Preeclampsia, a leading cause of maternal and fetal morbidity and mortality, is a hypertensive disorder of pregnancy characterized by new-onset hypertension after 20 weeks of gestation (systolic blood pressure >140 mmHg and diastolic blood pressure >90 mmHg), and clinical signs secondary to either hematopoietic, renal, hepatic, pulmonary, or neurological compromising (Burton et al., 2019; ACOG, 2020). The incidence of severe preeclampsia in the United States has increased approximately 6.7-fold over the course of 3 decades (Annath et al., 2013). Correspondingly, the prevalence of obesity, a major risk factor for preeclampsia, has also increased globally in the same period, with a substantial rise in the population of overweight children and adolescents (Ng et al., 2014; Olson et al., 2019). Importantly, transgenerational effects of maternal obesity and preeclampsia have been gradually unraveled, including long-term cardiovascular, metabolic, neurological and endocrine dysfunctions in offspring gestated in an obesogenic and hypertensive uterine environment (Wu et al., 2017; Fox et al., 2019; Lu and Hu, 2019).

While many investigations have focused on mechanisms underlying the risk of adverse cardiometabolic consequences in offspring from obese and preeclamptic mothers, the potential long-term reproductive outcomes remain largely unexplored (Wu et al., 2017; Fox et al., 2019; Lu and Hu, 2019). Sex-specific abnormalities in pubertal development may occur in children born from preeclamptic pregnancies (Ogland et al., 2011; Alsnes et al., 2016). In a population-based study, increased body mass index (BMI) and altered progression of pubertal development were described in daughters from preeclamptic pregnancies, with the latter being positively associated with the severity of maternal disease (Ogland et al., 2011). Notably, the pattern of pubertal development of girls exposed to preeclampsia in utero seems to differ from changes in puberty previously reported in girls affected by juvenile obesity born from uncomplicated pregnancies (Ogland et al., 2011). Peripubertal boys and girls exposed to preeclampsia in utero have also presented sexually dimorphic alterations in blood concentration of androgens at 11–12 years of age when compared to children born from uncomplicated pregnancies (Alsnes et al., 2016). Importantly, hyperandrogenism has not only been linked to altered female pubertal development, but also to cardiometabolic disorders, including insulin resistance, obesity, hypertension, and preeclampsia (Ogland et al., 2011; Mouritsen et al., 2015; Iwasa et al., 2017; Kumar et al., 2018; Zeng et al., 2020). Nonetheless, the specific pathways in the crosstalk between reproductive and metabolic abnormalities in offspring from preeclamptic mothers remain vastly speculative.

Kisspeptins are a family of small peptides encoded by the Kiss1 gene with sex-specific roles in reproduction and metabolism (Hussain et al., 2015; Dudek et al., 2018; Harter et al., 2018). Besides the central role of kisspeptins in the activation of the hypothalamic-pituitary-gonadal axis, this family of peptides appear to be important peripheral regulators of metabolism, energy expenditure, thermoregulation and adipogenesis (Hussain et al., 2015; Dudek et al., 2018; Harter et al., 2018; Izzi-Engbeaya et al., 2019; Hudson and Kauffman, 2022; Musa et al., 2022). In vitro studies suggest that kisspeptins may induce lipolysis and impair adipocyte glucose uptake in rodents and humans (Hussain et al., 2015; Pruszynska-Oszmalek et al., 2017; Dudek et al., 2018; Hudson and Kauffman, 2022). Interestingly, a sexually dimorphic metabolic phenotype has been reported in mice lacking functional kisspeptin receptor gene (Kiss1r) (Tolson et al., 2014; Tolson et al., 2016; Tolson et al., 2019). Namely, global Kiss1r knockout (KO) female mice display increased body weight, adiposity, serum leptin concentration and impaired glucose tolerance when compared to wild type littermates. Conversely, global Kiss1r KO males have similar body weight and glucose homeostasis as wild type counterparts (Tolson et al., 2014; Tolson et al., 2016; Tolson et al., 2019). Studies in gonadectomized rats suggest that both sex steroid hormones and nutrition modulate adipose tissue Kiss1 (Brown et al., 2008). Therefore, similar to the central nervous system, we speculate that kisspeptins are also the “missing link” in the crosstalk between reproduction and metabolism in peripheral tissues (Hussain et al., 2015; Dudek et al., 2018; Harter et al., 2018; Izzi-Engbeaya et al., 2019; Hudson and Kauffman, 2022; Musa et al., 2022).

