Bioengineering models of female reproduction.

One unique feature of the female reproductive system is that no reproductive organ functions alone. For example, the ovarian follicle development and ovulation are highly regulated by the pituitary hormones of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The ovarian hormones of estradiol and progesterone control the downstream reproductive track organs. As we described above, we have used the transwell insert or the microfluidic system to co-culture ovarian tissues and fallopian tube epithelium and demonstrated the crosstalk between these two female reproductive organs [ 59 , 53 ]. Furthermore, we collaborated with bioengineers and created a novel microfluidic device that can interconnect five different organs in one platform, which is termed ‘EVATAR’ ( Figure. 6 ) [ 53 ]. The five organs we cultured using EVATAR were human liver spheroids, mouse ovarian explants, human fallopian tube epithelium, human endometrium, and human cervix tissues. The EVATAR interconnected these reproductive and non-reproductive tissues through embedded microfluidic channels and the universal culture medium. The results generated from this bioengineered female reproductive system-on-a-chip indicated that the dynamic fluidic flow maintained the viability of all culture tissues for 28 days and the downstream reproductive track tissues responded to the upstream ovarian hormones. Additionally, compared to ovarian tissue culture alone, the co-culture system also produced a more physiologically relevant human 28-day menstrual cycle hormone profiles. Another important application of the microfluidic system is to introduce liver metabolism for drug screening or toxicity testing. In our previous works using EVATAR [ 53 ], the liver spheroids remained viable and secreted albumin over the entire 28 days culture period with other co-cultured female reproductive tissues; however, further studies are necessary to determine whether the co-cultured liver spheroids were able to metabolize the hormone and other secreted factors from reproductive tissues and whether the reproductive tissues can influence the metabolic activities of co-cultured liver tissues. A number of clinical drugs or environmental chemicals require liver metabolism to exhibit therapeutic or toxic effects. For example, cyclophosphamide, a widely used chemotherapeutic drug, needs to be metabolized to phosphoramide mustard to exhibit both anti-cancer effects and ovarian toxicities. Di(2-ethylhexyl)phthalate (DEHP), a plasticizer and well-identified EDC, only exhibits female reproductive toxicities when it is metabolized to the Mono-(2-ethylhexyl) phthalate (MEHP) [ 90 ]. The co-culture of liver and female reproductive tissues in a microfluidic setting will allow us to introduce pharmacokinetics or toxicokinetics of the tested compounds, providing a more complex and representative in vitro models.

There are limited ways to study female reproduction because of the complex interactions among cells, organs, hormones, and organ systems. Recent advances that use bioengineering methods to study female reproduction allow for a better understanding of the ovary, fallopian tube, uterus, embryo implantation, placenta, and reproductive disease. Bioengineering methods have been applied to study basic female reproductive biology, reproductive medicine, and toxicity screening. Hydrogel encapsulation, decellularized ECM scaffolds, 3D printing, microfluidic platform and other engineered advances indicate significant potentials to reconstruct and build artificial, functional, and implantable reproductive organs that may allow prepubertal girls and young adult women to restore their fertility and endocrine functions. Additionally, advances in in vitro systems like artificial wombs can help build better outcomes for preterm neonates. Furthermore, these engineered platforms can provide new knowledge on basic gynecologic cancer biology and pathology and for potential drug screening and development. While remarkable advancements have been achieved, most of these bioengineering models copy female reproduction at organotypic or cellular levels. Therefore, more in-depth analyses are required to investigate whether these bioengineering methods can recapitulate female reproduction at the molecular, genetic, and epigenetic levels, etc. For example, it is not well understood that whether the reconstructed ovaries using decellularized or 3D printed ECM scaffolds completely support the acquisition of oocyte transcriptome profiling to ensure its meiotic and developmental competence and whether the engineered oocyte or embryos fully preserve in vivo epigenetic reprogramming signatures. Compared to static culture environment, the microfluidic platform significantly promotes reproductive cell proliferation and differentiation. However, different microfluidic designed have been used and the precise microfluidic settings such as the fluid flow pattern, flow rate, and shear stress and whether these settings depend on cell/tissue types have not been well determined. Further, the safety assessment of applied biomaterials and fabrication methods also need to be considered when bioengineering female reproductive organs and function. In conclusion, although many endeavors are required in future studies, the intersection of bioengineering and female reproductive biology and medicine provides great potential to advance the knowledge of female fertility, genetic vulnerability, medications, environmental exposures and toxicities, aging, nutrition, and diseases.

