus, including the single hyoid bone and 27 specific skull components. These 27 centers were individually scored: the frontal (1), paired parietal (2), supraoccipital (1), interparietal (1), paired exoccipital (2), basioccipital (1), paired nasal (2), paired lacrimal (2), paired premaxillae (2), paired maxillae (2), paired zygomatic (2), paired squamosal (2), paired tympanic bullae (2), basisphenoid (1), presphenoid (1), paired alisphenoid (2), and the mandible (1). The number of ossification centers of the axial skeleton examined in the rat fetuses in this study are summarized in Table 1.

Table 1.

Total number of examined sites of the ossified axial skeleton bony centers of the control and aripiprazole treated groups in 20-day-old rat fetuses

Regions of axial skeleton examined	No. of examined bony centers/fetus	Total no. of ossified bones
		Control (n=39)	LDA (n=38)	HDA (n=45)	DHDA (n=29)
Skull (27) and hyoid body (1)	28	1,092	1,064	1,260	812
Ribs (13×2)	26	1,014	988	1,170	754
Sternum (5 sternebrae)	5	195	190	225	145
Vertebral centra (1)
Cervical C1–C7	0	0	0	0	0
Thoracic T1–T13	13	507	494	585	377
Lumbar L1–L6	6	234	228	270	174
Sacral S1–S4	4	156	152	180	116
Coccyx Co1–Co2	2	78	76	90	58
Vertebral arches (2 Rt and Lt)
Cervical C1–C7	14	546	532	630	406
Thoracic T1–T13	26	1,014	988	1,170	754
Lumbar L1–L6	12	468	456	540	348
Sacral S1–S4	8	312	304	360	232
Coccyx Co1–Co2	4	156	152	180	116
Total	148	5,772	5,624	6,660	4,292

The cervical centra are not included in the calculation in this study as they did not show ossification at the 20 days rat fetuses. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole; Rt, right; Lt, left.

Observed ossification patterns were classified into three distinct qualitative outcomes: complete, delayed, or absent. Indicators of delayed bone ossification were characterized by weak staining, small or dumbbell-shaped ossified centers. Furthermore, any observed structural or morphological abnormalities in ossification were documented.

The total number of the ossified centers in the whole axial skeleton in all groups was counted and their percentages were calculated. The correlation between the calculated FW/litter and their corresponding complete ossified centers/litter was evaluated.

Statistical analysis

Categorical variables were descriptively summarized using frequencies and corresponding percentages. All statistical analyses considered the dam as the experimental unit; therefore, the mean value of fetal parameters within each litter was used for group comparisons. The two-proportion Z-test was used to determine the difference between two proportions. ANOVA followed by Tukey’s post-hoc test was utilized to analyze fetal body weight. A scatterplot was used to measure the relationship between FW and the total number of complete ossifications of the axial skeleton in all four study groups: control, LDA, HDA, and DHDA. All collected data were performed utilizing the Statistical Package for Social Sciences, version 30 (IBM Co.), and Minitab Statistical Software Version 17. Statistical significance was established at P<0.05.

Results

A total of 151 rat fetuses on gestational day 20 were examined. The effects of prenatal administration of three doses of aripiprazole on their weights and on the ossification of their axial skeleton stained with Alizarin Red S were analyzed as follows.

Fetal weight

The FW was in the normal range in the control group. This was reduced in a dose dependent manner in other groups. There was a significant decrease in FW in both LDA and HDA groups when compared to control group (P<0.05). Moreover, FW in DHDA group showed a significant reduction when compared to control and LDA groups (P<0.05). On the other hand, there was no significant reduction between HDA and DHDA groups (P>0.05) (Fig. 1A).

Fig. 1.

