Mechanobiology of the female reproductive system.

The ovary has two major compartments, the cortex and medulla, which differ in their ECM composition and structure (Tables  1 and 2 ). 23 Decellularized human and bovine ovarian tissues exhibit radially aligned collagen fibers in the cortex, whereas the medulla is composed of a network of pores with anisotropic collagen fibers, suggesting differences between cortical and medullary ECM‐producing stromal cells. 23 The dominant paradigm is that the ovarian cortex, where primordial follicles reside during dormancy, is stiff versus the less dense medulla, where antral follicles vigorously remodel the ECM via proteolytic degradation as they reach the preovulatory stages. 24 The stiff cortex is considered to maintain quiescence of the immature primordial follicle pool. 24 , 25 Nagamatsu et al. showed that when whole mouse ovaries were incubated with a collagenase solution that digests ECM in the cortical region, translocation of FOXO3 into the cytoplasm of oocytes was observed at the edge of the cortical region, and some of the oocytes had started development into secondary follicles. 26 However, until recently, differences in the mechanical properties of the cortex and medulla had never been quantified. A very recent study mapped and quantified the mechanical microenvironment in mouse ovaries using colloidal probe atomic force microscopy. 27 The authors showed lower stiffness (Young's modulus, 2–3 kilopascals [kPa]) at the ovarian edge and center, which are dominated by extrafollicular ECM and higher collagen IV density, and the greatest stiffness (Young's modulus, ~7 kPa) in an intermediate zone dominated by large follicles with lower collagen IV density. 27 It has been postulated that the stiffest region of the ovarian microenvironment is the edge (cortex) of the ovary due to the high concentration of collagen. 24 However, there is an inverse relationship between collagen IV density and stiffness in mouse ovaries. 27 The prominence of ovarian cortical and medullary regionalization differs across species and is notably reduced in rodent ovaries compared with human ovaries. 28 The mouse is exceptional among mammals because its ovarian regionalization is much less relevant than that in other species. 28 Thus, findings in mouse ovaries may not be translated to human ovaries. 28 Large animal models, particularly bovine, equine, and ovine models, have been used to understand ovarian function in women. 29 Gargus et al. 30 assessed whole bovine ovaries ex vivo, using shear wave (SW) ultrasound elastography. They showed that the SW velocity was higher in the medulla than the cortex. 30 Although laboratory mice are not entirely suitable for investigation of mechanobiology in ovaries, recent studies in both murine and bovine ovaries have provided data contradicting the long‐standing dominant paradigm of a stiff cortex versus Less stiff medulla. 27 , 30 Summary of mechanical properties reported in the literature 27 2021 30 2020 No significant difference: just before ovulation Artificial increasing intrafollicular pressure by the injection of fluid: Not induce follicle rapture 33 1964 The average pressure: ~17 mm Hg No difference: pre‐ versus post‐coital follicles 34 1963 Intrafollicular pressure >30 cm H 2 O (~22 mm Hg) Artificial increasing intrafollicular pressure (>400 cm H 2 O [~294 mm Hg]): Not induce follicle rapture 1979 36 2001 44 2006 45 2016 Oocyte about 3–4 h after retrieval ZP outer layer of the immature MI is stiffer than that of mature MII ZP outer layer of “rejected” MII was softer than that of ‘‘suitable’’ MII Note: MII oocytes were classified as ‘‘suitable’’ and ‘‘rejected’’ according to their morphological characteristics 46 2016 C10 value (ZPSM): in the ranges of 0.20–0.30 kPa and 0.30–0.40 kPa: the highest implantation rates (6/12: 50% and 8/9: 88.8%, respectively) Outside this range: Average implantation rate: 6.70% 47 2020 Thawed 2PN stage zygotes 2PN stage zygotes Human Mouse Automated micropipette aspiration system The mechanical model (the modified Zener model): to extract mechanical parameters Prediction of blastocyst formation: In humans within hours after fertilization: >90% precision, 95% specificity and 75% sensitivity In mouse, 79% specificity and 76% sensitivity Prediction of live birth in mouse 45 2016 Mid‐stage blastocysts (E4.0): ~650 Pa Mature blastocyst stage (E4.5): ~1500 Pa 97 2018 Inner pressure Stiffness: trophoblasts and ICM Inner pressure at E4.5 is ~3.7 times that of E3.0 Stiffness: Trophoblast at E4.5 is ~1.6 times that of E3.0 ICM at E4.5 is ~3.6 times that of E3.0 98 2019 87 2020 87 2020 75 2014 77 2018 118 2019 Abbreviation: 2PN, two‐pronuclear; dpc, days post coitum; E, embryonic day; GV oocyte, germinal vesicle oocyte; ICM, inner cell mass; kPa, kilopascals; MI, metaphase I; MII, Metaphase II; ZP, Zona pellucida; Summary of manipulation of mechanical properties and/or mechanical stimulation reported in the literature Translocation of foxo 3 into cytoplasm in the oocytes at the edge of the cortical region Some of the oocytes started follicular growth to secondary follicles 26 2019 Follicles cultured in low shear elastic modulus (alginate: 0.7%): increased follicle growth, higher rates of antrum and theca layer formation and higher quality of oocytes The high level of androstenedione and progesterone: in the immature, two‐layered follicles cultured under increased matrix stiffness of alginate hydrogels (3%) 42 2007 50 2006 Oocytes during preincubation before IVF/ICSI A total of 450 retrieved oocytes from 32 IVF/ICSI cycles of 32 women A randomized controlled trial The rates of fertilization per mature oocyte and high‐grade cleavage‐stage embryo formation: no difference versus the control group The rates of blastocyst formation and of blastocysts graded 3BB or higher at day‐5: significantly higher in the TECS group than those in the control group (45.3% (67/148) versus 32.1% (51/159), (29.1% (43/148) versus 17.6% (28/159), respectively) 51 2013 Pronuclear embryo Group 1 (74 patients, n  = 148), without mechanical agitation) Group 