Animal models and alternatives in vaginal research: a comparative review

Abstract

While developments in gynecologic health research continue advancing, relatively few groups specifically focus on vaginal tissue research for areas like wound healing, device development, and/or drug toxicity. Currently, there is no standardized animal or tissue model that mimics the full complexity of the human vagina. Certain practical factors such as appropriate size and anatomy, costs, and tissue environment vary across species and moreover fail to emulate all aspects of the human vagina. Thus, investigators are tasked with compromising specific properties of the vaginal environment as it relates to human physiology to suit their particular scientific question. Our review aims to facilitate the appropriate selection of a model aptly addressing a particular study by discussing pertinent vaginal characteristics of conventional animal and tissue models. In this review, we first cover common laboratory animals studied in vaginal research – mouse, rat, rabbit, mini-pig, and sheep – as well as human, with respect to the estrus cycle and related hormones, basic reproductive anatomy, the composition of vaginal layers, developmental epithelial origin, and microflora. In light of these relevant comparative metrics, we discuss potential selection criteria for choosing an appropriate animal vaginal model. Finally, we allude to the exciting prospects of increasing biomimicry for in vitro applications to provide a framework for investigators to model, interpret, and predict human vaginal health.

Introduction

Women world-wide face a number of potential gynecologic issues over the course of their lifetime. In terms of issues specifically related to the vagina, women can develop a number of maladies. Between 15–50% of reproductive aged women are diagnosed with bacterial vaginosis, and up to 65% are diagnosed vulvovaginal candidiasis[1–3]. Additionally, rare congenital conditions such as Mayer-Rokitansky-Küster-Hauser Syndrome (1 in 4500 live births) or androgen insensitivity syndrome (2–5 in 100,000) result in vaginal agenesis[4–6]. Up to 50% of post-menopausal women experience symptoms of vaginal atrophy[7, 8] with up to 50% developing some degree of pelvic organ prolapse[9, 10]. Lastly, radiation therapy for gynecologic and colorectal cancer disrupts normal vaginal homeostasis[11, 12], Often treatment for these complications is limited or the treatment may lead to additional complications such as vaginal stricture and fibrosis[11, 12]. In order to facilitate innovation and novel treatment options to improve women’s vaginal and gynecologic health, the ability to model both healthy and diseased vaginal tissue is imperative.

Ideally, a vaginal tissue model captures all of the native complexity of the human vagina while providing the modular control over experimental parameters. New developments in in vitro model systems and computer modeling to study vaginal tissue abnormalities have led to exciting new findings related to vaginal toxicity and vaginal prolapse[13–15]. Commercial availability of human vaginal epithelial cells has also provided a new avenue to study vaginal health and disease[16]. Equally important has been the development of protocols to isolate primary vaginal cells from patient biopsies[17, 18]. While important advances, each have inherent limitations such as inability to simulate Valsalva pressures to the vagina, vaginal cultures lacking appropriate tensile strengths, cellular compositions or permeability properties, cost, patient availability, and multiple regulations. As such, mammalian vaginal tissue models can provide unparalleled insights into the anatomic, biologic, physiologic as well as hormonally responsive elements of the vagina.

The most suitable animal model of vaginal physiology and disease has yet to be established[19]. While literature searches for generic terms such as vagina or vaginal tissue yield almost 50,000 articles, more narrow searches like animal models for vaginal research yield significantly fewer articles (completed Aug 2020). There have been in-depth reviews written to evaluate models of vaginal irritation for toxicity studies[15] as well as the biomechanical properties of vaginal tissues as it relates to pelvic organ prolapse[20]. Here we will review the vaginal anatomy and physiology, as well as the cost of five different animals commonly used in vaginal research, including mice, rats, rabbits, minipigs, and sheep. We will also review basic human vaginal anatomy and how it compares to the different animal models. We provide a perspective of vaginal tissue engineering systems recent progress. Lastly, we will discuss the considerations and concessions investigators must make when choosing an animal model or tissue engineered model as well as the current state of the field. Our hope is that this comprehensive review can be used to aid and guide investigators in choosing and developing animal models which will be most useful in mimicking the human condition, in turn allowing accelerated innovation in the field of vaginal tissue impairments.

Key features in common animal vaginal models

While mouse, rat, rabbit, mini-pig, and sheep models are commonly used in vaginal research, these animal models do not fully encapsulate the complexity of the human vagina. In the following sub-sections, this review elaborates on species-specific vaginal properties. Each sub-section focuses on one species discussing the reproductive cycle and related hormones, basic reproductive anatomy, the comprising vaginal layers, the controversial developmental epithelial origin, and a brief discussion of the local microflora environment. We conclude each sub-section with a commentary on how the particular animal model relates to the human vagina. In the final sub-section, the human vagina is described underscoring the challenges in recreating a model embodying all of its characteristics.

Mouse

While the mouse model has many advantages such as relatively low cost, short generation time, prolific breeding, and generally well-established biological and genetic studies, particular structural differences between the mouse and human vagina may need to be considered before a murine model is selected[15, 21]. The gross anatomy of the female reproductive system of the mouse differs from human, however both exhibit similar histological features, cyclic estrus/menstrual cycles, and basic functions[22]. This section will specifically elaborate on the female mouse reproductive biology and discuss associated insight gained from previous studies.

The female mouse reaches sexual maturity between five to eight weeks. During the breeding period, the mouse undergoes cyclic changes called the estrus cycle approximately every four days. The estrus cycle can be distinguished as four separate stages: proestrus, estrus, metestrus, and diestrus[23]. Each stage is associated with specific features that can be used to identify which phase the mouse is in. The proestrus phase is characterized by active growth where eggs reach maturity in the ovarian follicles. There is thickening in the uterine mucosa, and a vaginal smear would show a similar ratio of nucleated epithelial cells to leukocytes. The estrus stage is also a proliferative phase lasting about one day. Anucleated squamous epithelial cells and very few leukocytes are present in a vaginal smear. During metestrus, the epithelium degenerates in a catabolic phase. The vagina is lined with keratinized epithelium and large numbers of leukocytes are present. Lastly, diestrus describes the quiescent phase in which the endometrium (uterine lining) collapses, and a vaginal smear exhibits a high number of leukocytes[24].