(A) Effect of prenatal exposure to aripiprazole on fetal body weight in the studied groups. All values represent mean±SD. *P<0.05 vs control group, and ^#P<0.05 vs. low dose aripiprazole (LDA) group, as determined by a one-way ANOVA followed by Tukey’s post-hoc test. (B) Effect of prenatal exposure to aripiprazole on the percentage of ossification of axial skeleton in the studied groups. All values represent percentage. *P<0.01 vs. control group, and ^#P<0.01 vs. LDA, HDA groups, as determined by Z-test. HDA, high dose aripiprazole; DHDA, double high dose aripiprazole.

Evaluation of the total number of ossified bony centers of the axial skeleton

The total number of complete ossified bony centers of the axial skeleton in the control group was 4,806 (83.27%). This was reduced in a dose dependent manner in other treated groups: 4,360 (77.52%), 4,982 (74.81%), and 2,924 (68.13%) in LDA, HDA, and DHDA groups respectively. Fig. 1B demonstrates the percentage of the total number of complete, delayed and absent ossified bones separately. There was a highly significant decrease in the percentage of complete ossified bony centers in both LDA, HDA, and DHDA groups when compared to control group (P<0.01). Moreover, complete ossified centers in DHDA group showed a highly significant reduction when compared to other two treated groups (P<0.01) (Fig. 1B).

Craniofacial and hyoid ossified bony centers

A total of 27 craniofacial bones were examined. A highly significant decrease in the number of completely ossified bony centers was detected in the HDA group compared to control and LDA groups (P<0.001). Furthermore, the ossified centers showed highly significant decrease in DHDA group compared to control and other treated groups (P<0.001) (Fig. 2, Table 2). Regarding the hyoid bone, there was highly significant decrease in the number of completely ossified bony centers of the hyoid body in the DHDA group compared to control group (P<0.001) and significant decrease in DHDA group compared to LDA (P<0.05) and no significant difference was apparent in ossification between control, LDA, and HDA groups (Fig. 2C, F, I, L, Table 2).

Fig. 2.

Ossification of the skull and hyoid bones. Control group (A–C): (A) showing normal ossification of parietal (P), interparietal (IP), supraoccipital (SO), occipital (O) bones; (B) showing presphenoid (PS) bone situated between the two alisphenoid bones (AS) at the base of the skull; (C) showing hyoid bone (h). Low dose aripiprazole group (D–F): (D) showing delayed ossification of the SO and IP; (E, F) showing presence of PS and h, respectively. High dose aripiprazole group (G–I): (G) showing delayed ossification of P, IP, SO; (H, I) showing absence of PS and h (arrows), respectively. Double high dose aripiprazole group (J–L): (J) showing marked delayed ossification of the P, IP, SO, O; (K, L) showing absence of PS and h (arrows), respectively. Note that (A, D, G, and J) showing posterior view of skull; (B, E, H, and K) showed magnified inferior view of base of the skull to identify the presphenoid bone ossifications (×2.25) and (C, F, I, and L) represent lateral view of the skull.

Table 2.

The number of ossified craniofacial and hyoid bony centers in 20 days rat fetuses following maternal ingestion of aripiprazole

Group	Craniofacial (27)				Hyoid body (1)
	Total examined bones	Complete ossification	Delayed and absent ossification		Total examined bones	Complete ossification	Delayed and absent ossification
Control (n=39)	1,053	990 (94.02)	63 (5.98)		39	14 (35.90)	25 (64.10)
LDA (n=38)	1,026	943 (91.91)	83 (8.09)		38	9 (23.68)	29 (76.32)
HDA (n=45)	1,215	1,091 (89.79)a)	124 (10.21)a)		45	8 (17.78)	37 (82.22)
DHDA (n=29)	783	657 (83.91)a,c,d)	126 (16.09)a,c,d)		29	2 (6.90)a,b)	27 (93.10)a,b)

Values are presented as number (%) or number only. Chi-square test. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole. ^a)P<0.001 compared to the control group. ^b)P<0.05 compared to LDA group. ^c)P<0.001 compared to LDA group. ^d)P<0.001 compared to HDA group.

