Intrauterine growth restriction (IUGR), the inability of fetuses to fully develop their potential growth, currently affects up to 10% of human pregnancies (1, 2). IUGR has been associated with acute medical complications, such as perinatal asphyxia by delayed pulmonary maturation and reduced surfactant production, hypothermia, prematurity, and death (3). Furthermore, studies of human populations and experimental animals exposed to suboptimal conditions during pregnancy have demonstrated a relationship between low birth weight and an increased risk of developing metabolic disorders (altered adipose tissue distribution, insulin resistance, diabetes, dyslipidemia, and metabolic syndrome), cardiovascular alterations (structural and functional changes in the heart and vasculature leading to increased lifetime risk of hypertension, ischemic heart disease, and stroke), and other failures in different organs like the lungs, kidneys, or intestine (4).

IUGR onset is mostly related to an inadequate intrauterine availability of nutrients and/or oxygen. IUGR is traditionally found in low-income countries and related to maternal malnutrition (e.g., socioeconomic disadvantage, food shortages, or inadequate diets) or maternal hypobaric hypoxia (e.g., pregnant women living in or visiting high altitudes). However, currently, there is a strong increase of IUGR incidence in developed countries, which is linked to placental factors (abnormal placental development and/or function and shortage of transfer of nutrients and oxygen to the fetus (5)). Such a condition, named placental insufficiency, represents one of the leading causes of IUGR cases nowadays (6).

The most worrying aspect is that placental insufficiency is expected to significantly increase in the near future since it is highly related to different pathological and behavioral factors (i.e., preeclampsia, obesity, diabetes, stress, malnutrition, sedentary habits, delays in childbearing age, pollution, and alcohol and tobacco consumption). Another worrying factor is that there are no actions with proven validity to prevent or improve placental insufficiency, so current treatments are just based on changes in lifestyle and diet. Hence, there is a strong need for preventive and therapeutic strategies. Such treatments should be based on either favoring the development of the placental/fetal tissues or favoring the processes of placental angiogenesis and vasodilation.

Development of fetal/placental tissues and, specifically, fetal growth and muscle development is activated, among other factors, by insulin and Insulin Growth Factor 1 (IGF-1) (7). IGF-1 signaling is essential during prenatal development because, in addition to Growth Hormone (GH), it regulates metabolic processes including protein synthesis and tissue development of the fetoplacental unit (8, 9). Moreover, links between deficiencies in the IGF-1 pathway and prenatal and postnatal consequences of IUGR have been clearly established (10, 11). Placental insufficiency is also related to vascular disorders of the organ (12).

Current evidence highlights the links between IUGR and deficiencies in proangiogenic factors, mainly in the vascular endothelial growth factor (VEGF) and the nitric oxide (NO) route. VEGF is considered a critical regulator of lipid metabolism, and suppression of the glycoprotein VEGF-D signaling leads to decreased expression of genes associated with triglyceride and cholesterol synthesis (13, 14). VEGF also promotes angiogenesis, vasodilatation, and vascular permeability (15) through production of NO and prostaglandin I2 (PGI2) (16). Thus, it has been described that the use of low-dose aspirin would be useful to counteract IUGR in case of placental insufficiency (17) because acetylsalicylic acid increases the production of PGI2. However, of the pros of routine administration of aspirin remain controversial, with many cons noted too. NO is the main vasodilator agent in the placenta, facilitating fetal homeostasis, development, and growth (18). The amino acid L-arginine, a substrate for the enzyme NO synthase (NOS), plays a key role in the production of NO and, therefore, maternal administration of arginine has been widely studied (19).

In any case, these therapies are based on oral administration of high doses of the substances to the mother to obtain high concentrations at systemic levels and, therefore, therapeutic concentrations at the fetoplacental level, which, moreover, may be confronted by inadequate placental transfer. Hence, there is a strong trend for research on other strategies, based on fetal-focused treatments, for administering treatments either directly to the fetus or through infusion of umbilical cord, amniotic sac, or placenta. Obviously, due to the high risk of fetal injury in case of direct administration or fluid/blood loss in case of administration in the umbilical cord, placenta, or amniotic sac (20), such research has to be performed in preclinical animal models.

Currently, studies on fetal-focused strategies are mostly based on the administration of gene therapies, specifically for inducing adenovirus-mediated (Ad) overexpression of growth factors, mainly IGF-1 and VEGF, in view of their importance as previously described. Adenoviral administration of IGF-1 has been found to increase pre- and post-natal offspring development and prevent liver, musculoskeletal, and cardiovascular dysfunctions associated with IUGR in mice, rabbits, and sheep (21, 22), while administration of VEGF has been found to increase uterine flow and improve fetal growth in sheep models (23).