Hormone signaling varies across the different estrus stages thereby functionally affecting the vaginal mucosa. Previous studies have shown that estrogen related hormones mediate tissue-level processes such as epithelial permeability[25], growth factor interactions[26], vaginal cell proliferation versus differentiation[27], and protection[28]. Though many groups have explored estrogen in the context of vaginal tissue, there remains much to be understood about the governing mechanisms of vaginal tissue repair and healing. Interestingly the vaginal tissue is capable of incredible transformation during the span of each cycle due to the changes of hormone levels. Higher levels of estrogen, during the estrus phase, results in reduced transepithelial resistance, or greater permeability, implying that the monolayer integrity is impacted. Since the barrier function of the vaginal epithelium is critical for protection against pathogen entry to the submucosa, understanding such hormonal effects may have vast implications on unintended treatment effects[25]. However, keratinized vaginal tissue during the estrus phase has reduced permeability and improved resistance to vaginal tissue damage[29]. Such nuances define mouse specific vaginal tissue.

The gross anatomy of the female mouse reproductive system is separated into two parts: the ovaries (contained in a fat pad called an ovarian bursa) and genital tract[24]. Not only are multiple follicles maturing simultaneously, but also multiple ova are released at ovulation lending to large litter sizes between 5–12 pups[30]. The genital tract comprises the bicornuate uterine tubes (oviducts leading to uterine horns), uterus, and vagina which remains independent from the urethral tract[24]. External to the reproductive tract is the vulva. The uterus has two separate uterine horns and has a body divided by a uterine veil. In the upper region of the uterine horns, there are many folds that gradually reduce when nearing the lower uterine body toward the cervix. Therefore, in the prevaginal portion, the cervical canal is double; but within the vaginal portion, there is a unique orifice. The copulatory organ, the vagina, describes the region between the cervix to the external vulva and is approximately 1 cm in length.

The uterine tube is composed of three layers: tunica mucosa, tunica muscularis, and tunica serosa. The tunica mucosa is the inner, lumen-facing layer subject to changes during the mouse’s hormonal cycle. Columnar epithelial cells are both ciliated and non-ciliated, the ratio of which is dependent on the stage of the estrus cycle. The uterine wall is also tri-layered with the tunica mucosa (or endometrium), tunica muscularis (or myometrium), and the tunica serosa (or perimetrium)[24]. In the endometrium, there are columnar epithelial cells and mucus-secreting, uterine glands contained in the lamina propria. However, the layer’s specific composition is related to the location along the uterine tract. Additionally, the simple columnar epithelium gradually transitions to stratified squamous epithelium. Also, the myometrium thins to an almost non-existent layer near the cervix. Interestingly, the overall endometrium morphology remains relatively the same throughout the estrus cycle. The tunica mucosa of the vagina is made up of stratified squamous epithelium and a fibrous lamina propria. Over the course of the estrus cycle, the epithelium changes vastly in response to the hormonal state from thick keratinized epithelium to a thin epithelium[24]. These are marked differences between the mouse and human responses during each phase of estrus. While the mouse endometrium remains relatively stable, the human endometrium responds differently to each stage. On the other hand, the mouse vaginal mucosa changes according to each estrus phase while the human vaginal mucosa maintains a stable morphology throughout[22].

Recently, there have been considerable advances in understanding the origins of vaginal epithelium[31]. The developmental origin of the vaginal epithelium has been under debate with many groups suggesting various degrees of contribution of Müllerian duct and/or urogenital sinus sources. Most mammals can trace female reproductive tract development to the embryonic Müllerian ducts. In the continuation of development, the Müllerian duct fuses with the urogenital sinus[32]. However, historic models to understand vaginal epithelial origins have relied on anatomical and histological inferences[33–35]. More recently, Kurita has found that vaginal epithelium is solely derived from Müllerian duct in the adult female mouse by using immunohistochemistry to label PAX2 and OSR1 which specifically target Müllerian and urogenital sinus epithelial cells respectively[36]. Results showed that the adult mouse vaginal epithelium was only positive for PAX2 (the Mullerian marker) potentially meaning that its development may be distinct from the human vagina[31, 36].

The murine vaginal microbiota exhibits distinct community populations that are dominated by one bacterial taxa as notable in humans. While there is likely variation across strains, vivaria, and vendors, there seem to be consistent bacterial presence from Staphylococcus, Enterococcus, and Lactobacillus. Interestingly, the commensal bacteria does not appear to vary across the estrus cycle[37].

Certain elements of the mouse female reproductive tract such as those discussed in this section bring into question its utility in vaginal research. Therefore it is critical to recognize and understand the mouse reproductive biology to provide context to each study.

Rat

Similar to the mouse model, rats have the potential to serve as a useful vaginal model due to their ease of access, low-cost, and similar reproductive cycle (to human). Many of the existing studies utilize female rat models (i.e. Wistar, Sprague Dawley) for understanding the vaginal environment[38–40] therefore this section will serve to describe anatomical, morphological, and functional characteristics of the rat vagina.

As with other species, the estrus cycle is associated with hormonal changes that manifest in morphological and functional differences in the vagina[41, 42]. Notably the epithelium is completely replaced every cycle and has an associated thickness with distinct stages of the cycle as well as specific regions of the tissue[41]. Each stage - proestrus, estrus, metestrus, and diestrus - presents characteristic cell types in a vaginal smear commonly used to identify the rat’s present point in the cycle as well as ancillary features (i.e. estrogen levels)[43]. Generally, leukocytes are low in proestrus and estrus however increase during metestrus and diestrus. The presence of leukocytes coincides with high progesterone giving rise to a neutrophil influx. Inversely, epithelial populations are highest during proestrus and estrus but decrease during metestrus and diestrus[42]. Specifically, during proestrus, a vaginal smear shows clumps of nucleated squamous epithelial cells. During estrus, the vaginal smear reveals an increase of large, cornified epithelial cells. In metestrus, there appears a high yield of leukocytes with some cornified epithelial cells. Lastly, a vaginal smear in diestrus contains leukocytes and few inconsistently shaped epithelial cells[43].