It was found that the most affected craniofacial bones that showed delayed or absent ossification pattern in the treated groups are the interparietal, supraoccipital, presphenoid, and parietal bones (Fig. 2, Table 3).

Table 3.

The most affected ossified craniofacial bony centers in 20 days rat fetuses following maternal ingestion of aripiprazole

Group	Interparietal			Supraoccipital			Presphenoid			Rt parietal and left parietal
	Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification
Control (n=39)	33 (84.62)	6 (15.38)		15 (38.46)	24 (61.54)		10 (25.64)	29 (74.36)		74 (94.87)	4 (5.13)
LDA (n=38)	29 (76.32)	9 (23.68)		5 (13.16)b)	33 (86.84)b)		4 (10.53)	34 (89.47)		70 (92.11)	6 (7.89)
HDA (n=45)	25 (55.56)b,d)	20 (44.44)b,d)		7 (15.56)a)	38 (84.44)a)		1 (2.22)b)	44 (97.78)b)		78 (86.67)	12 (13.33)
DHDA (n=29)	8 (27.59)c,f,g)	21 (72.41)c,f,g)		0 (0)c,d,h)	29 (100)c,d,h)		1 (3.45)b)	28 (96.55)b)		38 (65.52)b,e,g)	20 (34.48)b,e,g)

Values are presented as number (%). Chi-square test. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole; Rt, right. ^a)P<0.05 compared to control group. ^b)P<0.01 compared to control group. ^c)P<0.001 compared to the control group. ^d)P<0.05 compared to LDA group. ^e)P<0.01 compared to LDA group. ^f)P<0.001 compared to LDA group. ^g)P<0.05 compared to HDA group. ^h)P<0.01 compared to HDA group.

The number of complete ossifications of interparietal bone in HDA group showed highly significant decrease compared to control group (P<0.01) and significant decrease compared to LDA group (P<0.05). On the other hand, the ossification of DHDA group was the most affected among all groups and exhibited highly significant decrease compared to control and LDA groups (P<0.01) and significant decrease compared to HDA group (P<0.05) (Fig. 2A, D, G, J, Table 3).

In supraoccipital bone, there was a highly significant reduction in the number of complete ossified bones in LDA group (P<0.01) and a significant reduction in HDA group (P<0.05) when compared to the control. On the contrary, the DHDA group ossification was mostly affected and exhibited a highly significant reduction compared to control (P<0.001) and HDA groups (P<0.01) and a significant reduction compared to LDA group (P<0.05) (Fig. 2A, D, G, J, Table 3).

Regarding the presphenoid bone, there was a highly significant reduction in the number of complete ossified bones in both HDA and DHDA groups when compared to the control (P<0.01). In contrast, no significant difference was apparent in ossification among all treated groups (Fig. 2B, E, H, K, Table 3).

The parietal bones showed that there was a highly significant reduction in the number of complete ossified bones in the DHDA when compared to the control and LDA groups (P<0.01) and significant reduction compared to HDA group (P<0.05). No significant difference was noticed in ossification between the control, LDA and HDA groups (Fig. 2A, D, G, J, Table 3).

Vertebral column ossification

Ossifications of the centra and arches of the vertebrae in the different regions of vertebral column are divided into:

Vertebral centra ossification

Twenty-five vertebral centra were examined in each fetus (13 thoracic, 6 lumbar, 4 sacral, and 2 coccygeal). In all studied groups, evaluation of the cervical centra revealed no ossification and this is consistent with normal developmental timelines, as cervical centra do not normally ossify at this stage of development (Fig. 3A–D). A total of 3,775 ossified vertebral centra was examined in all groups (975 in control, 950 in LDA group, 1,125 in HDA group, and 725 in DHDA group).

Fig. 3.