However, experimental data on fetal-focused therapies have been only obtained by using highly invasive methods for inoculation of the Ad (exteriorization of the genital tract by laparotomy or catheterization), so the technique cannot be extrapolated to clinical practice yet. We hypothesize that adenoviral administration of IGF-1 and VEGF may be performed by ultrasound-guided injection using an Adenoviral vector 5, reaching similar effects on IUGR development to those previously described (21–23). Hence, using a rabbit model for pregnancy and IUGR, our main aims were to assess (a) the feasibility and efficiency of an alternative minimally invasive technique, the ultrasound-guided injection, for administering an Ad expressing a reporter gene (Ad5-βgal) at either placenta or amniotic sac and (b) possible effects of IGF-I or VEGF overexpression, induced with a single Ad administration with such a technique, on postnatal growth, organ architecture, and the metabolism of the offspring.

This study reports the results of three successive trials, in which a total of 176 rabbit fetuses from 26 multiparous New Zealand x California crossbreed females were involved. The first trial determined the safety and possible long-term effects of the ultrasound-guided fetal puncture by assessing pre and postnatal development of the offspring. The second trial evaluated the efficiency of the ultrasound-guided administration of an adenoviral vector expressing a reporter gene with βGalactosidase (Ad5-βgal), which is widely employed as a marker in a broad range of biological assays due to its ability to generate a chromogenic compound in the presence of an appropriate substrate (24, 25). The third trial assessed possible changes in the postnatal development of IUGR fetuses after adenoviral delivery of growth factors.

All the experimental procedures were carried out on the experimental farm of the Polytechnic University of Madrid (UPM, Madrid, Spain), according to the European Union Directive and the Spanish Policy for Animal Protection RD53/2013, which establishes the management of animals used in experimentation and other scientific purposes, including teaching. The Committee of Ethics in Animal Research of the Polytechnic University of Madrid (UPM, Madrid, Spain) approved the experimental procedure (Safety of intraplacental injection during the prenatal period in rabbits, with authorization date of December 22, 2020). All the does were fed ad libitum with a commercial diet containing 16% crude protein, 37% crude fiber, and 3.7% fat with a gross energy content of 2,400 kcal/kg (Nanta, Madrid, Spain) in individual cages under controlled conditions of temperature (18–24 °C), light (14 h with light and 10 h with darkness), and relative humidity (60–75% RH) and free access to drink water. The average gestational length in New Zealand x California crossbreed females is 31 days and the day of the artificial insemination was considered as Day 0 of pregnancy. Ultrasound-guided punctures and therapies were performed on Day 26 of pregnancy, just before the period of overt growth arrest in IUGR offspring, which becomes apparent at around 85–90% of the total length of pregnancy (26).

The results of the present study support the feasibility, safety, and efficiency of the ultrasound-guided administration of adenovirus-mediated gene therapies in a preclinical rabbit model. Our first trial confirmed the feasibility and safety, at both short- and long-term, of the ultrasound-guided puncture at either the placenta or the amniotic fluid; no deleterious effects were found when assessing the pre- and postnatal development of the offspring. The placental route was used in the subsequent trials, despite the lack of differences in feasibility and safety, because technicians were more confident when injecting in the placenta than when puncturing the amnios. The assessment of the efficiency of the ultrasound-guided administration of the adenoviral vector, performed in the second trial, showed a lack of tissue damage and a successful passage and expression of the transgene by Ad at the fetal organs. The third trial supports the efficiency of ultrasound-guided Ad-based gene therapies for preventing or alleviating IUGR-related events and sets the basis for further studies in the field. Most studies are focused on pregnancy and the prenatal period (5); this study is one of the few to evaluate long-term effects (up to 135 days postnatal) on multiple systems (growth, metabolism, and tissue histology).

We choose a rabbit model in the present study because it is considered one of the most suitable and robust models for reproductive preclinical research due to its placental physiology, which more closely resembles that of humans compared to other commonly used laboratory animals. Rabbits, unlike rodents, possess a discoid and hemodichorial placenta, mirroring the early gestational phase of the human placenta (27), as opposed to the hemotrichorial structure observed in rodent models (28). Moreover, the larger fetal size of rabbits when compared to rodents facilitates the administration of intrauterine therapies and the real-time monitoring of fetal development with clinical ultrasound equipment. Additionally, their high fertility rate enables a reduction in the number of pregnant females required, thus aligning with the 3Rs principle of animal experimentation—Replacement, Reduction, and Refinement (29). Such advantages are further supported by the docile temperament of this species, which favors the use of non-invasive imaging techniques (30).

The ultrasound-guided transplacental technique used in the present study induced no harm to mother and fetuses and only a minimal injury to pregnancy structures (puncture point at the placenta) when applied. Previous protocols make necessary the exteriorization of the uterus to enable direct intramural administration of therapies (18, 31), so they required one or more laparotomies during gestation, which implies major surgical burden for both dam and fetus. The intraplacental route has demonstrated promising outcomes in various animal models (16, 26); however, it also implies a laparotomy. Ultrasound-guided intragastric injection techniques (24) are feasible only in sheep fetuses older than 55 gestational days, prompting consideration of more invasive alternatives such as X-ray-guided catheterization via the femoral artery to access the uteroplacental circulation.