The female rat features a similar reproductive tract to the mouse with paired ovaries and oviducts that connect to bicornuate uterine horns. The uterine horns come together to form the vagina. The vagina leads to an external orifice, the vulva, separately from the urethral orifice maintaining two distinct lumens[44–46]. The vagina itself is between 15–20 mm in length with a distended diameter between 3–5 mm. Many folds are present dorsally to increase distensibility. Vaginal tissue is composed of a mucosa, muscularis, and adventitia layer. The lumen facing mucosa, consisting of stratified epithelium, is subjected to cyclic changes. An exception is near the vulva where the epithelium is perpetually cornified. The underlying lamina propria contains fibroblasts and a collagen rich environment. The muscularis layer consists of smooth muscle cells which are regionally variant. The anterior smooth muscle is thick and forms a mesh-like network longitudinally and obliquely. However, the smooth muscle diminishes gradually toward the distal (vulvar) end[43]. Sexual maturity aligns with the female rat’s first vaginal opening accompanied by the first proestrus usually 33–42 days after birth[41]. After this point, female rats exhibit cyclic reproductive stages every 4–5 days in the estrus cycle[47]. The gestation period is between 21–23 days producing litters between 6–13 pups[48].

While the rat vagina serves as a useful model for demonstrating cyclical changes, it also provides a framework for studying vaginal tissue due to its similarities in gross anatomy[43], structural/biomechanical properties[38, 49–51], and wound healing[52]. The vaginal epithelium is known to vary across the cycle, and these changes are closely associated with hormonal fluctuations linked to each stage. Therefore many studies investigate estrogen relationships with vaginal tissue[51, 53]. As with the estrus stage, estrogen treatment in ovariectomized rats induces epithelial cornification and thickening of the vaginal epithelium. Additionally, the vaginal muscalaris in estrogen treated groups features well organized smooth muscle bundles which allow for recovered distensibility demonstrated by decreased vaginal stiffness[51]. In a separate study, the ratio of collagen III to collagen I influenced vaginal compliance. Relatively high collagen III subtype is found in healthy rat vagina, thus providing high compliance, just as found in healthy human vagina. However, estrogen withdrawal from rat ovariectomy resulted in increased collagen I subtype thereby increasing vaginal wall stiffness[54]. In these cases, ovariectomized rats might provide parity to post-menopausal women experiencing decreased estrogen levels. For wound healing studies in the rat vagina, groups have explored the balance of matrix metalloproteinase (MMP) degradation and synthesis[52] and macrophage polarization[55] after vaginal injury. It was found that even if the epithelium heals, the underlying muscularis remains compromised thus diminishing vaginal wall biomechanical properties. By applying broad spectrum MMP inhibitors, wound healing improves and vaginal strength is recovered[52].

The embryology of the rat vagina appears to have Mullerian duct and urogenital sinus origins. The rat’s vagina forms from Mullerian duct fusion[56]. However, some studies suggest that the urogenital sinus epithelial cells proliferate to develop into the lower portion of the vagina[57]. As with the vaginal embryology of other species, the origins proposed warrant further exploration for a conclusive developmental understanding.

While the estrus cycle influences the vaginal flora much like in humans, the presence of specific bacteria like gram-negative rods, streptococci, and Bacteroidaceae members in rat vagina may differ from humans[58]. Interestingly, this dynamic vaginal microbiome can be influenced by various stimuli (i.e. neural electrical stimulation) in terms of diversity rather than abundance [59]. Generally for humans, a less diverse vaginal microbiome is considered more stable and thus healthier [60]. Since groups have shown that the microbiome can be manipulated into a more stable bacterial population, rats vaginal microflora may serve as a relevant comparison if focusing in this area [59].

The structural similarities of the vaginal wall between rat and human allow investigators to observe effects of their studies on the rat vagina and potentially extrapolate to the human. While there is a substantive knowledge base on rat vaginal properties, there remains many questions that may impact the utility of this model. As with other species, the rat vagina has several regional differences, perhaps of various embryonic origin, that require further investigation. These few known and perhaps more unidentified key differences in the vaginal environment might influence the appropriateness of the rat model for human relevance.

Rabbit

The rabbit serves as the gold standard model for FDA mandated preclinical assessment of vaginal irritation therefore such studies often use rabbit vaginal tissue models[15]. Common breeds for these studies include New Zealand White[61] and European (O. cuniculus)[62] rabbits. Both structurally and functionally, the female rabbit differs from the female human. This section will detail some of the anatomical and functional features that are specific to the rabbit.

Rather than an estrus cycle, female rabbits exhibit induced ovulation where 10–13 hours post mating, the female will ovulate. Alternatively, ovulation can be induced by a luteinizing hormone injection[48]. While there may not be an estrus cycle, there is an associated cycle of receptivity about every 5–6 days. This period of receptivity is manifested by a coloration shift of the vulva. When the vulva is enlarged and red/purple, the female rabbit is more receptive to mating[63], although rabbits are capable of and sometimes do mate at any time. Unlike mammals that experience an estrus cycle, rabbit vaginal smears do not provide useful information about receptivity. Rabbits are prolific breeders reaching sexual maturity by approximately 4–6 months (however, larger breeds take longer to reach sexual maturity by around 5–8 months). The gestation period is dependent on specific breeds but usually lasts between 30–32 days resulting in litters anywhere between 4–12 young. Female rabbits have a reproductive life of about 3 years[64].

The female rabbit features completely paired organs for the ovary, oviduct, and uterus. In contrast to many other mammalian species, the uterus is completely paired not featuring any kind of partial fusion. The bicornuate, coiled uterus (better characterized as didelphys uteri) also comprises paired cervixes that separately open into a single, elongated vagina approximately 13–14 cm in length [65]. The urethra also connects to the vagina near the midway of its vaginal canal.

