Maternal effects of RUPP

Table 1 shows the maternal effects of RUPP in rats.

Table 1 Maternal parameters and reproductive outcomes of sham and RUPP (preeclampsia) groups

No significant differences in maternal body weight (p = 0.49) and 24hUP (p = 0.38) were found between the sham and RUPP pregnant rats before the surgical procedures (GD13). Four days (GD18) after the RUPP or sham surgeries, BP and 24hUP were significantly elevated in RUPP rats compared to the sham group (p = 0.001 and p < 0.001, respectively). At GD20, the maternal body weight of RUPP rats was significantly lower than sham-operated animals (p = 0.001).

At the time of surgical procedures (GD14), no significant difference was found in the number of fetuses between the sham and RUPP groups. After parturition, assessment of litter size and the number of male and female pups revealed no significant differences between sham and RUPP groups. However, the mean percentage of stillbirth ratio was significantly higher in the RUPP group than in the sham group (8.71% vs 3.03%, p = 0.042).

Effects on offspring weight gain

The body weight of newborns was measured weekly from PND1 to PND56 (Fig. 2A). A three-way repeated measure ANOVA found that the main effects of group [F(1,160) = 381.2, p < 0.001], sex [F(1,160) = 67.54, p < 0.001] and time [F(7,160) = 27.48, p < 0.001] on body weight were all significant. Accordingly, the body weight of rat pups increased with age, and male rats were heavier than females. RUPP pups had a lower weight from PND1–PND42 than sham offspring. There were significant interactions between time × sex [F(7,160) = 37.95, p < 0.001], with females weighing less than males, and time × group [F(7,160) = 53.35, p < 0.001], with RUPP rats of both sexes had a lower body mass than their peers in the sham group from PND1 until PND42. Specific pairwise comparison showed that only male offspring of RUPP dams had higher body weight than male pups of the sham group at PND49 (p = 0.047) and PND56 (p = 0.016). However, it was not statistically sex-specific, because sex × group [F(1,160) = 0.54, p = 0.46] and time × sex × group [F(7,160) = 1.04, p = 0.404] interactions were not significant.

Fig. 2

Effects of maternal reduced uteroplacental perfusion (RUPP) on offspring body mass growth (A) and visceral fat mass (B). Over 56 postnatal days, all pups gained weight. In both groups, males weighed more than females. The body mass of RUPP pups (both sexes) was significantly lower than their sex-matched peers in the sham group from birth until postnatal day 42. Male offspring of RUPP dams were heavier than male offspring of the sham group at postnatal days 49 and 56, but the effect was not a sex difference (group × sex interaction: p > 0.05). At postnatal day 60, male rats of the sham group showed a higher visceral fat mass than females, but in RUPP offspring, no difference was found by sex. Male offspring of the sham group had a higher visceral fat index than males of the RUPP group. Results are expressed as mean ± SD. (n = 6). The body mass data were analyzed by a repeated measure three-way ANOVA (time, sex, group) and Tukey’s post hoc test. *p < 0.05, RUPP males vs. Sham males and #p < 0.05, RUPP females vs. Sham females. The data of PND60 visceral fat mass index were analyzed by two-way ANOVA (sex and group) and Tukey’s post hoc test: ***p < 0.001; (a) main effect of time; (b) the main effect of sex

In addition, the data of visceral fat mass measured at PND60 were analyzed by two-way ANOVA. The main effect of the group was significant [F(1, 20) = 29.35, p < 0.001], with RUPP rats having lower visceral fat mass than sham rats (Fig. 2B). The main effect of sex was also significant [F(1, 20) = 16.12, p < 0.001], with males having more visceral fat mass than females. The group × sex interaction was also significant [F(1, 20) = 5.509, p = 0.029]. Male rats in the RUPP group had lower visceral fat mass than male rats in the sham group (p < 0.001), whereas no similar difference was found in female rats (p = 0.16).

Effects on blood glucose and insulin

Biochemical and hormonal parameters including circulatory glucose, insulin, glucagon and ghrelin were measured at PND14 and PND60. No differences were observed between the studied groups on blood glucose at different ages [F(1, 5) = 1.44, p = 0.28] or by sex [F(1, 5) = 2/92, p = 0.068] (Fig. 3A).