Ossification of cervical and upper thoracic vertebrae: The cervical and upper thoracic centra seen in anteroposterior view in (A) control, (B) low dose aripiprazole (LDA), (C) high dose aripiprazole (HDA), and (D) double high dose aripiprazole (DHDA) groups. The cervical and 1st thoracic centra are absent in all groups (braces). In (B) the 2nd thoracic centrum is delayed (arrow). In (C) the 2nd thoracic centrum is absent (arrow). In (D) the 2nd and 3rd thoracic centra are absent (arrows) (×0.65). Ossification of cervical arches are seen in lateral view in (E) control, (F) LDA, (G) HDA and (H) DHDA groups. In (E) the 7 arches showed complete ossification (bracket). In (F) delayed ossification is seen in the middle three arches (bracket). In (G) delayed ossification in the middle 4 arches in the form of separation between anterior and posterior parts of the arches is seen (bracket). In (H) marked delayed ossification of cervical arches (bracket) with very small ossified posterior parts in the last three arches (×0.65).

In the thoracic region, the centra showed a highly significant reduction of the number of complete ossified centra in LDA, HDA, and DHDA groups when compared to the control group (P<0.001). Additionally, the DHDA showed a highly significant decreased ossification when compared to LDA group (P<0.01) and showed a significant decrease when compared to HDA group (P<0.05) (Figs. 3, 4, Table 4).

Fig. 4.

Ossification of the lower thoracic, lumbar, sacral, and coccygeal vertebrae. (A) Control group showing complete ossification of lower thoracic, lumbar, sacral, and upper 2 coccygeal centra and arches. (B) Low dose aripiprazole group showing delayed ossification of two lumbar centra, one delayed and two absent sacral centra, and all coccygeal centra are absent. The 3rd sacral arch is delayed (arrow), and the remaining sacral and coccygeal arches are absent. (C) High dose aripiprazole group showing marked delayed ossification of thoracic (circle), and lumbar (black arrows) and 1st sacral centra (white arrow) and absence of remaining sacral and coccygeal centra (bracket). The 2nd sacral arch is delayed, and the remaining sacral and coccygeal arches are absent (bracket). (D) Double high dose aripiprazole (DHDA) group showing significantly delayed ossification of lower thoracic, 1st lumbar centra and absence of the remaining lumbar, sacral, and coccygeal centra. The lower 2 lumbar and 1st sacral arches are delayed, and the remaining sacral and coccygeal arches are absent (bracket) (×0.65). The delayed ossified centra are small, faint in color or dumbbell shaped.

Table 4.

Effects of maternal aripiprazole ingestion on ossified vertebral centra in 20 days rat fetuses

Group	Thoracic (13)			Lumbar (6)			Sacral (4) and coccygeal (2)
	Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification
Control (n=39)	388 (76.53)	119 (23.47)		217 (92.74)	17 (7.26)		34 (14.53)	200 (85.47)
LDA (n=38)	283 (57.29)b)	211 (42.71)b)		185 (81.14)b)	43 (18.86)b)		24 (10.53)	204 (89.47)
HDA (n=45)	315 (53.85)b)	270 (46.15)b)		204 (75.56)b)	66 (24.44)b)		21 (7.78)a)	249 (92.22)a)
DHDA (n=29)	173 (45.89)b,c,e)	204 (54.11)b,c,e)		106 (60.92)b,d,f)	68 (39.08)b,d,f)		4 (2.30)b,d,f)	170 (97.70)b,d,f)

Values are presented as number (%). Chi-square test. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole. ^a)P<0.05 compared to control group. ^b)P<0.001 compared to the control group. ^c)P<0.01 compared to LDA group. ^d)P<0.001 compared to LDA group. ^e)P<0.05 compared to HDA group. ^f)P<0.01 compared to HDA group.

In the lumbar region, the centra demonstrated a highly significant reduction of the number of complete ossified centra in LDA, HDA, and DHDA when compared to the control group (P<0.001). Furthermore, the DHDA showed highly significant decrease in ossification when compared to control, LDA (P<0.001), and HDA groups (P<0.01) (Fig. 4, Table 4).