The female reproductive system is composed of the female gonads, the ovaries, and the female reproductive track, which includes the fallopian tubes (termed oviducts in non-primate species), uterus, uterine cervix, and vagina ( Figure. 1 ). It functions to provide hormonal support and anatomical structure for the production of new offspring. In addition to fertility, it is also important for women’s systemic health because hormones secreted from the ovaries contribute to the general health of their endocrine, cardiovascular, skeletal, and immune systems, etc [ 1 ]. Abnormal female reproduction is caused by a number of factors including genetic vulnerability, medical treatments, environmental exposures, age, nutrition, and diseases. In the United States, about 16.2% of married women within reproductive age (15–49 years old) have impaired fecundity, 8.8% of them are diagnosed as infertile, and 12.7% of them have received fertility treatments such as in vitro fertilization (IVF) [ 2 ]. Moreover, female reproductive diseases such as the endometriosis and polycystic ovary syndrome (PCOS) are becoming more and more prevalent [ 3 ]. Furthermore, gynecologic cancers do not only affect women’s fertility but also threaten their lives. For example, ovarian cancer is the fifth leading cause of cancer death among women and cervical cancer is the fourth most frequent female cancer disease [ 4 ]. The female reproductive system is also one of the major off-targets of clinical drugs. Both chemotherapy and irradiation have been demonstrated to exhibit highly detrimental effects on the ovaries and increase childhood and young adult female cancer patients’ risks of ovarian failure, early menopause, and infertility [ 5 – 7 ]. Our recent studies also suggested that doxorubicin (DOX), a commonly used chemotherapeutic chemical, permanently altered the uterine response to estrogen, an ovarian steroid hormone, indicating that anticancer agents can also directly target the uterus to impair female reproductive health and fertility [ 8 ]. In addition to pharmaceutical compounds, increasing evidence suggests that the environmental chemicals, particularly those identified as endocrine disrupting chemicals (EDCs) such as the bisphenols, phthalates, and flame retardants, etc, can also cause female reproductive toxicities. Thus far, due to the ethical concerns of using human subjects to study female reproduction, particularly when women are pregnant, the majority of the explorations for female reproductive physiology, pathology, and toxicology have been through in vivo animal models and two-dimensional (2D) cell or tissue culture models. However, the complexity and species difference of the female reproductive system in humans makes it difficult to compare to those of animals. For example, the average ovarian cycle is 4–5 days for rodents but 28 days for humans, and the average of gestation period is 20–23 days for rodents but 40 weeks for humans. Moreover, in vivo animal models are time consuming and costly, and it is unethical to sacrifice a large number of animals for human benefit. Regarding 2D culture models, the cells cultured on flat plastics or glass lose their 3D architecture as well as the physical or biochemical contacts with other cells in vivo . For instance, ovarian cell lines, including both somatic cells and oocytes, have been cultured in vitro [ 9 – 11 ]. However, these individual cell lines lack the 3D cell/tissue architecture and also the bidirectional communications between somatic cells and their enclosed oocytes [ 12 – 16 ], which are required for supporting normal ovarian development and functions [ 17 – 21 ]. These facts indicate that there is an urgent and unmet need to develop representative and effective models for studying human female reproduction. Bioengineering aims to use the principles and technologies of engineering to solve problems in biology and medicine. Over the past decades, the prodigious advancements of bioengineering (e.g. biomaterials, 3D printing, and microfluidics) allow us to study the female reproductive system and reproductive diseases in an entirely new way. Here, we review recent advances of bioengineering models of female reproductive tissues and functions, which provide us with encouraging research models to study the female reproductive science and medicine. We first discuss the research related to bioengineering and the ovaries, the primary female reproductive organs, which is followed by the downstream female reproductive tract including the fallopian tube, uterus, and placenta. Further, we reviewed current research that used bioengineering methods to study female reproductive diseases including endometriosis and gynecologic cancers.