2 (74 patients, n  = 148), with mechanical agitation Significantly higher percentage of excellent (grade A) and good (grade B) quality embryos at the 4–6 blastomere stage was observed in Group 2 compared with Group 1 (90.1 ± 1.7% versus 77.9 ± 4.4%, mean ± SD) The percentage of embryos at different blastocyst stages was 10% higher in Group 2 than that in Group 1 (14.1 ± 2.8 versus 4.5 ± 1.7). Significantly higher pregnancy rate in Group 2 versus Group 1 (78.4 ± 3.2% versus 50.1 ± 4.9%: transfer of day‐3 embryos, 72.2 ± 1.5% versus 33.2 ± 2.4%: transfer of day‐5 embryos) 52 2011 Constriction: mimicking peristaltic muscle contractions of the inner muscle of the oviduct Shear stress Microfluidic platform: Embryos: cultivated in constricted channels, with different constriction widths of the channel (150 and 160 µm) versus in a straight channel (control) Incubated on a tilting machine, which made a cyclic ± 10° tilt over 1 min to provide embryo movement via gravity for 44 h 53 2009 Successful cleavage from the non‐stimulated area and from mechanically stimulated area for 2 s Squeezed embryos were underdeveloped in the mechanically stimulated area for 12 s 54 2011 Microelectromechanical systems (MEMS): Four groups: (1) microdrop (control), (2) microdevice under static medium conditions, (3) microdevice under dynamic medium conditions at flow rate 0.1 µl/h and 4) at flow rate 0.5 µl/h, for 72 h 55 2002 Caused lethality within 12 h for E3.5 embryo The E2.5 embryo was more sensitive to shear stress than the E3.5 embryo 49 2006 Computer‐controlled microfluidic culture platform Periodic fluid pulses of media (0.135 Hz) for 96 h 57 2010 58 2013 EWOD (electrowetting‐on‐a‐dielectric) platform Controlled droplet velocity in dynamic (1 time (15) s/0.5 h, 60 voltage or 1 time (15 s)/2.0 h) EWOD chip or in a static EWOD chip (control) 59 2015 IVM oocytes IVF was performed either in a 4‐well dish (control) or in the oviduct‐on‐a‐chip device A microfluidic “oviduct‐on‐a‐chip platform”: perfusion: 5 μl h −1 The average shear stress exerted on the embryos: 0.70 ± 0.46 dyne cm −2 60 2018 Embryos cultured on 3D type I collagen gels increased in 2‐cell, blastocyst, and hatching frequency and TE cell number Fetuses derived from embryos cultured on 3D type I collagen gels had a greater placental weight at E12.5 157 2012 Little adverse effect on implantation and the early pregnancy phase of the uterus DVE‐related markers, Cer1 and Hex: not expressed at E5.75. At E6.5, salbutamol‐treated embryos expressed Cer1, an AVE marker, and T, a primitive streak marker, at a normal position 87 2020 90 2013 96 2016 By leaky cell junctions Bb(−): a selective inhibitor of myosin II ATPase activity) By reduced cortical tension (heterozygous [Myh9+/−] embryos derived from m−/− Myh9+/− or m+/−Myh9+/−) 97 2019 Increased cavity growth Decreased ratio of ICM to trophectoderm cells Reduced actomyosin contractility: Bb(−) Heterozygous (Myh9+/−) embryos that have reduced cortical tension Reduced fluid influx: recombinant GST‐C‐CPE fusion protein (an inhibitor of claudin4 and claudin6, Tight‐junction inhibition) Ouabain (a inhibitor of Atp1 Hypertonic) A higher ratio of ICM to trophectoderm cells Occasionally did not spatially segregate into the inner and outer cells, as shown by Cdx2‐positive cells in the ICM Significant reduction in the fluorescence levels of both EPI (Sox2) and PrE (Gata4) markers Significantly higher degree of overlap between EPI and PrE populations 99 2019 PrE specification levels are significantly reduced, while EPI specification levels are maintained Significantly higher degree of overlap between EPI and PrE populations 104 2021 63 2005 Cyclic stretch induced the secretion of IGFBP‐1 from decidualized ESs No effect on non‐decidualized ESs 64 2006 62 2020 omputed PWSSs (<0.05 kPa) for 0 (control), 60, and 120 min EEs: in direct contact with the shearing fluid. Increased F‐actin polymerization in EEs and MSMCs More pronounced in the EEs, than in the inner layer of MSMCs 65 2020 153 2019 158 2017 159 2020 Fusion ratio of vCTBs cultured on normal tissue stiffness (1.3 kPa): nearly twofold higher compared with those cultured on stiff substrates (7 kPa) BeWo cells grown on composite hydrogel substrate consisting of parallel soft (1.3 kPa) and stiff (17.4 kPa) strips: fusion ratio on both soft and stiff regions was significantly lower than on uniformly soft substrates 160 2020 Abbreviation: 3D, three dimensional; Atp1, a Na+/K+‐ATPase activity; AVE, anterior visceral endoderm; DVE, distal visceral endoderm; E, embryonic day; ECM, extracellular matrix; Epi, Epiblast; GST‐C‐CPE, glutathione S‐transferase (GST) tagged C‐terminal fragment (C) of clostridium perfringens enterotoxin (CPE); ICSI, intracytoplasmic Sperm Injection; IGFBP‐1, Insulin‐like growth factor‐binding protein 1; IL‐8, interleukin 8; IVF, In vitro fertilization; IVM oocytes, In vitro matured oocytes.; kPa, kilopascals; PrE, primitive endoderm; TE, trophectoderm; vCTBs, villous cytotrophoblasts. Numerous studies over time have shown that both mechanical and enzymatic factors are potentially involved in the mechanisms underlying follicle rupture during ovulation. 31 , 32 However, the precise underlying mechanisms remain to be clarified. 31 , 32 More than 50 years ago, the hydrostatic pressure within graafian follicles in rabbits was determined by direct cannulation using micropipettes. 33 , 34 Rondell 33 showed no significant measurable difference just before ovulation. Furthermore, artificially increasing intrafollicular pressure by fluid injection did not induce follicle rupture. 33 Tension‐length diagrams for the elastic elements of the follicular wall showed that a large increase in extensibility precedes rupture. 33 He concluded that such a change in the physical characteristics of the follicular wall may be involved in the mechanism of ovulation. 