Introduction
Pharmaceuticals and personal care products such as antibiotics, hormones, anti-inflammatory drugs, antiepileptic drugs, and pesticides have received growing attention in recent years as contaminants have been shown to have negative effects on fertility (Liu et al., Reference Liu, Li, Sessions, Rickords, White and Bunch2008; Hart, Reference Hart2016). Chiu et al. (Reference Chiu, Williams, Gillman, Gaskins, Minguez-Alarcon, Souter, Toth, Ford, Hauser, Chavarro and EARTH2018), showed that eating fruits and vegetables with high amounts of pesticide residue such as found for strawberries, spinach, peppers, or grapes may reduce women’s chances of conceiving and bearing children. Therefore, toxicity tests should be used to ensure adequate protection for human health and the environment against the adverse effects of exposure to various toxic products (Holmes et al., Reference Holmes, Creton and Chapman2010). Toxicological tests are an important part of the safety assessments put in place to regulate these products. Therefore whenever new products such as medicines, pesticides and food additives intended for human or animal use are introduced onto the market, studies must be carried out to predict the toxicological risks of the new substances. Current methods used to report the toxicological risks of chemical substances depend largely on the animal models used over the past 50–60 years. There is no ideal species to use for these tests, however the use of dogs and non-human primates in xenobiotic testing continues to be the norm. Pigs present a viable and perhaps more reliable model of non-rodent testing (Helke and Swindle, Reference Helke and Swindle2013). Few robust biomarkers specific for toxicant exposure are currently available to measure susceptibility or effect on the reproductive systems, there is expectation that post-genomic technologies will offer a new opportunity for identifying and verifying potential biomarkers (Rockett and Kim, Reference Rockett and Kim2005). At the present time, approaches to in vivo toxicity tests also depend on the observation of adverse biological responses in homogeneous groups of animals exposed to high doses of a given agent. However, the relevance of animal studies for risk assessment in heterogeneous human populations, even exposed to much lower concentrations, has been questioned as it remains unknown if results obtained can be extrapolated to humans. In addition, by using large numbers of animals, these studies are expensive and time consuming, as well as raising ethical issues as some in vivo practices may lead to suffering and death of the animals (Cruz, Reference Cruz2003). In this way, ways to evaluate the efficacy and toxicity of drugs have undergone reform, including evaluating replacement of in vivo testing by in vitro testing aimed at reducing animal numbers (Richmond, Reference Richmond2002). To this end, several models such as in cell, tissue or organ culture are being developed and used to replace in vivo experiments.
In vitro tests are more economical. Cells and tissues are easier to maintain in culture, require little physical space and can be used for various purposes such as preparing antigens and antibodies; producing vaccines; isolating microorganisms, particularly different types of viruses; and assessing the toxicity of various products (Cruz, Reference Cruz2003). In vitro culture of cells such as preantral follicles (primordial, primary and secondary) or artificial ovaries is an important tool for investigating basic mechanisms involved in gametogenesis (Arunakumari et al., Reference Arunakumari, Shanmugasundaram and Rao2010). As the events, factors and mechanisms involved in primordial to preovulatory follicle transformation are known and can be controlled in vitro, culture of preantral follicles, especially to secondary stage, may become an alternative way to preserve female fertility. In addition, in vitro investigation of the beneficial/toxic effects of drugs on ovarian follicles (reproductive female function) before use in experiments involving humans and/or animals is essential. That the use of human material for laboratory experimentation raises several ethical issues should also be considered (Guerreiro et al., Reference Guerreiro, Lima, Carvalho, Rodrigues, Castro, Campello, Pessoa, Gadelha, Figueiredo, Bordignon and Rodrigues2016).
Specifically, with regard to reproductive function, there is currently great concern about the effect of exposure to different compounds. Many products can affect the reproductive system and cause infertility or fetal malformation (Faqi, Reference Faqi2012). Regarding the female reproductive system, cases of infertility due to toxicity can be triggered by premature ovarian failure (POF) or early menopause (amenorrhea due to cessation of ovarian function before the age of 40; Goswami and Conway, Reference Goswami and Conway2005). For this reason, toxicological tests are required in investigative processes, during product development and up to registration and commercialization (Stokes, Reference Stokes2002; Meyer, Reference Meyer2003).