There are several considerable differences between the rabbit and human vagina. The majority of the rabbit vaginal epithelium consists of a thin layer of columnar epithelial cells. Perhaps due to this structural difference, the vaginal surface is highly susceptible to irritability to a greater degree compared to the thicker layer of human vaginal epithelium composed of stratified squamous epithelial cells[15, 66]. Moreover, the vaginal epithelium presents little to no secretion[58].

The rabbit vagina has several regional differences functionally and morphologically. The vagina is divided into three sections (upper, middle, and lower) with location-dependent abundance in smooth muscle arrangement as well as contraction/relaxation responses. The upper and middle regions of the vagina are highly associated with smooth muscle lending to strong contractile responses. On the other hand, the lower region contains smooth muscle bundles interspersed with sinusoids allowing for mediated relaxation[67]. Additionally, the mucosal layer thickens while the external layer inversely thins from pelvis to perineal regions of the vagina which could be associated with the varying contractile capacity[62].

As for embryologic origins, the vagina develops from multiple sources. The upper portion is derived from a fusion of the Mullerian ducts whereas the lower region is formed from the urogenital sinus. Outgrowths from the corresponding sources join to comprise the fully developed rabbit vagina[68]. Moreover, the epithelia reflect these differences by exhibiting distinct morphological features (e.g. columnar, non-stratified in lower region)[68, 69]. The apparent composite nature of the vagina mirrors other mammalian species but likewise requires further studies as this topic has historically discrepant theories.

Different acidity levels from the lack of Lactobacilli might be another potential contributor to varying levels of vaginal tissue sensitivity[15, 58]. In many other mammals, the estrus or estrus-like cycle greatly influences the vaginal microflora. However, because the female rabbit does not possess these cyclic stages, they do not generally harbor vaginal flora. Noguchi and colleagues found no isolated bacteria from 90% of the vaginal specimens [58].

While the rabbit vagina serves as the standard model for determining vaginal irritation, this discussion raises several considerations for a scientific researcher to pursue this animal model. The lack of an estrus cycle and normal microflora may have tremendous implications on its relevance to the human vagina. For example, the vaginal tissue permeability may be dependent on the concurrent estrogen levels that would otherwise vary across a cycle³⁴. However, perhaps a rabbit may serve as a stable comparison model removing the hormonal fluctuations (and downstream changes) that occur alongside a cycling animal model.

Minipig

Pigs provide another potential animal model for studying human diseases and wound healing. They have become a promising alternative to other non-rodent models including dogs or non-human primates due to their small size and similarities to human, as well as having fewer ethical concerns than non-human primates[70]. Pigs have been used to study and advance the fields of neurology, diabetes, wound healing, and are widely used in toxicology studies[70–72]. Pigs and pig tissue are readily available from various farms and slaughterhouses, which while cost effective, come with a variety of issues including potential for infection and high variability as well as the ergonomic and housing difficulties of using an animal with an average weight of >300 kg[73]. Because of this, minipig use has gained traction. There are several breeds of minipig available for biomedical research including Gottingen, Yucatan, Sinclair, and Hanford[74]. The Gottingen is the smallest, with an adult average weight between 30–45kg, and is the one most widely used, and therefore will be the main focus of this review[70, 73, 74].

The Gottingen species was developed in the 1960’s at the University of Gottingen in Germany and are out-bred in barrier facilities for research purposes, decreasing variability and chances of disease and infection[70]. Gottingen minipigs reach sexual maturity around 4–5 months if they are in the presence of a boar and by 6–7 months without a boar present[75]. At this age they are between 12 and 14kg[75]. Once they reach sexual maturity, they continuously ovulate and have an average of a 21-day cycle[76, 77]. The reproductive cycle in minipigs is divided into two stages: estrus, when a sow is receptive to sexual behavior, and non-estrus[78]. Alternatively, the estrus cycle can be divided into a follicular phase (5–6 days long) and a luteal phase (15–17 days long) based on hormone levels and the histologic appearance of the ovaries[78]. Unlike in human reproduction, ovulation is considered day 0, occurs during the midpoint of the follicular phase, and correlates with the lowest progesterone levels[76, 78]. Just prior to ovulation and at the beginning of the follicular phase (day 18–21 of previous cycle) estrogen and follicle stimulating hormone peak and rapidly decrease while progesterone peaks during the luteal phase[76, 77]. Both vaginal swab cytology smears and circulating progesterone levels can be used to determine what stage of the estrus cycle a minipig is in, which may influence research results[79, 80].

Anatomically, Gottingen minipigs have a bicornate uterus, a 7.5 cm cervix length which has several folds of mucosal tissue, and a vaginal tract of 13–14 cm in length[78]. Similar to other animal models mentioned above, minipigs have a single UG sinus with the urethra opening into the vaginal canal on the ventral surface approximately 1/3 to 1/2 way from the introitus to the cervix[78, 81]. At the histologic level, the vagina consists of a non-keratinized epithelial layer above the lamina propria, which consists of loose connective tissue, fibroblasts, and blood vessels, and finally a smooth muscle layer[78, 82]. While other reproductive organs undergo major histologic changes during the estrus cycle, the vagina undergoes more subtle changes[76]. The basal layer remains steady throughout the cycle, consisting of cuboidal to columnar cells, followed by up to 10 intermediate levels[76, 83]. During the follicular phase of the reproductive cycle and prior to ovulation, the luminal epithelium of the vagina consists of stratified squamous epithelium, that become flattened after ovulation[76, 83]. During early luteal phase, the superficial layer contains apoptotic cells and granulocytes infiltrate[76, 83]. While apoptotic bodies remain into late luteal phase, granulocytes are no longer present[76, 83].

Little research into the embryologic origin of the minipig vagina has been completed. From work published in the early 1930’s, the upper vagina was formed by fusion of the Mullerian ducts while the lower vagina was formed from the Wolffian ducts[84]. This is contradictory to the current thought that in mammals, including pigs, the vagina is formed from fusion of Mullerian ducts alone, with Wolffian ducts regressing[85]. It is currently unknown if the entire vagina is Mullerian origin, or if the urogenital sinus contributes.