In the sacral and coccygeal regions, the centra revealed a significant reduction of the number of complete ossified centra in HDA group when compared to the control group (P<0.05). Moreover, the DHDA group showed a significant decrease in ossification when compared to control, LDA group (P<0.001) and HDA groups (P<0.01). No significant difference was detected between LDA and control groups (Fig. 4, Table 4).

The delayed and absent ossifications of the vertebral centra in the aripiprazole treated groups are found in the cranio-caudal regions namely in the upper 3 thoracic vertebrae and in the coccygeal, sacral, and lower 2 lumbar vertebrae. However, few delayed centra appears also in the lower thoracic vertebrae. The delayed ossified vertebral centra appear most commonly in the form of dumbbell shape (Figs. 3, 4).

Vertebral arches ossification

Sixty-four ossified vertebral arches (right and left for each vertebra) were examined in each fetus (14 cervical, 26 thoracic, 12 lumbar, 8 sacral, and 4 coccygeal).

A total of 9,664 vertebral arches were examined in all groups (2,496 in control, 2,432 in LDA group, 2,880 in HDA group, and 1,856 in DHDA group).

In the cervical region, the 14 cervical arches showed a highly significant decrease in the number of complete ossified centers in LDA group when compared to the control group (P<0.001). Moreover, there was a highly significant decrease in the ossification in HDA group when compared to the control and LDA groups (P<0.001). Additionally, DHDA group exhibited a highly significant decrease when compared to the control and other treated groups (P<0.001) (Fig. 3E–H, Table 5).

Table 5.

Effects of maternal aripiprazole ingestion on ossified vertebral arches in 20 days rat fetuses

Group	Cervical (7×2)			Thoracic (13×2)			Lumbar (6×2)			Sacral (4×2) and coccygeal (2×2)
	Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification		Complete ossification	Delayed and absent ossification
Control (n=39)	498 (91.21)	48 (8.79)		1,014 (100)	0 (0)		464 (99.15)	4 (0.85)		144 (30.77)	324 (69.23)
LDA (n=38)	446 (83.83)c)	86 (16.17)c)		982 (99.39)a)	6 (0.61)a)		432 (94.74)c)	24 (5.26)c)		88 (19.30)c)	368 (80.70)c)
HDA (n=45)	360 (57.14)c,d)	270 (42.86)c,d)		1,162 (99.32)b)	8 (0.68)b)		510 (94.44)c)	30 (5.56)c)		100 (18.52)c)	440 (81.48)c)
DHDA (n=29)	152 (37.44)c,d,e)	254 (62.56)c,d,e)		746 (98.94)b)	8 (1.06)b)		290 (83.33)c,d,e)	58 (16.67)c,d,e)		34 (9.77)c,d,e)	314 (90.23)c,d,e)

Values are presented as number (%). Chi-Square test. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole. ^a)P<0.05 compared to control group. ^b)P<0.01 compared to control group. ^c)P<0.001 compared to the control group. ^d)P<0.001 compared to LDA group. ^e)P<0.001 compared to HDA group.

In the thoracic region, ossifications of 26 thoracic arches were examined in each fetus. Ossification of all thoracic arches was complete in the thoracic arches of the control group. However, the number of complete ossified thoracic arches significantly decreased in the LDA group (P<0.05) and a highly significant decrease (P<0.01) in both HDA and DHDA groups when compared to the control group. No significant difference was noticed among the 3 treated groups.

In the lumbar, sacral, and coccygeal regions, 12 lumbar, 8 sacral, and 4 coccygeal vertebral arches were evaluated in each fetus. Both LDA and HDA groups demonstrated a highly significant decrease in the number of complete ossified arches when compared to the control (P<0.001). There was a highly significant decrease in DHDA group when compared to the control and other treated groups (P<0.001) (Fig. 4, Table 5).

Delayed ossification of the vertebral arches in the cervical region appears as non-fusion of anterior and posterior segments of the arch or complete absence of its posterior part. The most common delayed arches in the 3 treated groups were bilateral and usually in 2–6 cervical vertebrae (Fig. 3E–H). Also, absence and delayed arches appear in the caudal region in the coccygeal and sacral vertebrae in the form of weak staining of a small center. These findings were seen mainly in sacral arches 3 and 4 and coccygeal 1 and 2 (Fig. 4B–D).