This review presents relevant general information on toxicological tests, with an emphasis on reproductive toxicology and women’s health, and more specifically on alternative toxicological testing with a view to reduce the number of animals involved in experimental research.
Toxicological tests
According to Corrêa et al. (Reference Corrêa, Alonzo and Trevisan2003), toxicology is the science of adverse effects caused by interaction between chemical substances and living organisms or biological systems. Toxicological tests have, as their main objective, the prediction of possible adverse effects following humans or animal exposure to a particular chemical. As a result of this exposure, the degree of toxicity can vary from light, such as ocular irritation, to severe irritation, which can lead to permanent incapacitation of an organ (Stokes, Reference Stokes2002). For female reproductive fitness, some chemical classes such as alkylating agents (cyclophosphamide; Lande et al., Reference Lande, Fisch, Tsur, Farhi, Prag-Rosenberg, Ben-Haroush, Kessler-Icekson, Zahalka, Ludeman and Abir2017) anthracycline (doxorubicin; Roti Roti et al., Reference Roti Roti, Leisman, Abbott and Salih2012, Reference Roti Roti, Ringelstetter, Kropp, Abbott and Salih2014) can deplete ovarian follicles and alter steroidogenesis, leading to impaired ovarian function and infertility. These include, but are not limited to, environmental, industrial, chemotherapeutic and xenoestrogenic chemicals (Hoyer and Sipes, Reference Hoyer and Sipes1996). Two experimental models used to study the adverse effects of product exposure are in vivo and in vitro tests. These tests allow quantitative and qualitative evaluation of the effects of exposure and are briefly described below.
In vivo tests
Every year, millions of animals worldwide are used as experimental models. These animals include mice, rats, hamsters, rabbits, fish (zebrafish and trout), birds (mainly chickens), guinea pigs, amphibians (frogs), different species of primates, dogs, and cats, among others and are used in scientific research (Giridharan, Reference Giridharan2000) to understand the effects of medical and surgical procedures, for the production of vaccines and antibiotics, and for the diagnosis and treatment of disease (Hendriksen, Reference Hendriksen2007). The most stringent tests performed on animals are reserved for medicines and foods. Series of tests performed can last from days to years, for example for general toxicity, eye and skin irritation, mutagenicity, carcinogenicity and teratogenicity (Creton et al., Reference Creton, Dewhurst, Earl, Gehen, Guest, Hotchkiss, Indans, Woolhiser and Billington2010; Vandebriel and Van Loveren, Reference Vandebriel and Van Loveren2010).
About 5000 animals are used for each chemical assay, for example (Abbott, Reference Abbott2005; Creton et al., Reference Creton, Dewhurst, Earl, Gehen, Guest, Hotchkiss, Indans, Woolhiser and Billington2010).
In the USA in 2004, the number of animals used in the scientific research was approximately 12 million, but some estimates say that this number could be up to 25 million (USDA Annual Report Animal Usage Statistics, 2014). Conversely, in the European Union in the same year, 3.87 million animals were used in research, representing a decrease of 6% (254,000 fewer procedures) compared with the 2013 (UK Home Office, 2014). This reduction is directly related to strict measures that Europe has adopted in recent years regarding the use of animals in research.