In addition to both macroscopic and microscopic differences in animal models, increasing evidence of the importance of the microbiome indicates these differences must also be considered. In the case of the Gottingen minipig, the vaginal microbiome is largely made of unassigned species of Gammaproteobacteria, Enterobacteriaceae, and Clostridiales[86]. Unlike in humans, the microbiome is relatively stable throughout the estrus cycle and results in a largely neutral vaginal pH (7–7.2)[78, 79, 86].

When compared to rodent models, the minipig is more costly in both initial procurement, housing, and veterinary services. The knowledge and skills required to successfully complete minipig studies are also less likely to be widely known[87]. In addition to being regulated by the PHS policy (The Guide), pigs are regulated by the USDA Animal Welfare Act, meaning there are additional regulations placed on them compared to rodent species[88]. Despite this and due to the similarities with human anatomy and physiology the minipig is a valuable option for studying vaginal wound healing. There are a limited number of genetically modified minipigs available currently, however with the increase in CRISPER/Cas9, more are likely to become available, further increasing the advantages of minipigs in research[89].

Sheep

Increasingly, sheep have become the selected animal model for reproductive research due to their similarities in anatomical size, fetus to pelvis ratio, and spontaneous development of pelvic-organ-prolapse. Investigators primarily interested in biomechanical studies may consider this animal model as appropriately addressing their research question. Sheep are additionally advantageous because they are readily available, relatively inexpensive, and docile (lending them easy to handle)[19]. However, there are some notable differences such as a single urogenital sinus that may impact one’s consideration for utilizing the sheep model[90]. In this section, we will review the ewe’s basic reproductive anatomy and characteristics as well as their physiological vaginal properties to better capture the potential benefits and shortcomings of the sheep for a vaginal model.

While the estrus cycle matches the four phases of other non-human mammals, the sheep’s estrus cycle is seasonally influenced typically occurring naturally in the fall within North America[91]. The number of light hours governs hormonal release which in turn regulates the estrus cycle (which can perhaps be controlled tightly in an indoor laboratory facility). Within this seasonal influence, sheep are polyestrus. The length of the full estrus cycle averages about 17 days with the estrus stage lasting between 24 to 36 hours. Without a ram present, signs of the ewe’s estrus cycle may not be detected. Sheep typically reach sexual maturity between 5 and 12 months of age. Their gestation length varies between 142–152 days (or about 5 months), and they typically give birth to one lamb though up to three is not uncommon. Sheep have a reproductive life span of approximately 5.5 years[92].

The female sheep, or ewes, feature a reproductive anatomy similar to humans in that their paired ovaries ultimately connect to a single uterine body before linking to the cervix and vagina. There are indeed bicornate uterine horns; however, a single uterine body is present and is the dominant location of fetal development[90]. Like rabbits and pigs, the urethra joins the vagina near the introitus to make a common reproductive and urinary tract. The cervix to vagina ratio is fairly high with the cervix length only a few centimeters shorter compared to the vaginal length (up to 8 cm – cervix; 10–12 cm – vagina).

Histological morphology of the sheep’s vagina features similar layers to the female reproductive tract. The sheep epithelium consists of stratified squamous epithelial cells and an underlying submucosa rich with vasculature much like the human vaginal layers albeit with a thinner epithelium. Interestingly, the sheep’s vaginal epithelium can serve as a predictive toxicology screening model due to such similarities. Vincent, et al. has even shown that the sheep’s vaginal epithelium mirrors the sensitivity of female epithelium in its thinning response to increasing doses of an agent, BSK (benzalkonium chloride), known to disrupt vaginal epithelium in humans[93]. Even though rabbit vaginal irritation models are currently the regulatory standard, the vast differences in sensitivity between those species (rabbit comprising only a single layer of columnar epithelial cells vs. relatively thick epithelium in human) may consequently rule out valuable pharmaceutical candidates. Thus, a few groups have turned to sheep as an alternative model for more relevant testing of contraceptives[94] and vaginal microbicides for prevention against infections like HIV[93, 95].

On a biomechanical level, the sheep’s pelvic floor and specifically the vagina offer many similarities to women. Since vaginal wall properties are impacted by life events (i.e. puberty, vaginal delivery, menopause), the sheep can provide a parallel model environment in terms of some of these life events[19]. With lower amounts of estrogen associated with menopause, women’s vaginas have stiffer mechanical properties; sheep likewise exhibit stiffer vaginal properties in ewes with low levels of estrogen (from oopherectomy) attributed to higher levels of collagen content[19]. However, mechanical properties like viscoelasticity may not be sensitive to strain which differs from women’s vaginal tissue[20]. Additionally, Rubod, et al. found no differences in biomechanical behavior in the anterior versus posterior vaginal walls which may or may not parallel women’s vaginal tissue (challenging to gather large, intact, and healthy human samples), but can certainly aid in negating location sampling as a potential contributor to biomechanical influences. This finding is particularly interesting due to the bipedal versus quadrupedal nature of the respective species. Nevertheless, there is evidence to suggest that orientation does matter as vaginal tissue is anisotropic therefore directionality of stretching will influence mechanical behavior[96]. Additionally, researchers are increasingly enthusiastic about the sheep model because of their capacity to spontaneously develop pelvic organ prolapse (POP). As such, investigators studying POP-related mechanisms and treatments should consider the sheep as a useful model[20].

The embryonic origins of the vagina differ regionally where the upper vaginal epithelium is of Mullerian origin and the lower vagina merges gradually into urogenital sinus-derived epithelium[97]. However, these findings are dated and would merit further investigation if this holds true given the controversy in vaginal development.

Though the epithelium generally serves as the first line of defense against infection, the vaginal commensal environment plays a critical role in preventing microbial penetration into underlying tissues[90]. In contrast to the dominant microflora present in women, ewes present low amounts of lactobacilli resulting in a more neutral vaginal environment (6.7 + − 0.38 pH)[98]. Therefore, understanding the needs of one’s study, whether that be providing the complementary acidic environment or appropriate epithelial thickness, ought to influence the utilization of this sheep vaginal model.