Ossification of sternum

The sternum was evaluated for ossification of its five bony centers, sternebrae. The number of complete ossified sternebrae was significantly decreased in the LDA and HDA groups when compared to the control (P<0.05). The DHDA group demonstrated a highly statistically significant decrease in ossification when compared to control (P<0.001), LDA (P<0.01), and HDA (P<0.001) groups (Fig. 5, Table 6).

Fig. 5.

Ossification of sternum in all groups. (A) Control group showing normal ossification of two sternebrae and one delayed ossification: small (arrow). (B) Low dose aripiprazole group showing normal ossification of one sternebra and one delayed ossification: small and bifid (arrow). (C) High dose aripiprazole group showing marked delayed ossification of two sternebrae: faint color, small and bifid, (arrows). (D) Double high dose aripiprazole group showing severely delayed ossification of one sternebra: small and bifid (arrows) (×2.25)

Table 6.

Total number of ossified centers of sternum and ribs in 20 days rat fetuses following maternal ingestion of aripiprazole

Group	Sternum (5 sternebrae)				Ribs (13×2)
	Total examined bones	Complete ossification	Delayed and absent ossification		Total examined bones	Complete ossification
Control (n=39)	195	35 (17.95)	160 (82.05)		1,014	1,014 (100)
LDA (n=38)	190	18 (9.47)a)	172 (90.53)a)		988	988 (100)
HDA (n=45)	225	24 (10.67)a)	201 (89.33)a)		1,170	1,170 (100)
DHDA (n=29)	145	3 (2.07)b,c,d)	142 (97.93)b,c,d)		754	754 (100)

Values are presented as number (%) or number only. Chi-square test. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole. ^a)P<0.05 compared to control group. ^b)P<0.001 compared to the control group. ^c)P<0.01 compared to LDA group. ^d)P<0.001 compared to HDA group.

Ossification of ribs

In each fetus, 26 ribs were examined. Control and other three treated groups revealed that 100% of the ribs are completely ossified (Table 6).

Correlations between FWs and number of ossified bony centers of the axial skeleton

The Pearson correlation analysis demonstrated a strong, positive, and a statistically significant relationship between FW and the total number of complete ossifications in all four study groups: control, LDA, HDA, and DHDA (r=0.923, 0.977, 0.980, and 0.991; P<0.001), respectively (Fig. 6).

Fig. 6.

Correlation between fetal weight and complete ossification of axial skeleton among the studied groups. (A) Control group. (B) LDA group. (C) HDA group. (D) DHDA group. LDA, low dose aripiprazole; HDA, high dose aripiprazole; DHDA, double high dose aripiprazole.

Discussion

The development of the fetal skeleton provides a reliable indicator for assessing the degree of its gestational growth [36, 37]. The regulatory factors of axial skeletal ossification are not yet fully elucidated. Genetic alterations, whether via spontaneous mutation or triggered by chemical exposure, can compromise structural patterning and lead to various developmental skeletal defects [38]. Fetal osteogenesis could be also compromised through maternal exposure to teratogenic agents, nutritional deficiencies, or gestational infections [3, 39]. This study was conducted to evaluate the axial skeleton ossification including craniofacial bones, hyoid bone, vertebral column, ribs and sternum in preterm rat fetuses after maternal exposure to three different doses of aripiprazole. To achieve this objective, we categorized the ossification pattern into, complete, delayed or absent.

In this study we do not consider absent ossification to be a permanent abnormality; rather, it may reflect a transient developmental delay in ossification caused by the drug [4, 40]. These delays often “catch up” during the rapid growth period of lactation if the underlying cause of growth restriction is removed. The compensatory mechanism phenomena stated that any unossified center during intrauterine development might be restored and become complete postnatally [11, 38].