In in vivo tests, the whole animal or its organs and tissues are used. The animals are euthanized using previously established protocols. Frequently, animals that survive pre-clinical tests are sacrificed at the end of an experiment to avoid pain and discomfort (Rusche, Reference Rusche2003). In some cases, for example in lethal dose (LD50) tests, the animals die as a result of the experiment. Pain, anguish, and death experienced by animals during scientific experiments is a matter of constant debate. Animal welfare advocates state that animals have the right not to feel pain or distress and therefore their use in experimentation is unethical and must be reduced or even prohibited (Rollin, Reference Rollin2003). The usefulness of in vivo toxicity tests is controversial, as many animal toxicity tests do not accurately reflect toxicity in humans, making the results inaccurate (Wilson-Sanders, Reference Wilson-Sanders2011). An example is thalidomide (a drug used as a sedative or anti-inflammatory) testing in rodents (Lenz, Reference Lenz1988; Saldanha, Reference Saldanha1994). In these studies thalidomide was shown to be a low-risk drug regarding intoxication and side effects, however in the 1960s it was responsible for the congenital malformation of thousands of children. Similarly, the Draize test in rabbits for ocular and cutaneous irritation by substances, especially cosmetics, has low reproducibility and highlights concerns about extrapolation of test results in animals to humans (Quantin et al., Reference Quantin, Thélu, Catoire and Ficheux2015). In vivo tests have some advantages and disadvantages. Depending on the species used, in vivo toxicity testing is generally seen as the most relevant predictor of human health effects, as tests include a measure of absorption, distribution, metabolism and excretion, all of which could modulate the toxicity of the sample (Blaise and Férard, Reference Blaise and Férard2005). Disadvantages of in vivo testing such as interspecies extrapolation (Martignoni et al., Reference Martignoni, Groothuis and De Kante2006), sensitivity (Asano and Cotruvo, Reference Asano and Cotruvo2004), artefacts and confounding factors (Postma et al., Reference Postma, de Valk, Dubbeldam, Maas, Tonkes, Schipper and Kater2002), ethical and costs (Balls et al., Reference Balls, Blaauboer, Fentem, Bruner, Combes, Ekwall, Fielder, Guillouzo, Lewis, Lovell, Reinhardt, Repetto, Sladowski, Spielmann and Zucco1995) are apparent.
In recent years, alternatives to animal experimentation have been sought through the development of toxicological tests based on the 3Rs principle (from English initials of its main objectives: reduction, refinement and replacement). A recent update of the 3Rs principle is described on Directive 2010/63/EU from the European Parliament and Council. The principle was put forward by Russell and Burch (Reference Russell and Burch1992) and published in The Principles of Humane Experimental Technique.
The 3Rs principle simultaneously ensures the highest standards of safety for consumers and patients and respect for the environment and animal welfare (National Research Council (USA) Institute for Laboratory Animal Research, Reference National Research2004). Reduction can be understood by decreasing the number of animals in a single test or instead of using animals in all experimental phases, they would only be used in the final stages. Refinement involves implementing animal care and treatments to minimize any pain or suffering (Richmond, Reference Richmond2002; Doke and Dhawale, Reference Doke and Dhawale2013). Finally, Replacement is characterized by the non-use of animals. Different techniques are used in this approach, such as in vitro culture of cells and tissues. Briefly, this principle relates to reduction in the number of animals used in research, improvement in conduct of studies, reduction in animal suffering to the minimum possible, and searching for alternative methods that, in the end, replace tests in live animals. Therefore, the main purpose of the 3Rs principle is to serve as a unifying concept, a challenge and an opportunity for scientific, economic and humanitarian benefits (Balls et al., Reference Balls, Van ZelleR and Halder2000).
There is now a consensus that animal studies should be conducted only when: (1) the objective is of justifiable importance; (2) there are no valid alternative methods; (3) all relevant reduction and refinement strategies have already been identified and implemented; (4) the design and conduct of the study minimizes damage caused to animal welfare, not only in relation to the number of animals used, but also in relation to pain and suffering caused; and (5) there is maximum scientific benefit (Richmond, Reference Richmond2002). Therefore, if the in vivo tests do not fit into any of the above-mentioned conditions, in vitro tests are suggested.
In vitro tests
In vitro studies, the non-use of animals in toxicological tests, provide important ways to understand the risks of certain chemicals and predict their possible effects on humans (Broadhead and Combes, Reference Broadhead and Combes2001). Over the last few decades many studies have developed ways to use living tissues and cells from mammals, lower organisms and inert substrates for toxicological testing (Chamberlain and Parish, Reference Chamberlain and Parish1990).