Taken together, the sheep appears to provide a suitable vaginal model for many reproductive investigations. In this section, we cover details of the anatomical similarities that are beneficial for a large, relatively inexpensive animal model. When it comes to addressing surgical techniques, developing size-matched medical devices, studying certain biomechanical properties, these similarities allow for predictive interpretation. However, there are few notable differences such as the microflora and specific aspects of biomechanical properties that may deter its use.

Human

As one of the primary goals of basic science research is to discover how the human body responds and recovers from various physiologic processes, insults, and injuries, it is vital that any animal model mimic, as best as possible, human physiology. The above sections have outlined the estrus cycle, reproductive tract anatomy and physiology, with specific focus on the vagina, and pros/cons of several animal models. This section will provide a description of the female human reproductive cycle, a brief description of the reproductive tract, and an in-depth look at the vagina, both macroscopically and microscopically for comparison.

Human females reach sexual maturity around the age of 11–12 and continue to cyclically ovulate[99]. Each menstruation cycle is an average of 28 days and consists of a follicular phase and luteal phase, with ovulation occurring between the two phases[99, 100]. The reproductive cycle and related hormones are under control of the hypothalamic-pituitary-gonad axis[101]. During the follicular phase, gonadotropin releasing hormone from the hypothalamus stimulates the pituitary to release follicle stimulating hormone (FSH) and luteinizing hormone (LH)[100, 102]. FSH and LH signal for a subset of primordial follicles within the ovary to begin the maturation process[99]. FSH is highest in the first week of the follicular stage and has a small peak just prior to ovulation[100]. LH is relatively stable with the exception of a large peak which stimulates ovulation[100]. As the ovarian follicle matures, estrogen is produced and released, peaking just before ovulation[100, 102]. Following ovulation is the luteal phase consisting of an increase in ovarian progesterone production, from the corpus luteum which peaks around day 21–23 of a the 28 day cycle[100]. If the released egg is not fertilized, the corpus luteum wanes, progesterone production decreases, and FSH levels begin to rise signaling for menses to start[99, 102]. This cyclical hormone release continues every 28 days until a woman reaches menopause between the ages of 45–55 (average age of 51 years)[99].

The reproductive tract of female humans consists of two ovaries, approximately the size of almonds, which release a mature egg once during ovulation of each reproductive cycle that is then taken up by the fallopian tubes and deposited into the uterus[100]. If fertilization occurs, an embryo attaches to the uterine wall to develop[99]. If fertilization does not occur, the excess uterine lining that was built up in response to estrogen and progesterone during the reproductive cycle is shed[99, 100]. The uterus narrows at the base to form the cervix, which opens into the vaginal lumen that then leads to the outside of the body at the vaginal introitus[99, 100].

The macroscopic shape and size of the human vagina has been characterized using impression casting and imaging including magnetic resonance imaging (MRI)[103, 104]. The linear length is 6–10 cm, with the anterior vaginal wall measuring 6–7 cm and the posterior vaginal wall measuring 8–10 cm[104, 105]. This is due to the curved shape of the vagina as it passes beneath the pubic bone and into the pelvic cavity[105]. Near the cervix the vaginal diameter is larger, 3–5 cm, and narrows as it reaches the introitus, 1.5–2.5 cm[105, 106]. At the microscopic level, vaginal tissue in menstruating females has a substantial (average of 28 layers) epithelial layer of non-keratinized stratified squamous epithelium which is fully replaced every 96 hours[107]. As cells move from the basal layer towards the lumen, the nucleus is lost and become terminally differentiated[107]. Unlike rodents, they do not become keratinized, but cornified and contain large quantities of glycogen deposits[107]. In pre-pubertal and post-menopausal women, this layer is significantly thinner[108, 109]. Unlike the endometrial thickness that is influenced by fluctuations of hormones throughout the menstrual cycle, vaginal epithelial thickness is largely unaffected[108]. Deep to the epithelial layer is the lamina propria, consisting of connective tissue, numerous thin-walled (capillaries) blood vessels, fibroblasts, and immune cells including Langerhans cells, macrophages, dendritric cells, and lymphocytes[18, 110]. The extracellular matrix within the vagina is largely collagen, elastin, and glycosaminoglycans[111]. The epithelial layer and lamina propria together comprise the mucosal layer, with a muscularis layer directly below consisting of an outer longitudinal and inner layer of smooth muscle[99].

Human vaginal epithelium was originally thought to be mostly derived from Mullerian tissue[33]. However, Bulmer proposed that the epithelium was derived solely from urogeital sinus cells (Bulmer, 1957). Recent studies, using markers for Mullerian tissue (PAX2) and the urogenital sinus tissue (FOXA1), showed that at 12 weeks the vaginal plate is derived from Mullerian tissue. However, by 21 weeks the vaginal canal epithelium was replaced by FOXA1+ urogenital sinus derived cells[21, 31]. These subtle differences in embryologic origin across species could affect each animals response, particularly in the case of wound healing, and must be considered when choosing an animal model.

The vaginal microbiome in humans is largely composed of lactic acid producing Lactobaccilus sp.[112, 113]. While the relative composition of the microbiome changes over the course of the menstrual cycle, an acidic vaginal environment is maintained, pH 4.2–5[112, 114]. Disruption of this microenvironment contributes to bacterial vaginosis, urinary tract infection, and increases susceptibly to some sexually transmitted diseases[115, 116].

In vitro culture has gained popularity and allows study of human physiology without the use of animal models. In fact, there have been multiple reports of using primary human vaginal epithelial cells and fibroblasts to evaluate multiple cellular mechanisms including hormone regulation of extracellular matrix component, microbicide sensitivity, and immune responses to infectious diseases[117–119]. Both primary vaginal cell culture and use of immortalized cell lines have been described, with the ability to purchase cells for use[120, 121]. Similar to regulatory board approval for animal use (i.e. Institutional animal care and use committee, IACUC), if primary cells are used from patients, institutional review board (IRB) approval must be completed prior to any patient recruitment. While advances in human cell and tissue culture can provide valuable insights, animal models can provide essential knowledge in whole tissue response to insult/injury and choosing the right animal model is vital.