The dosing regimen in this study was strategically tiered to bridge the gap between clinical application and toxicological thresholds. The LDA (3 mg/kg) was calibrated to be nearly equal to the human MRTD, in contrast, the HDA (6 mg/kg) was set just below known teratogenic doses, while the DHDA (12 mg/kg) was selected at the minimal threshold for the documented teratogenic doses [25, 29]. Aripiprazole treatment was initiated on gestational day 6 and maintained through day 19, effectively covering both embryonic and fetal periods of the development in rats.

Delayed skeletal ossification and decreased FW were markedly observed after prenatal oral administration of 3, 10, and 30 mg/kg aripiprazole (1, 3, and 10 times the maximum recommended human dose [MRHD] on a mg/m² basis) in pregnant rats [41].

Maternal rabbits received oral aripiprazole doses of 10, 30, and 100 mg/kg/day during organogenesis period. These regimens provided systemic exposures equivalent to 2, 3, and 11 times the human MRHD based on the area under the curve [42]. Administration of 30 and 100 mg/kg aripiprazole is associated with reduction in FW and increased occurrence of skeletal malformations such as fused sternebrae. Furthermore, few skeletal variations appeared after usage of the dosage of 100 mg/kg [41].

In the current study, FW was reduced in all aripiprazole treated groups with highly significant reduction in DHDA group when compared to other groups. These findings are consistent with prior studies from our lab that reported the reduction of FW in a dose dependent relationship after different doses of aripiprazole administration [27, 29]. A recent study by Sarkar et al. [43] observed reduction in the chick embryonic growth parameters following exposure to aripiprazole at concentrations of 5 ppm and 10 ppm.

In the current study, a positive correlation was observed between FW and number of complete ossification centers in the axial skeleton in all groups which could be attributed to teratogenic effect of aripiprazole. Moreover, in our previous studies, aripiprazole affected nutritional status of the fetus through affection of the placental barrier [27] and lead to general growth restriction of fetuses as reported in our previous published study [29]. So, we suggest that the delayed ossification is due to both generalized growth restriction and a direct teratogenic effect of aripiprazole.

While limited numbers of studies explored the pathways through which aripiprazole cause many structural changes [27, 29], its influence on axial skeleton ossification is still poorly investigated. Oxidative stress stands as a cornerstone mechanism of teratogenicity, frequently disrupting normal ossification patterns [44, 45]. Evidence suggests that aripiprazole induces a distinct oxidative stress in various brain areas [46]. Moreover, aripiprazole at concentrations of 5 ppm and 10 ppm elicited a significant, dose-dependent elevation in alkaline phosphatase enzyme in chick embryos. This enzymatic surge reflects a profound disruption of the normal developmental biochemistry in the chick model [43]. This may explain the hazardous effect of aripiprazole on ossification detected in the present study.

In the present study, aripiprazole treatment significantly affected the ossification of craniofacial and hyoid bones in 20 days old rat fetuses in a dose dependent relationship. Of all the craniofacial bones examined the delayed or absent ossifications were highly noticed in parietal, interparietal, supraoccipital, and presphenoid as well as hyoid bones. In accordance with the findings of the current study, Sarkar et al. [43] reported significant morphological alterations in chick embryos following exposure to 5 ppm and 10 ppm of aripiprazole such as cephalic abnormalities and systemic embryonic degeneration, highlighting the risk of developmental toxicity of this medication. Conversely, Okab et al. [47] explored the teratogenic effects of other two atypical antipsychotic drugs, olanzapine and risperidone in a rat model. Maternal administration of olanzapine or risperidone from gestational day 4 through 19 and subsequent skeletal analysis via Alizarin Red staining confirmed that craniofacial ossification remained unaffected and no anomalies were detected in both treatment cohorts compared to the control group.