Wide varieties of human and animal cell lines from a wide range of tissues and organs are stored in cell banks, facilitating the development of many studies (Castro, Reference Castro1978). The American Type Culture Collection (ATCC), founded in the 1960s, is one of the pioneer banks for providing certified lineages (American Type Culture Collection, Reference American Type1994). Cell culture techniques have been widely used as they are economical, relatively easy to maintain and require little physical space. Cell survival and/or proliferation is evaluated by counting numbers of cells or using vital dyes (Husoy et al., Reference Husoy, Syversen and Jenssen1993).
Other in vitro assays use organs isolated from animals, such as assays with rabbit isolated eye (IRE; De Torres et al., Reference De Torres, Larrauri and Kunh1997), isolated chicken eye (CEET; Burton et al., Reference Burton, York and Lawrence1981), as well as bovine corneal opacity and permeability (BCOP; Burdick et al., Reference Burdick, Mason, Hinman, Thorne and Anseth2002). All of these methods were designed to quantitate the irritant potential of a product or a chemical after application using parameters such as corneal opacity, permeability, and hydration, or corneal thickness (Chamberlain et al., Reference Chamberlain, Gad, Gautheron and Prinsen1997).
In addition to using live cells, computational systems can also be used through the development of computerized databases and programmes that evaluate toxicity by determining structure− relationships, such as the quantitation structure−activity relationship (QSAR) test, which relates the physicochemical structure of a component to its toxicity (Toropov et al., Reference Toropov, Toropova, Raska, Leszczynska and Leszczynski2014).
Several methods have been suggested that avoid the use of animals in experimentation. These methods provide an alternative means of testing drugs and chemicals that may affect different cells, tissue and/or organs, such as those related to the reproductive system. Unlike male reproductive physiology, which has a continuous formation of gametes (spermatogenesis), women are born with a limited number of germ cells. Female gamete formation is defined as oogenesis (Pesty et al., Reference Pesty, Miyara, Debey, Lefevre and Poirot2007), which is a complex process regulated by many intra- and extra-ovarian factors. Oogonia, which originate from primordial germ cells, proliferate by mitosis and form primary oocytes that arrest at the prophase stage of the first meiotic division until they are fully grown. (Krysko et al., Reference Krysko, Vanden Berghe, D’Herde and Vandenabeele2008). Oocyte meiotic and developmental competence is gained in a gradual and sequential manner during folliculogenesis and is related to oocyte growth and the interaction with its companion somatic cells (McLaughlin and McIver, Reference McLaughlin and Mciver2009). Folliculogenesis is the physiological process of formation, activation, growth and maturation of ovarian follicles (Cortvrindt and Smitz, Reference Cortvrindt and Smitz2001).
Chemicals that destroy female gametes can lead to POF due to destruction of the follicular reserve (Hoyer and Sipes, Reference Hoyer and Sipes1996). Considering the great relevance of preserving female fertility, many studies have investigated reproductive toxicology in women.
Female reproductive toxicology
The focus of this review is the occurrence of adverse effects on the reproductive system due to exposure to chemical or physical agents (Eaton and Gilbert, Reference Eaton and Gilbert2013). The identification of the toxic potential to the reproductive system, as well as the mechanisms of action, are a major scientific challenge during chemical safety assessments. Reproductive toxicology is one of the most complicated domains of toxicology, due to the multiple organs and tissues involved, different modes of toxic action and dependence on the endocrine system (Lorenzetti et al., Reference Lorenzetti, Altieri, Arabi, Balduzzi, Bechi, Cordelli, Galli, Ietta, Modina, Narciso, Pacchierotti, Villani, Galli, Lazzari, Luciano, Paulesu, Spanò and Mantovani2011). In addition, some essential characteristics involving reproductive toxicology are completely different from all other areas of toxicology, as some adverse effects can only be observed in the generation next after parental crossing (Spielmann, Reference Spielmann2009).