33 Similarly, Espey and Lipner 34 also hypothesized that structural changes in the thecal wall under the force of steady pressure in the antrum induce ballooning and follicle rupture. Later, another group also showed by direct cannulation using micropipettes no increase in intrafollicular pressure as ovulation approached in pigs. 35 Intrafollicular pressure in many follicles was greater than 30 cm H 2 O (~22 mm Hg), and consistent with the findings by Rondell, 33 it was increased to over 400 cm H 2 O (~294 mm Hg) by antral injection of mineral oil without follicle rupture. 35 Therefore, it had been considered that intrafollicular pressure might not contribute to follicle rupture but rather to a change in the physical characteristics of the follicular wall under the force of a steady pressure in the antrum. 33 , 34 , 35 However, the method used in those previous studies might not be capable of detecting subtle changes in intrafollicular pressure. In a later study, use of a servo‐null micropipette system, which detects very rapid and subtle changes within very small fluid‐filled spaces, showed a significant increase in intrafollicular pressure from the preovulatory phase (16.6 ± 1.0 mm Hg, diameter 1045 ± 84 µm) to the late ovulatory phase (23.9 ± 1.9 mm Hg, diameter 1071 ± 18 µm) in rat ovaries after hCG stimulation. 36 Those authors speculated that the increased intrafollicular pressure, along with enzymatic degradation of the ECM, may be important for follicle rupture before ovulation. 36 Further studies are required to investigate the role of mechanical and biochemical cues in follicle rupture during ovulation, using large animal models such as cows, horses, and sheep, which have been validated for their use in evaluating ovarian function in women. 29 The cortex of human ovaries in polycystic ovarian syndrome (PCOS) is harder than that of normal ovaries. 8 , 24 , 37 However, it remains inconclusive whether a difference in SW propagation exists in ovaries between women with PCOS and those with normal ovulation. 38 , 39 , 40 Previous studies using SW ultrasound elastography reported no significant differences in ovarian mechanical properties between PCOS and normally ovulating women. 39 , 40 However, those studies did not investigate the differences in the mechanical properties of the ovarian cortex separately from the medulla between women with PCOS and normally ovulating women. 38 , 39 , 40 In PCOS, intra‐ovarian hyperandrogenism may promote early follicular growth, leading to follicles sized 2 ± 5 mm. 41 Three‐dimensional (3D) culture of mouse follicles in alginate hydrogels resulted in high levels of androstenedione and progesterone in immature two‐layered follicles, whereas androgen accumulation was not as pronounced in mature multilayered follicles cultured under increased matrix stiffness in alginate hydrogels (3%). 42 There is a marked but temporary reduction in ovarian androstenedione secretion in PCOS patients after wedge resection. 43 These findings suggest that the harder cortex in PCOS ovaries than normal ovaries may promote early follicular growth via excess ovarian androstenedione. 42 , 43 Ovarian wedge resection may temporarily promote ECM degradation, resulting in a marked but temporary reduction in ovarian androstenedione secretion. 43 Murayama et al. 44 measured the Young's modulus of the zona pellucida during oocyte maturation, fertilization, and early embryonic development in mice using a micro tactile sensor. The zona pellucida softened during oocyte maturation from the germinal vesicle (GV) oocyte (Young's modulus, 22.8 ± 10.4 kPa) to metaphase II (MII; 8.26 ± 5.22 kPa) stage. 44 Then, the zona pellucida hardened at the pronuclear (PN) stage (22.3 ± 10.5 kPa) to a similar level as that of the GV oocyte, followed by gradual softening as the embryo developed from the 2‐cell (13.8 ± 3.54 kPa) to morula (1.88 ± 1.34 kPa) and early blastocyst (3.39 ± 1.86 kPa) stages. 44 Using an automated micropipette aspiration technique, Yanez et al. 45 also showed that oocytes soften as they mature from GV to metaphase I (MI) to MII. Other studies additionally demonstrated that human oocytes become softer during maturation, and an optimal stiffness range of the zona pellucida before fertilization is required for successful embryo implantation. 45 , 46 , 47 Using indentation measurements via atomic force spectroscopy, Andolfi et al. 46 investigated the mechanical properties of whole human oocytes approximately 3–4 h after retrieval under physiological conditions. Measurements were performed on immature (MI) and mature (MII) oocytes, and MII oocytes were further classified as ‘‘suitable’’ or ‘‘rejected’’ according to their morphological characteristics. 46 Those authors demonstrated that the zona pellucida outer layer is stiffer in immature (MI) than mature (MII) oocytes. 46 Furthermore, the zona pellucida outer layer of “rejected” MII oocytes was softer than that of ‘‘suitable’’ MII oocytes. 46 Another study measured the zona pellucida shear modulus of fresh human oocytes using computational methodology to calculate the mechanical parameters that govern zona pellucida deformation during a routine intracytoplasmic sperm injection (ICSI) procedure. 47 Then, those authors investigated the association between parameter C10, which represents the zona pellucida shear modulus, and implantation rate and showed that oocytes with C10 values of 0.20–0.30 and 0.30–0.40 kPa had the highest implantation rates (50% [6/12] and 88.8% [8/9], respectively). 47 Outside of these ranges, the average implantation rate was 6.70%. 46 , 47 After fertilization, optimal stiffness in the zygote is required for successful human embryonic development. 45 , 46 , 47 A study demonstrated that the viscoelastic properties of the zygote within hours after fertilization can predict blastocyst formation in humans and mice. 45 Appropriate changes in mechanical properties may be required to develop successfully, and too stiff or too soft zygotes may result in non‐viable embryos. 45 RNA‐seq data revealed that non‐viable human embryos exhibit altered expression of several genes involved in fertilization, which could impair cortical granule release and zona pellucida hardening, thus affecting the mechanical phenotype of the embryo. 