The Blood Pressure High Subline-5 (BPH/5) mouse is a well-established translational model of superimposed preeclampsia (Davisson et al., 2002; Sones et al., 2021). The BPH/5 mouse resulted from an eight-way cross of the mouse strains LP, SJL, BALB/c, C57BL/6, 129, CBA, RF, and BDP, followed by multiple generations of brother-sister matings (Schlager, 1973). BPH/5 females are spontaneously obese, dyslipidemic, hyperleptinemic, hyperphagic and hypertensive, a phenotype exacerbated during pregnancy (Davisson et al., 2002; Reijnders et al., 2019; Sones et al., 2021). Even though long-term adverse outcomes occur in both BPH/5 male and female offspring, a sexually dimorphic cardiometabolic phenotype has been elucidated (Sutton et al., 2017; Beckers et al., 2021). Interestingly, while the obese phenotype is perpetuated in BPH/5 female offspring, BPH/5 males do not present increased food intake, body weight, or reproductive white adipose tissue (rWAT) mass in comparison to controls (Sutton et al., 2017; Beckers et al., 2021). Importantly, the influence of obesity in the preeclamptic-like phenotype of the BPH/5 mouse model has been clearly established (Reijnders et al., 2019; Beckers et al., 2022). Of interest, maternal weight loss via a pair-feeding paradigm not only attenuates the BPH/5 mouse maternal inflammatory milieu and pregnancy outcomes, but also improves long-term consequences in the offspring in a sex-specific manner (Reijnders et al., 2019; Beckers et al., 2022).

In this study, the BPH/5 mouse model was utilized to further investigate long-term reproductive outcomes of in utero exposure to maternal obesity and a preeclampsia-like syndrome. It was hypothesized that BPH/5 progeny would present abnormal pubertal development when compared to C57BL/6 (C57) control mice. Furthermore, the sexually dimorphic obese phenotype of the BPH/5 mouse was further explored, and a potential link between nutrition, rWAT kisspeptin expression and sex steroid hormone profile was explored, utilizing the well-established BPH/5 pair-feeding paradigm (Reijnders et al., 2019; Beckers et al., 2022).

In this study, the BPH/5 mouse model was used to further investigate transgenerational reproductive and metabolic outcomes of a preeclampsia-like syndrome. Herein, we provide evidence of sex-specific abnormalities of pubertal development in offspring prenatally exposed to an obesogenic and hypertensive uterine environment. In accordance with previous studies, the day of vaginal opening and balanopreputial separation were selected as clinical signs of onset of puberty (Lapatto et al., 2007; Caligioni, 2009; Novaira et al., 2014). While BPH/5 female offspring presented marked precocious pubertal development, age at balanopreputial separation was not different between BPH/5 male offspring and C57 controls. Accordingly, serum testosterone concentration and testicular wet weight were also similar between age matched BPH/5 and C57 males. As one could anticipate based on their younger age, BPH/5 females had lower body weight than C57 at the day of vaginal opening. Notably, however, peripubertal BPH/5 females presented similar rWAT/body weight ratio as age-matched C57, further highlighting the role of adiposity in pubertal development.

The correlation between body weight, body composition and puberty has long been recognized (Baker, 1985). Specifically, a minimum body weight and fat mass are required for onset and progression of pubertal development (Baker, 1985; Rosenfield et al., 2009). In peripubertal girls, normal age at onset of puberty ranges from 8–13 years old, and the normal progression of pubertal development is breast development, termed thelarche, followed by axillary and pubic hair growth (pubarche) and onset of menses (menarche) (Rosenfield et al., 2009; Ogland et al., 2011). There is a rising incidence of early onset of puberty in girls, which has been closely associated to juvenile obesity (Jasik and Lustig, 2008; Rosenfield et al., 2009). Notably, however, while obesity per se is associated with hastened thelarche, daughters from preeclamptic pregnancies may present an abnormal progression of pubertal development, with pubarche preceding thelarche, particularly in offspring from severe preeclampsia (Jasik and Lustig, 2008; Ogland et al., 2011). To date, the mechanisms and clinical significance of those findings are not known.