Most protocols for reproductive toxicology involve the use of live animals. However, over the past few years, a wide range of in vitro models have been developed to detect the teratogenic effects of chemicals including tests that use dissociated cells from the brain and from buds of the embryo members of rats (Hartung et al., Reference Hartung, Bremer, Casati, Coecke, Corvi, Fortaner, Gribaldo, Halder, Hoffmann, Roi, Prieto, Sabbioni, Scott, Worth and Zuang2004; Hareng et al., Reference Hareng, Pellizzer, Bremer, Schwarz and Hartung2005; Luciano et al., Reference Luciano, Franciosi, Lodde, Corban, Lazzari, Crotti, Galli, Pellizzer, Bremer, Weimer and Modina2010) or culture of whole rat embryos (Bremer and Hartung, Reference Bremer and Hartung2004).
Alternative in vitro tests to evaluate female reproductive toxicity
Some in vitro systems can be used to evaluate reproductive toxicity in women such as culture of isolated ovarian follicles, embryos, ovary (whole organ or only part of the tissue) and embryonic stem cells, which will be described as follows.
Culture of embryos, oocytes, isolated ovarian follicles, follicular cells
Culture of isolated secondary ovarian follicles allows evaluation of the effect of toxic substances on follicular development, so that oocytes from these follicles can be matured and fertilized in vitro. Therefore, the effect of substances on characteristics related to oocyte maturation such as meiotic spindle formation, chromatin damage, gene methylation, as well as on embryonic development can be evaluated (Zhang and Liu, Reference Zhang and Liu2015; Bouwmeester et al., Reference Bouwmeester, Ruiter, Lommelaars, Sippel, Hodemaekers, Van den Brandhof, Pennings, Kamstra, Jelinek, Issa, Legler and van der Ven2016; Žalmanová et al., Reference Žalmanová, Hošková, Nevoral, Adámková, KottT, Šulc, Kotíková, Prokešová, Jílek, Králíčková and Petr2017). Follicles can be isolated enzymatically or mechanically to obtain an intact oocyte/cell complex of the granulosa/theca cell (Cortvrindt et al., Reference Cortvrindt, Smitz and Van Steirteghem1996). In vitro follicle culture systems differ in terms of the species used, the follicular stage in which the culture process is initiated, the type of culture system (96-well plates or droplets covered with mineral oil), and composition of the medium and final parameters evaluated (Rose et al., Reference Rose, Hanssen and Kloosterboer1999). Characterization of this system is extremely important to enable comparison between in vivo and in vitro systems (Smitz and Cortvrindt, Reference Smitz and Cortvrindt2002).
Siddique et al. (Reference Siddique, Sadeau, Foster, Feng and Zhu2014) evaluated the secondary exposure of mice to condensed cigarette smoke and benzo(a)pyrene; results showed that both substances induced oxidative stress in ovarian follicles. Another study evaluated the effect of cadmium chloride on the development of secondary follicles of rats and found changes in follicular growth, differentiation and steroidogenesis (Wan et al., Reference Wan, Zhu, Zhu, Ma, Zheng, Wang, Liu and Zhang2010).
Some authors have used oocytes only to evaluate the effect of various substances such as cadmium (Leoni et al., Reference Leoni, Bogliolo, Deiana, Berlinguer, Rosati, Pintus, Ledda and Naitana2002; Beker, et al., Reference Beker, Gröllers-Mulderij, Snel, Jeurissen, Stierum and Wolter-Beek2012), bisphenol A (Mlynarcikova et al., Reference Mlynarcikova, Nagyova, Fickova and Scsuková2009) and nicotine (Vrsanska et al., Reference Vrsanska, Nagyová, Mlynarciková, Ficková and Kolena2003) on maturation, fertilization and embryonic development of different species. In these studies, oocyte maturation was sensitive to exposure to these drugs such that lower maturation rates and, consequently, lower fertilization rates were obtained. In a recent study, Ferris et al. (Reference Ferris, Mahboubi, Maclusky, King and Favetta2016) evaluated exposure of bovine oocytes to 30 ng/ml bisphenol A during oocyte maturation. The authors observed a reduction in the rates of cleavage and formation of blastocysts, and an increase in the rate of apoptosis and proportion of female blastocysts. Another study evaluated the effect of fluoride on fertilization and subsequent embryonic development of mice. After overexposure of oocytes to fluoride, a reduction in ATP production and mitochondrial membrane potential was observed, which influenced the rates of fertilization and embryonic development (Liang et al., Reference Liang, Zhao, Ock, Kim and Cui2016).