Fig. 3

Effects of maternal reduced uteroplacental perfusion (RUPP) on offspring blood glucose (A), insulin (B), glucagon (C) and ghrelin (D) on postnatal days 14 and 60 (PND14 and PND60). In the male offspring of the sham group, insulin levels were upregulated from PND14–PND60, but insulin levels of the male offspring of RUPP dams did not change. At PND14, only female pups in the sham group had higher glucagon levels than males, and RUPP pups exhibited lower glucagon levels than their sex-matched counterparts in the sham group. Over time glucagon levels of sham offspring (both sex) were decreased, while in RUPP rats (only males), circulatory glucagon was increased. At PND60, male offspring of the RUPP group had higher glucagon levels than females. At PND14, male pups (both groups) exhibited higher ghrelin levels than females, but at PND60, only male rats of the RUPP group had higher ghrelin than females. RUPP pups (both sexes) had lower ghrelin than their peers in the sham group at PND14, but only female rats in the RUPP group showed lower ghrelin levels than the sham group at PND60. Over time, ghrelin levels were unchanged in the sham group, but its levels were upregulated in RUPP pups (both sex). Results are expressed as mean ± SD. (n = 6). Tukey’s post hoc test: **p < 0.01;***p < 0.001; (a) main effect of time and (b) main effect of sex

At PND14, no differences were observed by group [F(1, 5) = 0.9366, p = 0.377] and sex [F(1, 5) = 3.599, p = 0.116] in circulatory insulin concentrations. However, at PND60, there was a statistically significant interaction between the effects of group and sex on circulatory insulin levels [F(1, 5) = 12.23, p = 0.017], with sham males having higher insulin levels than RUPP males (p = 0.005), RUPP females (p = 0.002), and sham females (p = 0.007). Assessment of insulin change over time (PND14–PND60) showed that only male offspring from the sham group displayed a significant change (p = 0.0008) (Fig. 3B).

Effects on circulatory glucagon and ghrelin

At PND14, significant effects of the group [F(1, 5) = 148.3, p < 0.001] and sex [F(1, 5) = 13.08, p = 0.015] were found on plasma glucagon levels. RUPP offspring had lower glucagon levels than the sham group, and males had lower glucagon levels than females (Fig. 3C). However, the group × sex interaction was not significant [F(1, 5) = 0.9834, p = 0.366]. At PND60, female rats exhibited lower glucagon levels [F(1, 5) = 28.27, p = 0.003] than males and no difference was observed between the studied groups [F(1, 5) = 0.2522, p = 0.633]. The group × sex interaction was not significant [F(1, 5) = 1.553, p = 0.267]. Assessment of glucagon change over time (PND14–PND60) showed that glucagon levels in the sham group (both sexes) were significantly downregulated at PND60 (p = 0.005 in males and p < 0.001 in females). Conversely, male offspring from RUPP dams exhibited an elevated trend of circulatory glucagon (p = 0.003). No significant glucagon change was observed in female RUPP pups from PND14–PND60 (p = 0.13).

At PND14, significant effects of group [F(1, 5) = 163.1, p < 0.001] and sex [F(1, 5) = 32.18, p = 0.0024] were found on circulatory ghrelin levels (Fig. 3D). Females had lower ghrelin levels than males, and RUPP offspring exhibited lower ghrelin levels than the sham group. The interaction of group × sex was not significant [F(1, 5) = 6.305, p = 0.053]. At PND60, there was a statistically significant interaction between the effects of group and sex on circulatory ghrelin levels [F(1, 5) = 20.73, p = 0.006], with RUPP females having lower ghrelin levels than sham females (p = 0.003), RUPP males (p = 0.0007), and sham males (p = 0.0005). Assessment of ghrelin change over time (PND14–PND60) showed that ghrelin levels were not changed in both male (p = 0.56) and female (p = 0.42) rats of the sham group. On the other hand, ghrelin levels were significantly upregulated in both males (t = 8.95, p = 0.001) and females (t = 10.13, p = 0.0006) of the RUPP group.

Effects on HOMA-IR and IPGTT

At PND60, HOMA-IR was varied according to sex [F(1, 5) = 22.51, p = 0.005], with females showed lower HOMA-IR than males (Fig. 4A). However, no difference was observed between the studied groups [F(1, 5) = 0.4605, p = 0.52] and the interaction of sex × group was not significant [F(1, 5) = 3.723, p = 0.11].