An adverse intrauterine environment has an important role in postnatal offspring outcomes of preeclampsia. Prenatal starvation and fetal growth restriction may lead to altered in utero metabolic programing, excessive compensatory growth, obesity, and adverse cardiovascular outcomes later in life (Jain and Singhal, 2012; Jensen et al., 2015). Correspondingly, children born from preeclamptic pregnancies often have higher BMI postnatally, and higher risk of developing obesity (Ogland et al., 2011; Yang et al., 2021). High rate of BMI increase from 6 to 12 years of age has been associated with increased risk of cardiovascular disease (Yuan et al., 2020; Yang et al., 2021). Additionally, the risk of preeclampsia is reportedly 3-fold higher in obese women (i.e., body mass index >30 kg/m²) when compared to lean counterparts (Mbah et al., 2010). Since daughters from preeclamptic women are more prone to develop preeclampsia (Arngrimsson et al., 1990), it is speculated that fetal growth restriction, excessive compensatory growth and obesity in girls born from preeclamptic mothers may contribute to self-perpetuation of this syndrome.

Adverse fetal programing seems to be recapitulated by the preeclamptic-like BPH/5 mouse model. When reciprocal breeding crosses of BPH/5 and C57 pairs were performed, only embryos gestated in BPH/5 dams presented delayed embryonic development (Sones et al., 2016). Additionally, BPH/5 offspring are affected by intrauterine growth restriction, evidenced by fetal demise, smaller litter sizes, and marked decrease in birth weight when compared to C57 controls (Davisson et al., 2002). Postnatally, excessive compensatory growth from birth to early adulthood has been reported in BPH/5 females, but not males (Sutton et al., 2017; Beckers et al., 2021). Specifically, while BPH/5 females are smaller than C57 controls at postnatal day 1, no difference in body weight is seen at 3 weeks of age, and higher body weight is displayed by 8-week-old BPH/5 females (Sutton et al., 2017). It is therefore speculated that higher growth rate and adiposity in BPH/5 females from birth to early adulthood may trigger earlier onset of puberty. Leptin, a metabolic hormone mainly derived from white adipose tissue and placenta, has an important role in metabolism and pubertal development, providing cues to the hypothalamic-pituitary-gonadal axis (Kiess et al., 1999; Messager et al., 2005; De Bond et al., 2016). Hyperleptinemia has been demonstrated in non-pregnant and pregnant BPH/5 females, but does not seem to occur in males, which is in agreement with the sexually dimorphic reproductive phenotype reported herein (Sutton et al., 2017; Reijnders et al., 2019; Beckers et al., 2021). Nonetheless, further investigation of serum leptin concentration and hypothalamic leptin signaling in the peripubertal BPH/5 mouse is warranted.

Hypoestrogenism and hyperandrogenism have been described in women carrying preeclamptic pregnancies and in daughters gestated in a preeclamptic uterus (Ogland et al., 2011; Alsnes et al., 2016; Berkane et al., 2018; Kumar et al., 2018; Keya et al., 2019). A protective metabolic role of estrogens in females is widely recognized, with low estrogens directly associated with increased body weight and adiposity (Leeners et al., 2017). Notably, high testosterone levels in females may further disrupt mechanisms that prevent hyperphagia and adiposity, leading to exacerbated food intake, leptin resistance, and obesity (Iwasa et al., 2017; Leeners et al., 2017). Additionally, hyperandrogenism seems to be associated with pubarche superseding thelarche in girls born from preeclamptic women (Mouritsen et al., 2015; Alsnes et al., 2016). Although 17β-estradiol and testosterone were below the assay detection limit in peripubertal females in this study, BPH/5 females presented higher reproductive tract/body weight ratios at the day of vaginal opening. It remains to be determined if the increased reproductive tract weight of peripubertal BPH/5 females is due to a sex steroid hormone imbalance or uterine inflammation, as suspected in adult BPH/5 (Sutton et al., 2017). Considering the previously reported irregular estrous cyclicity of adult BPH/5 females and hypoestrogenism during proestrus, this estrous cycle stage was selected for further sex steroid hormone profile characterization in BPH/5 female offspring (Sutton et al., 2017). Interestingly, hyperandrogenism accompanies hypoestrogenism in proestrual BPH/5 mice. The source of excessive androgens in girls born from preeclamptic mothers and in preeclamptic-like BPH/5 females is yet to be fully elucidated. Abnormal in-utero programing of steroidogenic enzyme activity, particularly aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, is speculated in preeclamptic offspring, and may also occur in the BPH/5 mouse model (Mouritsen et al., 2015; Alsnes et al., 2016; Berkane et al., 2018; Noyola-Martinez et al., 2019). Importantly, a metabolic-endocrine interplay is emphasized by the attenuation of hyperandrogenism in proestrual BPH/5 females after 1 week of dietary restriction.