45 Non‐viable mouse embryos had abnormalities in cortical granule release and speculated that abnormalities in oocyte softening and maturation before fertilization and/or in cortical granule exocytosis at fertilization may result in different mechanics between viable and non‐viable embryos. 45 A limitation of that study was that the mechanical properties were measured in the whole embryo. 45 The embryo is a mechanically heterogeneous structure consisting of relatively soft cells surrounded by a stiff zona pellucida. Thus, the contributions of separate structures (zona pellucida and cytoplasm) to the bulk mechanics and developmental potential of the embryo remain to be clarified. 45 Blastocyst hatching, which is a crucial step in embryo implantation, was shown to be driven by mechanical forces in mouse embryos. 48 Using a hydrogel deformation‐based method, the hydraulic pressure inside blastocysts was measured. 48 The blastocyst was mathematically modeled as a thin‐walled pressure vessel. 48 The mouse blastocysts were cultured in polyacrylamide hydrogels with cylindrical cavities from the early cavity stage at ~3.5 days and during embryonic development; the hydraulic pressure and volume of the blastocysts increased, causing mechanical stretching and consequently decreased thickness of the zona pellucida. 48 This model demonstrates that volumetric expansion, rather than pressure generation, induces zona pellucida rupture and embryo hatching. 48 The mathematical model estimated that zona pellucida thickness decreases to one‐third of its original thickness when embryo hatching occurs. 48 Segmental muscular contractions in the oviduct and mechanical agitation of oviduct fluid by epithelial cilia result in mechanical effects on embryos, including shear stress by tubal fluid flow, compression by peristaltic tubal wall movement, and kinetic friction forces between the embryo and cilia. 49 Consequently, embryos are continually moving preimplantation, which provides a homogenous environment. Thus, dynamic culture platforms are required to provide an in vitro environment that recapitulates the in vivo growth environment more closely. 49 To mimic mechanical stimuli similar to those in the fallopian tube (shear stress, compression, and friction forces), different culture systems have been developed. Matsuura et al. developed the tilting embryo culture system (TECS) to move embryos along the bottom of a dish at a rate of ~1 mm/min via the fluid motion of microdroplets. 50 They showed that the development rate of mouse blastocysts in the TECS was significantly better than that of static control blastocysts. 50 The TECS produced more high‐grade blastocyst of fresh human embryos compared with a control static culture system. 51 Isachenko et al. 52 showed that mechanical micro‐vibration at 44 Hz/h over 5 s intervals significantly increased the developmental competence and quality of human embryos in vitro. Mechanical micro‐vibration of embryos in vitro resulted in a significantly higher pregnancy rate, compared with no micro‐vibration, after transfer of day‐3 embryos (78.4 ± 3.2% vs. 50.1 ± 4.9%) and day‐5 embryos (72.2 ± 1.5% vs. 33.2 ± 2.4%) in women. 52 As both studies used conventional culture dishes placed on a moving platform inside an incubator, 51 , 52 clinical implementation of the techniques can be achieved easily. Further studies in human embryos may be required to investigate whether a significantly higher live birth rate can be achieved using this approach. Kim et al. 53 compared the development rates of embryos cultivated in a straight channel versus constricted channels with different constriction widths (150 and 160 µm) to mimic peristaltic muscle contraction. Bovine embryos were incubated on a tilting machine, with a cyclic ±10° tilt over 1 min, to move the embryo via gravity. 53 They showed significantly more two‐cell embryos developing into eight‐cell embryos when cultivated in constricted channels with widths of 150 (37.8 ± 11.0%, mean ± SD) and 160 µm (56.7 ± 13.7%) compared with straight channels (23.9 ± 11.0%). 53 However, in their preliminary experiments, only ~10% of embryos cultivated in the constricted channels with a 140 µm width developed until the eight‐cell stage, and many did not exhibit a healthy morphology. 53 Then, the same group evaluated the amplitude of the pressure and duration of the stimulus on the development of bovine embryos, to investigate the effect of defined embryo culture conditions on stimulation of the embryo in the fallopian tube. 54 They developed a microfluidic platform with varying patterns of compressive force via Polydimethylsiloxane membrane deformation independently controlled by a micro‐modulated syringe pump. Bovine embryos were stimulated using a maximum pressure of 150 Pa over a duration of 12 s or 2 s. 54 After culturing in the device for 3 days, successful cleavage of non‐stimulated embryos and of embryos mechanically stimulated for 2 s was observed. 54 On the other hand, the embryos mechanically stimulated for 12 s were underdeveloped. 54 These findings clearly showed that both the amplitude of the pressure and duration of the stimulus are significant factors affecting bovine embryonic development in vitro. 53 , 54 Advances in microfluidic systems have enabled the development of automated approaches to achieve precise fluid flow. 49 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 In the early 2000s, studies showed that excess shear force may be harmful to mouse embryonic development. 49 , 53 , 54 , 55 Hickman et al. 55 developed a silicon‐glass microdevice for in vitro manipulation of preimplantation embryos using microelectromechanical system technology. They evaluated the effects of dynamic medium conditions on the development of mouse embryos cultivated under four different conditions: microdrop (control), microdevice under static medium conditions, and microdevice under dynamic medium conditions at flow rates of 0.1 and 0.5 µl/h. Two‐cell mouse embryos were cultured for 72 h. 55 Significantly higher proportions of blastocysts and morulae and lower proportions of abnormal and eight‐cell embryos were observed in embryos cultivated in the microdevice under static medium conditions compared with dynamic medium conditions. 