Although engineered tissues, organs and organ system models have been applied for a variety of research, studies regarding bioengineering and female reproductive diseases remain limited. Several studies have been published using microfluidics and scaffold engineering to understand endometriosis [ 80 , 81 ] and reproductive cancers [ 82 , 83 ], though none to study polycystic ovary syndrome (PCOS). Endometriosis occurs when endometrial tissues are outside of the uterine cavity. It is one of the most common gynecological diseases in women of reproductive age worldwide [ 84 ]. An in vitro model was used to investigate the cell interactions between endometrial stromal cells (ESCs) and human peritoneal mesothelial cells (HPMCs) that are similar to endometriosis conditions [ 80 ]. Microfluidic channels and cover slips were used to observe interactions between ESCs and HPMCs, mimicking the physiological processes of peritoneal endometriosis. Control HPMCs resisted introduction of ESCs from both control and endometriotic individuals. However, HPMCs from endometriotic individuals were unable to resist ESCs from both normal and endometriotic individuals. This study developed an adaptable and simple in vitro method for real-time monitoring of interactions between ESCs and HPMCs. This approach can be used for demonstrating interactions among three or more types of cells and for investigating organ development of other diseases. The data suggest that endometriosis is related not only to the condition of endometrial cells, but also to the locations where there is the disease. Another study used the microfluidic system to investigate the drug pathologies and biomarkers for those with endometriosis vs. those without [ 81 ]. Authors aimed to evaluate an in vitro model that was designed to look at inflammation and could be applied to endometriosis tissues. Results showed that the developed droplet-based microfluidic platform allowed for the observation of hundreds of protease enzyme activity reactions for hours, creating physiologically relevant differences in controls and those with endometriosis [ 81 ]. Traditional cell cultures fail to repeat the natural tumor microenvironment. Recently, functional 3D in vitro models have been investigated and engineered for studying cervical cancers [ 82 ]. Normal epithelial and immortalized cervical epithelial carcinoma cell lines were used to mimic 3D artificial normal cervical and cervical cancerous tissues. Human skin cells were used as a scaffold for both models. Results indicated that the created 3D in vitro cervical cancer model showed stratified epithelial layers and expressed the same types and patterns of differentiation marker proteins as seen in corresponding in vivo tissue in either normal cervical or cervical cancerous tissues. Additionally, other 3D microfluidic systems have been investigated to mimic ovarian cancer environments [ 83 , 85 , 58 ]. For example, a microfluidic platform of the peritoneum was constructed to mimic ovarian cancer spheroids in the peritoneal cavity with mesothelial cells under hydrodynamic conditions. The interactions between cancer cells and mesothelial cells were analyzed. This model can help future researchers understand mechanisms of metastatic progression and assist with therapeutic development [ 83 ]. High-grade serous carcinoma (HGSC), which is the most common type of ovarian cancer, originates in epithelial cells in the fallopian tube. Several studies use oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human carcinoma [ 86 , 87 ]. These in vitro models can allow for the study of biomarkers for early detection of cancer and for improved therapeutic treatments. Theory shows that cortical inclusion cysts (CICs) in the ovary play a role in HGSC progression. Other studies use human samples to engineer an in vitro model that mimics the size, shape, and extracellular matrix properties of CICs [ 88 , 89 ]. Taken together, these engineered platforms can provide new knowledge on basic gynecologic cancer biology and pathology and for potential drug screening and development.