Studies on the direct effect of exposure of embryos to different substances are mostly carried out using chick embryos (Gao et al., Reference Gao, Li, Zhang, Liang, Chen, Zhang, Chuai, Bao, Wang and Yang2016; Strojny et al., Reference Strojny, Grodzik, Sawosz, Winnicka, Kurantowicz, Jaworski, Kutwin, Urbańska, Hotowy, Wierzbicki and Chwalibog2016) and fish, mainly zebra fish (Zhang et al., Reference Zhang, Ji, Yan, Lu, Lu and Zhao2016; Hu et al., Reference Hu, Guo, Zhao and Fu2017). Zhang et al. (Reference Zhang, Ji, Yan, Lu, Lu and Zhao2016) evaluated the effect of 4-hydroxychlorothalonil metabolite from the chlorothalonil fungicide on zebrafish embryos and revealed that 4-hydroxychlorothalonil exhibited potent effects on endocrine disruption, causing negative effects on embryonic development.
In addition to the culture of oocytes and embryos, the in vitro culture system of granulosa cells or luteal cells has also been used and allows evaluation of how specific substances can act specifically on these structures and therefore determine the possible mechanism of action of the substance causing cellular toxicity (Sun et al., Reference Sun, Betzendahl, Shen, Cortvrindt, Smitz and Eichenlaub-Ritter2004). Twu et al. (Reference Twu, Srinivasan, Chou, Wu and Chiu2012) in a study with goats used corpus luteum cells to evaluate the effect of cantharidin and norcantharidin, natural toxins produced by Chinese beetles (Mylabris phalerata or Mylabris cichorii). According to these authors, these toxins are responsible for accidental intoxication in humans and animals, as well as used in folk medicine to induce abortion. Within the study, the authors observed that both substances inhibited the production of progesterone and that cantharidin inhibited steroidogenesis by reducing the expression of StAR protein, important for this process.
Culture of ovarian tissues or whole ovary
Culture of part (fragments) or whole ovary allows evaluation of different parameters in a controlled way and, therefore, has the potential for more complete reproductive toxicity studies (Stefansdottir et al., Reference Stefansdottir, Fowler, Powles-Glove, Anderson and Spears2014). This type of culture is already well established for different species such as mice (O’Brien et al., Reference O’Brien, Pendola and Eppig2003), rats (Cain et al., Reference Cain, Chatterje and Collins1995), cows (Jimenez et al., Reference Jimenez, De Azevedo, Silveira, Penitente-Filho, Carrascal-Triana, Zolini, Araujo, Torres and Gonçalves2016), sheep (Cavalcante et al., Reference Cavalcante, Gouveia, Barberino, Lins, Santos, Gonçalves, Celestino and Matos2015), and goats (Faustino et al., Reference Faustino, Rossetto, Lima, Silva, Saraiva, Lima, Silva, Donato, Campello, Peixoto, Figueiredo and Rodrigues2011) to evaluate the positive effects of various substances on development and oocyte and follicular function within the ovary itself. The numbers of studies using in vitro models are still low compared with in vivo studies. Various substances have already been tested in ovarian tissue to evaluate negative effects on female reproduction (Table 1), in particular:
∙ 4-vinylcyclohexene diepoxide (VCD), used commercially as an intermediate and chemical reactive diluent for diepoxide and epoxy resins (Huff, Reference Huff2001);
∙ 7,12-dimethylbenzyl(a)anthracene (DMBA), produced from burning organic compounds (cigarette smoke; Gelboin, Reference Gelboin1980);
∙ monoethylhexylphthalate (MEHP), found in many everyday products such as PVC, plastic bags, food packaging, cosmetics and industrial paints (Hauser et al., Reference Hauser, Duty, Godfrey-Bailey and Calafat2004; Heudorf et al., Reference Heudorf, Mersch-Sundermann and Angerer2007);
∙ bisphenol A, present in several plastics in common human contact (Ikezuki et al., Reference Ikezuki, Tsutsumi, Takai, Kamei and Taketani2002).