55 The authors proposed two potential explanations for this result: (1) both flow rates used were too high for mouse preimplantation embryos, and (2) necessary compounds in addition to waste products might have been washed away. 55 Xie et al. 49 showed that shear stress at 1.2 dynes/cm 2 using a rotating wall vessel caused lethality in embryonic day (E) 3.5 (early blastocyst stage) mouse embryos within 12 h. E2.5 (compacted 8‐cell/early morula stage) embryos were more sensitive to shear stress than E3.5 embryos. 49 They speculated that the experimental shear stress at 1.2 dynes/cm 2 is presumably 10‐fold greater than that of embryos in the oviduct. 49 Furthermore, the maximum velocity duration of embryos in the oviduct was shorter than 12 h. 49 It is presumed that the shear stress acting on embryos in the oviduct in vivo has low velocities or high velocities but short durations that prevent harm to the embryo. 55 After these earlier studies showing the harmful effects of excess shear force on mouse embryonic development, 49 , 53 , 54 , 55 Heo et al. evaluated periodic fluid pulses in medium at physiological frequencies (0.135 Hz in rabbit oviduct 56 ) using a computer‐controlled microfluidic culture platform in mouse embryos. 57 The blastocyst developmental stage as well as the rates of embryo implantation and ongoing pregnancy was significantly higher under dynamic microfunnel culture conditions than static conditions (microdrop control and microfunnel static control). Embryo implantation and ongoing pregnancy rates under dynamic microfunnel culture conditions more closely resembled the rates obtained from in vivo blastocysts. 57 Esteves et al. further showed that one‐time pulsatile delivery of medium in a nanoliter chamber at 3 days postcoitum (dpc; morula stage) may benefit mouse embryonic development. 58 They showed that a transient (1–2 min) but much higher flow rate (4–50 μl/h; maximum shear stress: 0.17 dynes/cm 2 ) than that applied by Hickman et al. 55 (0.1 µl/h or 0.5 µl/h) had no influence on embryo preimplantation rates, and more importantly, this one‐time medium refreshment increased birth rate (28.8%) compared with that without medium refreshment (16.9%). 58 Another group evaluated mouse embryonic development in an electrowetting‐on‐a‐dielectric substrate. 59 The droplet velocity was controlled in the range of 0.3–1 mm/s to mimic the movement of a cleaved embryo in the oviduct. 59 Embryonic development from the 4‐ or 8‐cell blastocyst stage in dynamic culture with a droplet velocity (15s/0.5 h, 60 V or 15 s/2.0 h, 60 V) was compared with the development of embryos in static culture. 59 Ferraz et al. designed a microfluidic oviduct‐on‐a‐chip device with uniform shear stress across the entire epithelial layer under perfusion (5 μl/h); bovine oviduct epithelial cells cultured in this device maintained their morphology and function, similar to in vivo oviduct epithelial cells. Using in vitro matured oocytes, in vitro fertilization (IVF) was performed either in a four‐well dish or inside the microfluidic device. 60 The average shear stress exerted on the embryos was 0.70 ± 0.46 dynes/cm 2 . Both cleavage (56.0% vs. 84.4%) and 8–16‐cell embryo formation (36.7% vs. 53.7%) rates were lower on the microfluidic chip than on the four‐well dish. 60 Nearly half of the mature oocytes/embryos had escaped through the pillars and subsequently either were lost during perfusion or became trapped between the pillars, which resulted in developmental arrest. 60 Furthermore, embryos trapped between the pillars were exposed to a maximum shear stress of 2.06 dynes/cm 2 , greater than the stress (1.2 dynes/cm 2 ) that caused lethality within 12 h in E3.5 (early blastocyst stage) mouse embryos. 60 Thus, this oviduct‐on‐a‐chip system did not mimic the in vivo oviduct environment. 60 Three types of uterine peristaltic contractions can be distinguished during the menstrual cycle: cervico‐fundal contractions, fundo‐cervical contractions, and isthmical contractions. 61 Cervico‐fundal contraction waves are seen during the follicular and luteal phases of the cycle, with a maximum frequency occurring during the preovulatory phase. 61 Maximum‐frequency fundo‐cervical contraction waves (fundo‐cervical peristalsis) are seen during the late menstrual period, after which they decrease progressively and disappear nearly completely following ovulation. 61 In vitro studies showed that mechanical strain is transduced into biochemical signals in the endometrium. 62 , 63 , 64 , 65 Kim et al. 62 showed that mechanical strain mimicking the peristaltic motion of the uterine smooth muscle layer via cyclic tensile stretch in vitro upregulated alpha‐smooth muscle actin expression and enabled the endometrial stromal cells to acquire contractility. These findings suggest that the endometrium passively acquires new contractile functions in vivo under mechanical stimulation by the myometrium. 62 Myometrial contractions deform the endometrial layer and induce peristaltic intrauterine fluid flow. 65 Another study investigated the effects of peristaltic intrauterine fluid flow on the polymerization of F‐actin filaments in endometrial epithelial cells co‐cultured on top of myometrial smooth muscle cells, using a commercially available human endometrial epithelial cell line, RL95‐2, and uterine muscle cells. 65 In contrast to the study by Kim et al., 61 the endometrial epithelial cells were in direct contact with the shearing fluid. 65 The cyclic shear stress reached values as high as 0.045 Pa. 65 F‐actin polymerization showed a more pronounced increased in the endometrial epithelial cells but was also increased in the inner layer of myometrial smooth muscle cells that were not in direct contact with the shearing fluid. 65 That study clearly showed that mechanical force can be applied at a distance and transmitted through another structure, which is a typical example of mechanical signaling. 