There were no other short- or long-term consequences on either the offspring or the mother after the use of ultrasound-guided transplacental technique. Occurrence of long-term effects on fetal development has not been assessed in other techniques previously reported [i.e., oral (1, 2), subcutaneous (5), intravenous (6, 10, 32), or intra-amniotic routes (6, 11, 17)]. Concurrently, maternal safety has not been assessed in the different preclinical models of therapeutic interventions intended to mitigate fetal programming [i.e., sheep (11, 18, 19), guinea pigs (20), rats (1), or rabbits (7, 22)].

The use of our ultrasound-guided technique for the administration of adenoviral vectors was found to be successful, with passage and expression of the transgene at the fetal organs and significant positive effects on organ architecture, post-natal growth, and metabolic traits of IUGR offspring. These effects were found to be more robust when using Ad-VEGF than Ad-IGF-1, which may support these treatments based on improving angiogenesis; the fetal or intraplacental administration of VEGF specifically represents a promising strategy for preventing or counteracting IUGR effects. However, we should highlight that the present trial is only a preliminary study and the results obtained are only the basis for further research on different therapeutic agents and treatment protocols, bearing in mind that only a single administration was used.

In any case, it is noteworthy that the administration of Ad-βGal showed the highest expression of the transgene in the fetal intestine and lungs, followed by the kidneys, liver, heart, and placenta. We can hypothesize that fetal intestine and lungs are particularly exposed to Ad-based therapies because of their relatively low vascularization during intrauterine life (32); a decreased perfusion in these regions likely facilitates vector retention and accumulation, thereby enhancing transgene expression. Blood in the fetal circulatory system is preferentially directed through the ductus arteriosus, bypassing the pulmonary circuit; hence, only a small fraction of total blood volume (~6% of total volume) reaches the lungs. Similarly, fetal intestinal circulation receives a reduced blood flow (~7% of total volume) due to the minimal digestive activity during gestation.

Afterwards, the assessment of postnatal features in the offspring showed that, even in the case of a single Ad administration, like in the present study, the VEGF-based therapy evaluated in this study was effective in mitigating IUGR-related impairments. At a systemic level, the therapy administration mitigated the effects of undernutrition on fetal growth; i.e., the reduction in body weight of offspring which were found in the IUGR group of our study as in previous studies (33). At a local level, the effects of therapy administration were found particularly within the lung and intestinal tissues, which were also the tissues with higher expression in our second trial. Our findings are especially important because neonatal health critically depends on the proper maturation of both the respiratory and gastrointestinal systems, which are essential for adequate breathing and oxygenation, postnatal nutrient absorption, and immune defense and are compromised in IUGR offspring. In this way, early intestinal development is vital for optimal gastrointestinal functionality; current research has established a connection between IUGR and reduced nutrient digestibility, as well as increased microbial fermentation in the intestine, in pig models (34, 35). In such models, a 15–20% reduction in the number and length of intestinal villi found in IUGR, together with alterations in the balance between mitosis and apoptosis within the intestinal crypts and villi, have been found to affect nutrient absorption and immune protection in piglets (36, 37). It is noteworthy that, in our present study, the offspring in the IUGR-IGF and IUGR-VEGF groups showed intestinal remodeling with increased villi number; we have to note that the concomitant decrease in villous length has been previously described in previous studies on the physiological dynamics between epithelial turnover and intestinal villous length (38). Overall, our results suggest that the proposed therapies may exert structural remodeling effects in intestinal and pulmonary tissues, potentially contributing to improved functional outcomes in IUGR-compromised fetuses. Hence, these findings pave the way for future studies that include digestibility assays, which may provide valuable insights into intestinal functional development and nutrient absorption capacity and provide improvement in the effects of fetal inflammatory response syndrome (39).

The assessment of postnatal metabolic features in the present study, as in the histological study, were significantly weakened by the lack of plasma and tissue samples in the control groups due to animal welfare limitations imposed by our Ethical Committee, which estimated that our research group had enough comparative data on normal birth-weight, IUGR, and supplemented offspring from previous studies (22, 34, 40, 41). Hence, the present study was based on possible improvement of the IUGR condition by the proposed therapies. In fact, our results clearly showed that the administration of VEGF during intrauterine life demonstrated a protective effect against IUGR-related alterations—again, greater than in the case of IGF-based therapy. Blood HDL concentrations were higher while LDL-c, triglycerides, lactate, and urea were lower after both treatments, but VEGF-based therapy resulted in a more pronounced reduction in NEFA levels. Free fatty acids, or NEFAs, are protein-bound lipid biomolecules that represent approximately 98% of total serum fatty acids and constitute the principal building blocks of triglycerides. The serum fatty acid profile is widely recognized as a biomarker for assessing the risk of various metabolic and cardiovascular disorders (42). NEFA release is typically stimulated by lipoactive hormones, including epinephrine, norepinephrine, glucagon, thyrotropin, and adrenocorticotropic. In this regard, future research could benefit from quantifying the circulating levels of these hormones to elucidate possible variations in endocrine responses associated with VEGF or IGF-1 treatment, thereby contributing to a more comprehensive understanding of their metabolic effects.

Our present study indicates the feasibility and safety of the ultrasound-guided administration of adenovirus-mediated gene therapies and will aid further investigations involving multiple and/or higher doses in order to achieve significant therapeutic outcomes that could be the basis for translational research into clinical practice.