Kisspeptins are considered the “gatekeepers” of the hypothalamic-pituitary-gonadal axis, since hypothalamic kisspeptin signaling is critical for pubertal development and reproduction in males and females (de Roux et al., 2003; Funes et al., 2003; Seminara et al., 2003; Lapatto et al., 2007; Tassigny et al., 2007). Besides their role in the central nervous system, kisspeptins seem to have important roles in peripheral metabolic tissues, including sex-specific regulation of adiposity (Hussain et al., 2015; Dudek et al., 2018; Wang et al., 2018; Izzi-Engbeaya et al., 2019; Hudson and Kauffman, 2022). Altered adipose tissue Kiss1 expression has been previously associated with obesity in rats and humans (Brown et al., 2008; Cockwell et al., 2013). Furthermore, studies using Kiss1r KO mice have shown that impaired kisspeptin signaling leads to increased body weight, adiposity, and hyperleptinemia in adult female mice, along with decreased energy expenditure and impaired glucose tolerance (Tolson et al., 2014; Tolson et al., 2016; Tolson et al., 2019). Conversely, Kiss1r KO males display normal body weight and glucose homeostasis when compared to WT littermates (Tolson et al., 2014; Tolson et al., 2019). Herein, we have shown a sexually dimorphic Kiss1/Kiss1r dysregulation in the rWAT of preeclamptic-like BPH/5 offspring and hypothesize that rWAT kisspeptin downregulation is linked to obesity in BPH/5 females.

A series of in vitro studies using mouse (3T3-L1) and rat adipocytes have shown that kisspeptin-10 inhibits cell proliferation and viability, reduces the intensity of intracellular glucose uptake and triglyceride synthesis, and stimulates basal lipolysis (Pruszynska-Oszmalek et al., 2017). Histomorphometry of adult BPH/5 rWAT is suggestive of adipocyte hypertrophy in females when compared to males (Parlee et al., 2014). Further mechanistic investigations are warranted to confirm if lower levels of kisspeptin-10 in BPH/5 female offspring are associated with adipocyte glucose uptake, lipogenesis, and adipocyte engorgement. Although the upstream regulators of adipose tissue kisspeptin expression are poorly understood, studies suggest that adipose tissue Kiss1 expression is influenced by nutrition and the sex steroid hormone milieu (Brown et al., 2008). Namely, exogenous administration of testosterone and 17β-estradiol to gonadectomized male and female rats, respectively, increased adipose tissue Kiss1 expression (Brown et al., 2008). Additionally, while 6 h of fasting led to adipose tissue Kiss1 upregulation, Kiss1 downregulation occurred in rats after 19 days of high fat diet (Brown et al., 2008). Accordingly, both serum testosterone and rWAT Kiss1 were ameliorated in a cohort of BPH/5 females subjected to dietary weight loss compared to ad libitum-fed counterparts. Hence, we speculate that rWAT Kiss1 downregulation in BPH/5 females may result from an abnormal sex steroid hormone profile from puberty onward, somewhat “closing the circle” between reproduction and adipose tissue regulation in a preeclampsia-like syndrome. While mice globally lacking either functional Kiss1 or Kiss1r have impaired pubertal development (i.e., hypogonadotropic hypogonadism), the BPH/5 mouse may be a suitable additional model to further study the crosstalk between reproductive hormones, adipose tissue kisspeptin dysregulation and adiposity (de Roux et al., 2003; Funes et al., 2003; Seminara et al., 2003; Tassigny et al., 2007; Tolson et al., 2019).

•In summary, beyond the long-term adverse cardiometabolic outcomes, offspring born to obese and preeclamptic-like BPH/5 dams also display sex-specific reproductive abnormalities during pubertal development and early adulthood. In BPH/5 female offspring, the crosstalk between metabolic and reproductive abnormalities seem to involve altered levels of sex steroid hormones and adipose tissue kisspeptin signaling, which may be consequences of abnormal fetal programming. Excitingly, the preeclamptic-like BPH/5 mouse model closely recapitulates clinical findings of children exposed to a hypertensive uterine environment and may be a suitable model to unravel the mechanisms underlying this altered phenotype.