Effects of different substances evaluated in vitro on ovary of different species
In addition to these substances, there are commercial chemotherapies such as docetaxel, doxorubicin (DXR), paclitaxel (PTX) (Guerreiro et al., Reference Guerreiro, Lima, Carvalho, Rodrigues, Castro, Campello, Pessoa, Gadelha, Figueiredo, Bordignon and Rodrigues2016) and substances derived from plants with antitumor effects, such as oncocalyxone A (Leiva-Revilla et al., Reference Leiva-Revilla, Lima, Castro, Campello, Araújo, Celestino, Pessoa, Silveira, Rodrigues and Figueiredo2016), the ethanolic fraction of Auxemma oncocalyx (Leiva-Revilla et al., Reference Leiva-Revilla, Lima, Castro, Campello, Araújo, Celestino, Pessoa, Silveira, Rodrigues and Figueiredo2016) and frutalin (Soares et al., Reference Soares, Costa, Vasconcelos, Ribeiro, Souza, Silva, Van den Hurk and Silva2017).
Most toxicology studies using this type of strategy focus on female mice and rats models to assess the adverse effects of substances on reproductive function and fertility. Because large numbers of animals are required for reproductive tests, these species have been used due to ease of handling and spacial considerations. In addition they have been very well characterized, anatomically, physiologically and genetically and have short life cycles (gestation, breastfeeding, and puberty; Harkness and Wagner, Reference Harkness and Wagner1993; Santos, Reference Santos2002). Rodent and human reproductive physiology differ in many respects, however, such as ovarian size and consistency, follicular distribution, oocyte diameter, luteal body signalling (Betteridge and Rieger, Reference Betteridge and Rieger1993; Ménézo and Hérubel, Reference Ménézo and Hérubel2002), as well as duration of folliculogenesis (Ménézo and Hérubel, Reference Ménézo and Hérubel2002; Smitz and Cortvrindt, Reference Smitz and Cortvrindt2002). Conversely, domestic animals such as goats can be excellent experimental models for the human species, as they have similar ovaries in size and consistency and in other aspects such as follicular diameter and folliculogenesis (6–7 months) to humans (Smitz and Cortvrindt, Reference Smitz and Cortvrindt2002; Baldassarre, Reference Baldassarre2008). Besides this similarity, goat ovaries can easily be obtained in slaughterhouses. Such access would spare the lives of 1000s of laboratory animals destined for in vivo experiments (Figueiredo et al., Reference Figueiredo, Rodrigues, Amorim and Silva2008). Consequently, goat ovarian tissue has been used in vitro to evaluate different drug toxicities, as demonstrated by Guerreiro et al. (Reference Guerreiro, Lima, Carvalho, Rodrigues, Castro, Campello, Pessoa, Gadelha, Figueiredo, Bordignon and Rodrigues2016). In this study, the authors evaluated the toxicity of two antineoplastic drugs, DXR and PTX, widely used in the treatment of different types of cancer, during in vitro culture of goat ovarian tissue (data presented in Table 1). The authors observed deleterious effects of these drugs such as oocyte retraction, nuclear pyknosis germ cell and large disorganization of granulosa cells. In addition, PTX was more toxic to preantral follicles than DXR. In another study, Leiva-Revilla et al. (Reference Leiva-Revilla, Lima, Castro, Campello, Araújo, Celestino, Pessoa, Silveira, Rodrigues and Figueiredo2016) used in vitro culture of goat ovarian tissue as a strategy to evaluate the toxicity of two substances with antiproliferative effects, oncocalyxone A and the ethanolic fraction of Auxemma oncocalyx. Both substances adversely affected follicular development in a dose-dependent manner.