Selection Criteria in Choosing Animal Models for Vaginal Health Research

Many things must be considered when choosing an animal model for research, including anatomic and physiologic similarities to human; cost, regulations, and available expertise; reagents; and transgenic lines. The mouse has become a popular choice due to the low cost and availability of species-specific reagents and transgenic strains. However, in regard to vaginal research, the small size, keratinization of vaginal tissue limiting permeability and tissue damage, and differences in embryologic origins of the vagina may in fact limit the mouse’s usefulness as a model. Though rat vaginal tissue properties have been delineated substantively by previous researchers, locoregional, as well as embryologic tissue origination differences, combined with vaginal flora differences must be considered in study design. Those utilizing rabbit vaginas for drug studies or other investigations should factor in the clear histological differences between rabbit and human vaginal tissue. The rabbit vaginal epithelial layer is histologically distinct and therefore susceptible to greater permeability and irritability than the human vagina. Most importantly, lack of an estrus cycle and vaginal secretions, the vaginal pH, and microflora are distinct from human tissue. These factors warrant careful consideration when planning rabbit vaginal interrogations. The minipig has many advantages as a comparative model to human vaginal systems including vaginal size, estrus cycle similarity, histologic makeup, wound response, and genetic modifications. However, significant drawbacks including cost, government regulations, housing, and vet care as well as differences in the vaginal microbiome relative to humans should be factored into decision-making. The sheep has gained increasing attention as a reproductive model particularly to investigate biomechanical properties or vaginal device suitability due to their anatomical similarities. Additionally, specific issues like pelvic organ prolapse can occur spontaneously in sheep thus adding more value to this particular model in such studies. While these advantages prove the sheep’s utility in many cases, there are instances that require more paralleled aspects to human vaginal tissue such as the commensal environment which differ between these two species. Overall, investigators should evaluate the merit of each model as it pertains to their study and accessible working environment (i.e. veterinarian care, cost, experience).

As the selection criteria table for animal models in vaginal health research suggests, not one animal model fully embodies the human vaginal tract. Rodents are well established models in many biological aspects (with wide availability in transgenic lines) and thus represent a promising model for human vaginal tissue such as relative anatomical and epithelial similarity. Rodents exhibit estrus cyclicity albeit varied from women which results in fluctuations in hormones, pH, and microflora much like in women across their menstrual cycle. Investigations into vaginal wound healing and drug permeability importantly must capture this kind of cyclicity to account for changes in the vaginal environment during these changes. However, the comparative size is demonstrably smaller in rodents making studies in device development challenging in these models. Elaborating further, this small vaginal size in turn presents additional difficulties such as perturbing the tissue reliably for vaginal injury to study wound healing or expanding primary vaginal cells from relatively little tissue to study cell-cell interactions.

Animals that comparatively possess similar vaginal tract size to humans – rabbit, minipig, and sheep – offer relevant dimensions for researchers developing vaginal devices. Notably these animal models have a much higher associated cost. Between these larger animal models, the rabbit vagina resembles very little to the human vagina outside of its relatively similar size. Therefore, outside of physical device development, one must heavily consider many caveats when translating observations in the rabbit vagina to human. Without an estrus cycle (and related changes in hormones, microflora, and acidity), rabbits might not recapitulate the human vaginal environment. However, rabbit vaginal tissue represents the FDA golden standard for drug permeation/irritation studies[15] (perhaps as the worst-case scenario). Rabbit vaginal tissue has a very different, much thinner epithelial layer from human vaginal tissue; this golden standard begs the question: what drugs are we missing out on by limiting to this rabbit vaginal model? The other two animal models, minipig and sheep, display a myriad of similarities to human vaginal tissue. However, these research animals are costly and lack the breadth of previous scientific literature in this space. While we believe minipigs and sheep may provide an effective translatable model to human vaginal tissue, there are still questions concerning the microflora environment as well as the biomechanical properties inherent to a quadruped.

Alternatives to Animal Models

Animal models provide invaluable insight into the complex biological responses needed to assess the safety and efficacy of medical devices and therapeutics. However, each has limitations in fully replicating human vaginal responses to stimuli or injury. The anatomical and biomechanical variations of animal models can lead to difficulties in data interpretation that can be used to ultimately predict clinical outcomes. The anatomical differences of animal models as compared to humans in particular may lead to difficulties in assessing the responses of medical devices to the physiological loads that are experienced during resting and Valsalva. In addition to the structural differences between the animal and human vagina described earlier, it has been noted that muscular differences exist between humans and animal models such as the lack of bulbospongiosus muscle in rats[122]. The evolution to bipedalism has additionally resulted in well-developed pelvic floor muscles compared to quadrupeds[123]. These facets lead to a mechanical environment that is substantially different from humans. In addition, the tissue organization differences between animal models and humans present further challenges for clinical translation. The epithelium of animal models differs from human not only in thickness, but in organization. For example, the rabbit model is lined with columnar epithelium for two-thirds of the vagina compared to the stratified squamous epithelium of humans[15]. The rabbit is the standard species for vaginal irritation; however, the difference in the histology often provide an exaggerated irritation response as compared to the human vagina[124]. Similarly, the presence of keratinized epithelium in mouse and rat models may impact evaluations for irritation, permeability, and damage[15, 82]. Thus, if the evaluation of the device or therapeutic is strongly dependent on either the biomechanics or epithelial response, current non-primate animal models may not be sufficient for pre-clinical testing.

Significant progress has been made in in vitro cell culture and computer modeling providing researchers with alternatives to animal models. Similar to animal models, the selection of these alternative models requires consideration of what information each can provide relevant to human biomechanical evaluations and tissue responses. Here we will discuss promising alternatives within cell derived, tissue engineered models and refer readers to in silico alternatives in another review[125].