65 Clinical studies suggested that optimal cervico‐fundal contractions are required to guide spermatozoa to the ovum during ovulation, and an overall dampening of endometrial wave activity during the secretory phase is necessary for successful embryo implantation. 66 , 67 , 68 , 69 In infertile patients with endometriosis, the mean overall peristaltic activity was significantly increased compared with healthy controls during the late menstrual, early and mid‐follicular, and mid‐luteal phases. 61 IL‐8 may be involved in the pathophysiology of endometriosis. 70 IL‐8 protein expression was highest during the late secretory and early proliferative phase in the endometrium of women without endometrial disease. 70 IL‐8 stimulates endometrial cell proliferation and attachment to the ECM. 70 An in vitro study showed that cyclic stretch stimulated IL‐8 production in human endometrial stromal cells (ESs). 63 These findings suggest that the greater peristaltic activity in patients with endometriosis may induce greater IL‐8 production in the ESs of infertile patients with endometriosis. 63 Another study by the same group also showed that mechanical stretch induced the secretion of IGFBP‐1, a marker of decidualization, from decidualized ESs, but not from non‐decidualized ESs. 64 Those two in vitro studies suggest that optimal, well‐controlled uterine contractility throughout the menstrual cycle is required for normal endometrial physiology. 63 , 64 Submucosal and intramural leiomyoma cause pregnancy loss or infertility. 71 Cine magnetic resonance imaging (MRI) detected abnormal peristaltic patterns in patients with uterine leiomyoma (submucosal n  = 6/7, intramural and subserosal: n  = 3/4) in the mid‐luteal phase of the cycle. 72 , 73 A high frequency of uterine peristalsis (≥2 times/3 min) during the mid‐luteal phase in patients with intramural leiomyoma significantly decreased pregnancy rate ( n  = 0/22) compared with that of the low‐frequency group ( n  = 10/29). 74 The frequency of uterine peristalsis was normalized (0 or 1 time/3 min) after myomectomy in 14 out of 15 patients with intramural leiomyoma. 75 Abnormal uterine peristalsis during the mid‐luteal phase might be one of the causes of infertility in patients with submucosal and/or intramural leiomyoma. Previous in vitro studies suggested that mechanical uterine movements are transduced into biochemical signals in the endometrium. 62 , 63 , 64 , 65 However, comparison with precise in vivo measurements of mechanical stress by uterine peristaltic movements is ultimately required to validate these previous in vitro study findings. Using 3D multifrequency magnetic resonance elastography, a study showed lower elasticity of the endometrium during the secretory phase than proliferative phase (1.97 ± 0.34 vs. 3.34 ± 0.42 kPa, mean ± SD). 76 Because the ECM composition generally determines the stiffness of a tissue, 77 that finding suggests that endometrial ECM stiffness is lower during the secretory phase than proliferative phase. An in vitro study showed that the stiffness of human ESs decreased during in vitro decidualization. 78 They speculated that the reduced stiffness of ESs was most likely attributed to the cytoskeletal changes promoted by mesenchymal‐epithelial transition, including downregulated expression of vimentin, alpha‐smooth muscle actin and myosin light chain kinase, and F‐actin destabilization. 78 Myosin light chain kinase downregulation has also been shown to result in a decrease in MLC20 phosphorylation, thereby reducing the cell stiffness contributing to decidualized ES plasticity. 79 , 80 Thus, decidualized human ESs might be softer than non‐decidualized ESs. Furthermore, studies in both mice and non‐human primates suggested that changes in the surrounding ECM may differ between implantation and non‐implantation sites. 81 , 82 , 83 A mouse study showed specific localization of lysyl oxidase (LOX), an ECM‐shaping enzyme, in the stromal cells surrounding the implanting embryos. 83 LOX is a copper‐containing amine oxidase that catalyzes the covalent cross‐linking of collagen and elastin in the ECM. 84 The ECM, especially its interstitial collagens, exerts mechanical forces on surrounding cells, leading to the transduction of mechanical signals on the cell surface and the initiation of chemical cascades. 5 , 14 , 15 Thus, the ECM surrounding the implanted mouse embryos may increase matrix stiffness. In “fertile women” undergoing infertility treatment due to tubal ligation or male factor infertility, LOX expression in stromal cells was significantly higher during the mid‐secretory phase (7–10 days post‐ovulation) that is during the window of embryo implantation, than during the proliferative phase. 85 These findings suggest that the stiffness of human endometrial ECM during the implantation window may be modified. An in vitro study showed that the motility of human endometrial stromal cells is increased at the embryo implantation site. 86 The stiffness of cancer cells is inversely correlated with their migration and invasion through 3D basement membranes. 87 The decreased stiffness of decidualized human ESs may increase their migration capacity. In mice, blastocyst invasion and stromal cell migration were impeded by an inhibitor of Lox activity, β‐aminopropionitrile. 83 Further studies are required to investigate whether spatial changes occur in the stiffness of the human endometrial ECM at the implantation site, and whether such changes, as well as decreased stiffness of decidualized ESs, are required during the initial phase of human embryo implantation. There is very limited information about how intrauterine pressure produced by uterine smooth muscle contractions is produced and accurately adjusted, and how it contributes to mammalian embryogenesis. Ueda et al. investigated the effects of intrauterine pressure on early mouse embryonic development. 88 In mouse embryos, after fertilization from E3.5 to E6.5, the amplitude of intrauterine pressure was the highest and most frequent at E5.5, just after implantation. 