Ovarian tissue culture has the advantage of being able to evaluate the effect of substances on the pool of primordial follicles that makes up the ovarian reserve. However, this strategy is limited regards duration of culture time, as short periods may not be sufficient to ensure follicular development. Conversely, cell viability can be reduced with very long periods of culture (Alves et al., Reference Alves, Chaves, Rocha, Lima, Andrade, Lopes, Souza, Moura, Campello, Báo, Smitz and Figueiredo2013).
Validation of alternative methods
Although existing since 1991, the European Union (EU) Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) was formally created only in 2011. Due to increasing need for animal replacement through newly developed methods, and proposed by the EU, the creation of this laboratory occurred after large investment of time and resources to coordinate and promote the development, validation and use of alternative methods (Adler et al., Reference Adler, Basketter, Creton, Pelkonen, Van Benthem, Zuang and Andersen2011). In Brazil, the National Network of Alternative Methods for the Use of Animals (RENAMA) was recently created through Ordinance No. 491, 3February 2012, whose objective was the development of methods capable of reducing, refining or replacing animals as experimental models, based on the principles of Russell and Burch (Reference Russell and Burch1992).
Validation can be defined as the process by which the relevance and reliability of a particular approach, method, process, or assessment are established for a definite purpose (OECD, 2005). The relevance of a procedure refers to the scientific value and practical utility of the results obtained, whereas reliability is related to the reproducibility of these results within and between laboratories over time, in relation to a clearly defined and specific purpose (Balls et al., Reference Balls, Botham, Cordier, Fumero, Kayser, Koëter, Koundakjian, Lindquis, Meyer, Pioda, Reinhardt, Rozemond, Smyrniotis, Spielmann, Van Looy, Van Der Venne and Walum1990; Balls and Karcher, Reference Balls and Karcher1995). The initial focus on validation was the performance of alternative methods as assessed in multilaboratory studies, which usually involve analysis of chemical substances.
Validation consists only of one of the steps in the progress of a test, from its design to its regulatory application (Balls et al., Reference Balls, Blaauboer, Fentem, Bruner, Combes, Ekwall, Fielder, Guillouzo, Lewis, Lovell, Reinhardt, Repetto, Sladowski, Spielmann and Zucco1995). In fact, the validation process of alternative methods is aimed at verifying optimization, transferability, reproducibility, and relevance of the proposed method with the objective of being submitted to the regulatory agency. Once approved, the method becomes officially available for toxicological evaluation of raw material. The worldwide availability of validated methods occurs through the Organization for Economic Cooperation and Development (OECD) and Pharmacopoeias (ICCVAM).
Considering the information mentioned above, for a new method to be accepted it must be evaluated to establish its relevance for implementation and reliability. For this to be possible, it is necessary to harmonize validation processes through international committees (Schechtman, Reference Schechtman2002). Therefore, scientific validation of alternatives for assessing in vitro toxicity as a laboratory method for drug testing and its subsequent acceptance by government bodies responsible for drug release is critical and will have important positive consequences for animal welfare, as 1000s of animals will be spared in vivo tests.
Final considerations
Reproductive toxicology tests are extremely important because animals, including humans, are exposed daily to large numbers of chemicals that may have toxic effects on reproductive function. For women, these effects may cause POF, infertility or even become hereditary, and may result in serious consequences such as congenital malformation and deleterious effects on the offspring. However, most toxicity trials involve the use of live animals that has generated ethical problems in the scientific community. Therefore, it is necessary to invest in the improvement of in vitro culture techniques, especially ovarian tissue or even follicles isolated from the ovary that allows the ability to quickly and safely trace the effect of chemical compounds on ovarian function and fertility.
Financial support
The authors would like to thank the National Council for Scientific and Technological Development (CNPq Brazil) and Coordination of Improvement of Higher Level Personnel (CAPES) for financial support. Denise Damasceno Guerreiro is a recipient of a doctoral scholarship from Cearense Foundation for Scientific and Technological Development Support-(FUNCAP, Brazil). Ana Paula Ribeiro Rodrigues is a recipient of a grant from CNPq Brazil through projects 457226/2013-7 and 308071/2016-6.
Conflicts of interest
The authors declare no conflicts of interest.
Ethical standards
Not applicable.