Recent advancements in human primary cell culture and the commercial availability of human vaginal epithelium have permitted in vitro assessment of human vaginal responses. Additionally, the development of induced pluripotent stem cells offer another cell source with an exciting opportunity to provide an unlimited potential to become any type of cell that is perfectly patient-matched[126]. Compared to standard monolayer cell culture, 3-D culture enables more complex tissue-like constructs with improvements in cell-cell contact, intercellular signaling, and differentiation. These vaginal tissue culture systems can be utilized toward recapitulating human vaginal tissue complexity such as a highly differentiated stratified epithelium[15, 127]. Recent tissue engineered vaginal models have thus far utilized self-assembled cellularized monolayers of vaginal fibroblasts [128], porcine acellular extracellular matrix vaginal scaffolds [129], and other biologically-derived vaginal materials [130]. These constructs have been utilized for evaluations in HIV research, irritation, metabolism, permeability, toxicity, and drug screening[15]. The utility of these constructs have led to commercially available products from companies such as MatTek allowing for improved accessibility[131]. Although 3-D tissue constructs provide a promising avenue for assessment, the inherent lack of a complete and complex living system limit their utility. For example, the constructs lacks a functional immune response and vascular components that limit investigations into inflammatory responses[124]. Additionally, these constructs do not replicate biomechanical forces that may significantly alter cellular and tissue responses.

The development of biomechanical testing apparatuses would allow for more accurate simulations of vaginal anatomy and physiological forces. Ideally, the apparatus would allow for controllable and regionally distinct physiological loads, simulation of body fluids, and the ability to assess anatomical variations that exist among the population leading to improved clinical translation. Recently, Lazarus3D^™ generated a bench top model by 3D printing a pelvis from a biocompatible silicone that included the uterus, cervix and vagina based on a representative adolescent pelvic MRI images, Figure 3A. The design allows for the ability to inject fluids into the model cavities to simulate blood and vaginal secretion and a loop/peg locking system to simulate uterine/vaginal tilts (anteverted, retroverted, axial) mimicking anatomic variations. This anatomic model can then be encased in a pressurized acrylic casing that enables testing of device deployment (time, expansion) and retention under simulated Valsalva force conditions. Lazarus3D^™ has also created a more specific vagina model with additional pressure control using a pressure cuff, Figure 3B. Intra-vaginal pressure microsensors and transducers can be used to line the vaginal canal in both of these models and enable accurate, reliable, and continuous measurements of force applied from the stent to the vaginal walls. This feature allows for an improved capture of dynamic information not available in current testing systems. Although this represents a significant step toward producing a benchtop model, the complex regional variations in vaginal pressure profiles still need to be replicated to produce a more accurate simulation of the in vivo environment[132]. Additionally, biomechanical models are limited in evaluations where assessment of healing and tissue responses in a complex mechanical environment are necessitated. Similar to the many considerations that must be made when choosing an animal model, there is no perfect answer in alternative, whether that be benchtop modeling or in vitro modeling.

Fig. 3. Benchtop 3D printed pelvic model for biomechanical testing.

(a) The comprehensive pelvic model incorporates uterine, cervical, and vaginal cavities based on representative adolescent pelvic MRI images. As it is enclosed in acrylic housing, this model can be uniformly pressurized for experimental modeling. (b) Additionally, the vaginal cavity can be isolated as shown here to simulate external pressure applied to it in order to test and validate stent placements or other vaginal medical devices within the model.

Conclusion

There is a renewed focus on health research targeting the diseases and conditions that affect women (NIH). In conjunction with this interest, there is exciting acceleration of gynecologic and vaginal health research. The first step towards innovations in research and women’s health advancements is the experimental setup. To support translational endeavors in the gynecologic health research arena, animal vaginal tissue models are often required in order to draw conclusions regarding comparative human vaginal responses to stimuli or injury, test products, or evaluate devices. This review outlines the anatomy and physiology of five commonly-used laboratory animals and the major similarities and differences as compared to the human vagina. Although many aspects of the vaginal system are conserved across mammalian species, various differences exist between common laboratory animal models that must be considered. Increasingly, scientists and engineers are developing tissue engineered replacements to study the vagina. Indeed, to draw appropriate conclusions in gynecologic health research, the choice of the animal model or tissue engineered model is imperative as it will dictate the relevance of the disease or anatomic configurations relative to the human vaginal system. Balancing the anatomic, physiologic, and technical complexities of the animal model or tissue engineered model as it relates to the specific application is required to maximize the information relevant to the clinical outcomes under investigation. This definitive panoramic review of mammalian vaginal systems and consideration of tissue engineered alternatives will serve to guide translational researchers towards the optimal model to yield the most robust and impactful interpretation of their scientific conclusions.

Fig. 1. Vaginal anatomy across species.

The anatomical variations of the vagina is schematized with their associated relative size. Although rodent vaginas are much smaller than human vaginas, they similarly possess a unique orifice through which the vagina reaches the external introitus. As with human, the mouse and rat vaginas extend from the uterine body to the external opening dorsal to the urethra. In contrast, the urethra enters the vagina for a single urogenital sinus at the base of the vaginal tract for rabbit, pig, and sheep. Commonly used animal models possess bicornuate uterine horns. The sheep ewe notably does feature a single uterine body similarly to the uterus found in the human reproductive tract. However, the cervical region joining the uterine body to the vaginal cavity differs across species.

Fig. 2. Composition of vaginal tissue layers across species.

The histologic features of the vagina is schematized with the vaginal lumen facing up. Rodent epithelium is keratinized although the thickness varies with species as well as across the estrus cycle. The rabbit vagina is unique in that the epithelium is simple columnar and hyper-rugated compared to the other species. Pig and sheep have very similar histologic features, which are similar to human with the exception of epithelium thickness. The subepithelial layer of each species are similar in that they are vascularized, contain fibroblasts, immune cells, and extra cellular matrix, although the relative amount varies with mice and rabbits having less dense matrix. Below the submucosal space is smooth muscle and below that loose connective tissue or adventitia, which attaches the vaginal tissue to the surrounding structures.

Table 1:

Selection criteria for animal models for vaginal health research.

Animal Model	Comparative Size	Anatomical Similarity	Epithelial Similarity	Estrus cycle, pH, Microflora	Cost
Mouse	−	+	+	+	$
Rat	−	+	+	+	$
Rabbit	+	−	−−	−−	$$
MiniPig	++	++	++	+	$$$$
Sheep	++	++	++	+	$$$