Fig. 4

Effects of maternal reduced uteroplacental perfusion (RUPP) on offspring HOMA-IR (A), intraperitoneal glucose tolerance test (IPGTT) (B) and glucose clearance rate, as reflected by the area under IPGTT curves (C) on postnatal days 60. Male rats in the RUPP group exhibited a slower glucose clearance than male rats in the sham group. Results are expressed as mean ± SD. (n = 6). Repeated-measure ANOVA followed by Sidak’s post hoc test was used to analyze IPGTT data. In IPGTT curves (B): *p < 0.05, RUPP males vs. Sham males; #p < 0.05, RUPP females vs. Sham females. (b) show a significant main effect of sex. **p < 0.01

At PND60, the baseline blood glucose levels prior to IPGTT were comparable across the studied groups [F(3, 20) = 1.52, p = 0.23]. However, glucose concentration during the IPGTT varied significantly with time [F(3.376, 67.53) = 791.3, p < 0.001] and group [F(3, 20) = 3.626, p = 0.0308]. In addition, interaction of time × group was significant [F(12, 80) = 2.903, p = 0.002] (Fig. 4B). Significant differences of glucose levels between the studied groups were found at 60 min [F(1, 5) = 14.27, p = 0.0129] and 90 min [F(1, 5) = 9.25, p = 0.028] following 2 g/kg glucose administration. Sixty minutes after the glucose loading, both male and female rats of the RUPP group had higher glucose levels than their sex-matched peers in the sham group (p = 0.012, and p = 0.016, respectively). However, at the studied time point (60 min), the main effect of sex [F(1, 5) = 2.87, p = 0.15] and sex × group interaction [F(1, 5) = 0.04, p = 0.83] were not significant. At 90 min after the glucose administration, only male RUPP rats had significantly higher glucose concentrations than sham males [group × sex interaction: F(1, 5) = 11.14, p = 0.020].

The overall change in blood glucose levels during 120 min following glucose administration was measured as AUC (Fig. 4C). The AUC was varied according to sex [F(1, 5) = 97.74, p = 0.0002] and group [F(1, 5) = 69.46, p = 0.0004]. Females had lower glucose AUC than males, and the RUPP group had higher AUC than the sham group. The interaction of sex × group was also significant [F(1, 5) = 6.76, p = 0.048], with male rats of the RUPP group exhibiting a slower glucose clearance than male rats of the sham group (p = 0.010).

Effects on pancreatic morphology and insulin secretion

No differences were observed by sex [F(1, 5) = 0.004, p = 0.94] and group [F(1, 5) = 0.09, p = 0.77] in surface area of pancreatic islets and also in the positive immunoreactive area per islet area [Fsex(1,5) = 0.209, p = 0.66; Fgroup(1,4) = 0.05, p = 0.83] (Fig. 5A–C). However, There was an interaction between sex × group [F(1, 5) = 20.86, p = 0.006] on the intensity of insulin immunoreactivity. Male rats in the RUPP group exhibited lower insulin intensity than their peers in the sham group (p = 0.005) (Fig. 5D).

Fig. 5

Effects of maternal reduced uteroplacental perfusion (RUPP) on offspring pancreatic islets insulin immunohistochemistry (A) at the age of 2 months. Islet area (B), insulin-positive area per total islet area (C), and quantification of insulin reactivity by optical density per islet surface area (D). Male rats in the RUPP group exhibited lower insulin intensity than male rats in the sham group. Bars represent the mean ± SD for each group, n = 6. Two-way ANOVA and post hoc Tukey’s multiple comparisons: **p < 0.01; (b) main effect of sex

Effects on offspring blood pressure and 24hUP

At PND60, no significant differences were observed in heart rate [Fsex(1,5) = 2.94, p = 0.14; Fgroup(1,5) = 1.02, p = 0.35], systolic blood pressure [Fsex(1,5) = 0.62, p = 0.46; Fgroup(1,5) = 3.12, p = 0.13], and 24hUP [Fsex(1,5) = 6.40, p = 0.052; Fgroup(1,5) = 0.71, p = 0.43] (Fig. 6A–C) between the studied groups.

Fig. 6

Effects of maternal reduced uteroplacental perfusion (RUPP) on offspring heart rate (A), systolic blood pressure (B), and 24-h urine protein excretion (C) at the age of 2 months. No significant differences were found by group and sex. Bars represent the mean ± SD for each group, n = 6. Data were analyzed by two-way ANOVA