88 Mouse anterior‐posterior axis polarization is preceded by formation of the distal visceral endoderm (DVE). 89 , 90 The mouse anterior‐posterior axis is initially generated in a proximal‐distal (P‐D) direction at around 5.5 days post‐coitum (dpc). 89 , 90 Those authors 88 showed that adjusting the intrauterine pressure within the sealed space between the embryo and uterus contributes to early egg cylinder morphogenesis in a P‐D orientation. Then, they investigated the effects of relaxation of uterine smooth muscle contractions on mouse embryonic development. 88 Salbutamol, an activator of β2‐adrenoreceptor, was administered three times every 6 h from E5.0 to E5.5, and the embryos were fixed at E5.75. 88 Salbutamol had little adverse effect on implantation and the early pregnancy phase in the uterus. 88 However, in salbutamol‐treated embryos, the DVE‐related markers Cer1 and Hex were not expressed, and Lhx1 and Lefty1/2 were not properly induced, at E5.75, whereas at E6.5, the anterior visceral endoderm (AVE) marker Cer1 and primitive streak marker T were expressed at the normal sites. 88 Salbutamol‐treated embryos proceeded to undergo normal fetal development and delivery. 88 These findings suggest that intrauterine pressure is necessary for timely specification of the DVE. 88 By culturing mouse embryos immediately after implantation in microfabricated cavities in vitro, the same group showed that the mechanical environment outside of the embryo can direct selective elongation of embryos in the P‐D direction. 91 Furthermore, they showed that the mechanical forces induce a breach in the basement membrane barrier at the distal portion locally and induce transmigration of epiblast cells directly into the visceral endoderm. 91 By in vitro culturing of mouse embryos, they showed that confinement by a sufficiently stiff external environment is required for proper formation of the elongated egg cylinder shape and the DVE. 91 Stage 1 embryos (5.0 dpc) inserted into the cavities formed inside agarose gels of different stiffness levels. 91 Agarose gels with a stiffness over 22.94 kPa can promote the elongated shape of the egg cylinder as well as DVE formation, whereas agarose gels with a stiffness below 7.29 kPa cannot induce either an elongated egg cylinder shape or DVE formation. 91 Using atomic force microscopy, they validated that the in vivo stiffness of the decidual tissue surrounding the embryo, isolated at 5.5 dpc (23.26 ± 9.8 kPa, mean ± SD), is comparable with the gel stiffness sufficient for DVE formation. 91 Furthermore, impairment of cell proliferation in embryos treated with SB431542, an inhibitor of activin receptor‐like kinase receptor‐dependent signaling, resulted in failure of DVE formation. 91 These findings suggest that the mechanical cues arising from the appropriate physical growth of embryos in terms of size and the external geometrical constraints imposed by the uterine environment directly regulate the local breach in the basement membrane and transmigration of epiblast cells into the visceral endoderm at the distal tip, thereby establishing the mammalian primary body axis in the mouse embryo. 91 However, it remains to be clarified whether stimulated contractions and/or increased ECM stiffness at the site of embryo implantation in the human uterus occur after embryo implantation, and whether such external mechanical cues from the uterine environment are involved in human embryonic development, as shown in mouse embryos. The mammalian preimplantation embryo contains three cell lineages: pluripotent embryonic epiblast cells that generate embryonic tissues (EPI cells) and two extra‐embryonic lineages, the trophectoderm, which contributes to the placenta, and the primitive endoderm, which contributes mostly to the extra‐embryonic yolk sac. 89 , 90 , 92 The molecular mechanisms underlying the specification of these distinct lineages have been extensively studied in the mouse. 89 , 90 In the mouse, the first cell fate decision, in which outer cells are specified to differentiate into the trophectoderm and inner cells into the inner cell mass (ICM), involves differential Hippo signaling during compaction. 89 , 90 Differential FGF signaling during the blastocyst stage leads to the second cell fate decision, involving segregation of the EPI and primitive endodermal lineages within the ICM. 89 , 90 , 92 Comparatively little is known about the mechanisms of lineage specification in human embryogenesis. 90 , 92 Fertilized mouse eggs undergo three rounds of cleavage, followed by polarization concomitant with compaction at the eight‐cell stage. 89 From the eight‐cell stage onward, asymmetric division rounds result in generation of two distinct cell types: those located inside versus outside of the embryo. 89 Cells that inherit the apical domain differentiate into the trophectoderm, whereas cells that do not inherit the apical domain and are internalized become the ICM (precursors of the embryonic epiblast and extra‐embryonic primitive endoderm in mouse embryos). 89 Transcriptionally active Tead4 can induce the expression of Cdx2 and other trophoblast genes. 93 Nishioka et al. 93 showed that two components of the Hippo signaling pathway, Lats and Yap, are involved in the establishment of position‐dependent Tead4 activity and cell fate specification. In mouse embryos, Yap, the coactivator of Tead4, localizes to the nuclei of the outer cells, whereas in the inner cells, Yap is phosphorylated in the cytoplasm by Lats‐mediated inhibition of nuclear Yap localization. 93 It was shown that a combination of cell polarity and cell‐cell adhesion establishes position‐dependent Hippo signaling. 94 In the nonpolar inner cells, which have no contact‐free surface, the junction‐associated protein angiomotin localizes to adherens junctions (AJs), and cell‐cell adhesion activates the Hippo pathway. 94 In the outer cells, cell polarity disconnects the Hippo pathway from cell‐cell adhesion by sequestering angiomotin from adherens junctions, thereby suppressing Hippo signaling. 