In the present study, delayed or absent ossification of vertebrae including centra and arches were investigated in the three aripiprazole treated groups (LDA, HDA, DHDA) and all groups were affected. It was explored that there were highly statistical differences in the DHDA group when compared to control as well as LDA and HDA groups, and we concluded that these effects are dose dependent related. In agreement with the current study, Sarkar et al. [43], demonstrated that aripiprazole treated chick embryos showed significant embryonic changes, including reduction in somites number, neural tube defects and abnormal flexion of the vertebral column curvature. Consistent with the varied impact of antipsychotics on skeletal maturation, Okab et al. [47] reported that olanzapine and risperidone did not disrupt the general morphology of the vertebrae, however only one or two ossified caudal vertebrae appeared complete and the other ossified centers were absent. In agreement with Aliverti et al. [4], who reported that ossification of the vertebrae starts from the lower thoracic and upper lumber vertebrae then extends in cephalic and caudal directions, the current study revealed that the delayed and absent ossifications were found mainly in the cervical and caudal regions.

The preservation of normal rib ossification and morphology in the present study may be related to their developmental origin from the costal processes of the thoracic sclerotome. Notably, the vertebral arches—which also arise from sclerotomal derivatives—were similarly unaffected. This parallel is biologically meaningful, as both ribs and vertebral arches follow relatively stable and robust developmental pathways compared with other axial elements such as the vertebral centra or sternebrae, which undergo more complex processes including resegmentation and delayed ossification [48]. Selective effects on vertebral centra (bodies) while sparing (or less severely affecting) neural arches and ribs occur because the different parts of the vertebra arise from distinct subpopulations of the sclerotome, each with unique positions, signaling dependencies, and developmental timing. Aripiprazole may disrupt specific molecular gradients or cell behaviors that disproportionately impact the ventromedial sclerotome (which forms the centra) more than the dorsolateral or lateral portions (which form arches and ribs) [49].

The number of complete ossified bony centers of the sternum was significantly decreased in all aripiprazole treated groups when compared to the control. The DHDA was the most affected group. Parallel to our findings, the work of Okab et al. [47] highlighted the variable effects of antipsychotics on sternal ossification centers. While the risperidone group maintained a normal ossification pattern, the olanzapine group manifested incomplete ossification of the fifth sternebrae. Further investigation in a rabbit model revealed that maternal exposure to aripiprazole at dosages of 30 and 100 mg/kg is associated with skeletal abnormality in the form of fused sternebrae [41].

In the present study, reduction of FW in aripiprazole treated groups was accompanied by a parallel decrease in the number of complete ossified centers of the axial skeleton. Our data suggests that aripiprazole exerts prenatal growth restriction. This is consistent with the criteria proposed by Ariyuki et al. [40] who concluded that a decrease in FW is frequently accompanied by a reduction in ossification and serves as a reliable indicator for assessing the degree of intrauterine growth retardation. Previous study within our laboratory [27] revealed that aripiprazole administration induces significant degenerative alterations in placental morphology. These changes in the placental integrity compromises the maternal-fetal nutrient exchange, subsequently depriving the developing fetus of the essential metabolic precursors required for robust osteogenesis and resulting in the observed delays in skeletal ossification.

In alignment with Frost’s Mechanostat theory [48], bone development mechanism indicated that bone cell activity is modulated by mechanical bone demands. The mechanism of mechanostat-controlled fetal bone development [50] is associated with low bone mineral density at birth and impaired periosteal expansion. Since aripiprazole traverses the placental barrier and diffuses in fetal tissue [27], it is hypothesized that a reduction in prenatal physical activity might result in delayed bone development and ossification.

In conclusion, maternal exposure to aripiprazole during gestation resulted in delayed ossification and development of axial skeleton including craniofacial bones, vertebral column, and sternum in rat fetuses and their effects were in a dose dependent response. The clinical use of aripiprazole during pregnancy should be restricted to therapeutic dosages, as higher doses can cause fetal growth restriction and delayed ossification of axial skeleton. Future research should focus on the molecular mechanisms and consequences related to the detected prenatal impacts of aripiprazole.