94 However, Anani et al. showed that cell‐cell contact is involved in, but not essential for, the initiation of differential Yap localization in polar/apolar cells. 95 They showed that phosphorylated YAP (p‐YAP) is differentially regulated between polar and apolar cells prior to localization to the outer/inner positions as a result of higher cortical tension in apolar cells than polar cells. 95 Later, another group showed that the apical domain is required and sufficient to control the first cell fate decision. 96 Their findings suggested that the difference in cell‐surface contact is the crucial signal distinguishing the outer and inner positions, independent of Cdh1. 96 The apical domain emerges at the center of the contact‐free surface, and the apical domain induces asymmetric division: The apical domain is inherited differentially by two daughter cells, one with and one without polarity. 96 Apolar cells compete for a position inside the embryo, and those that are pushed out toward the surface assume a position outside of the embryo. 96 They showed that Cdx2, a master regulator of the trophectoderm in mice, is specifically upregulated only in polar daughter cells. 96 Their previous study also showed that the apical domain exhibits reduced cortical contractility and distinct actomyosin contractility, and tension between polar and apolar cells triggers their sorting into the inner and outer positions and regulates Yap localization. 97 Without contractile forces, blastomeres adopt an inner‐cell‐like fate, regardless of their position. 97 When contractile forces are blocked by treatment with blebbistatin, a selective myosin II ATPase inhibitor, phosphorylated Yap localization, and Cdx2 levels become homogeneous within doublets of 16‐cell‐stage blastomeres, regardless of their position. 97 These findings indicate that contractile forces are required for fate specification. 97 Altogether, these findings suggest that interactions among cell contact, polarity, and mechanics may transduce the signaling necessary for cell fate specification in mouse embryos. 93 , 94 , 95 , 96 , 97 Furthermore, the fluid‐filled lumen of blastocysts plays a key role in the control of embryo size and spatial allocation of the trophectoderm and ICM within cells, as demonstrated in mouse embryos. 98 In mice, mid‐stage blastocysts (E4.0) have a lumenal pressure of approximately 650 Pa, which increases to ~1500 Pa by the mature blastocyst stage (E4.5), which translates into a concomitant increase in cell cortical tension and tissue stiffness in the trophectoderm lining the lumen. 98 Wang et al. also measured the inner pressure and Young's modules of the trophoblasts and ICM of blastocyst‐stage mouse embryos using a laser‐assisted magnetic tweezer technique. 99 The inner pressure levels of the embryo and Young's modulus of the trophoblast were ~3.7 and ~1.6 times higher, respectively, at E4.5 than at E3.0. The Young's modulus of the ICM increased 3.6 fold from E3.0 to E4.5. 99 Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establish a positive feedback loop to promote lumen growth. 98 The presence of a cortical tension threshold leads to trophectoderm rupture and blastocyst collapse. 98 Tight junctions then reseal, and the blastocyst expands again, and the whole process repeats itself to enable size oscillations and size control. 98 Leaky cell junctions also lead to a reduced expansion rate and blastocyst size, and softening (or stiffening) of the trophectoderm shell leads to an increase (or decrease) in the expansion rate and size. 98 The blastocyst cavity shows an initial steady increase in volume. 98 Collapse events are rarely observed in the early‐ and mid‐blastocyst stages, suggesting that high cortical tension may be required to “prime” junctional rupture during mitosis. 98 Inter‐collapse intervals become more frequent toward the late blastocyst stage (E4.5). 98 Changes in cavity size or trophectoderm stiffness can independently affect lineage composition. 98 Embryos with a reduced cavity size occasionally do not spatially segregate into the inner and outer cells. 98 Furthermore, embryos under hypertonic conditions showed an increased frequency of an outer cell undergoing asymmetric division to generate one trophectoderm‐forming and one ICM‐forming cell. 98 Once the mouse embryo reaches the blastocyst stage at E3.5, mouse embryos undergo a second cell fate specification event, in which the ICM segregates into pluripotent epiblast and primitive endoderm (in humans, extra‐embryonic hypoblast). 89 Formation of a hollow cavity, the blastocoel, denotes the formation of the blastocyst, which by E4 (mouse) and E6 (human) is composed of three main tissues: epiblast, hypoblast, and trophectoderm. 89 , 90 , 92 Epiblast (Epi)‐primitive endoderm (PrE) specification and spatial segregation within the ICM are dependent on luminal expansion in mouse embryos. 100 Luminal deposition of FGF4 can partially rescue EPI‐PrE specification in embryos with reduced lumen expansion. 100 A potential explanation is that luminal expansion may increase the luminal FGF4 concentration to guide ICM differentiation. 100 These findings imply that chemical and physical cues are integrated to guide ICM self‐organization in the mouse blastocyst. 100 The molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. 90 , 92 However, studies showed significant differences in both the timing and expression pattern of transcriptional factors between mouse and human embryos. 89 , 90 , 92 In human embryos, lineage segregation between the ICM and trophectoderm occurs later than in mouse embryos. 89 , 90 , 92 At ~5 days post‐fertilization, the blastocyst segregates into two lineages. 90 , 92 In contrast to mice, strong nuclear expression of YAP is also detected in ICM cells during the early stage of blastocyst formation (E5), whereas during the late blastocyst stage, YAP nuclear localization is restricted to the trophectoderm, similar to mice. 101 In mouse embryos, polarization determines p‐YAP nuclear localization partly via mechanical control, as described above, 93 , 94 , 95 , 96 , 97 and thus controls the first lineage segregation. It is very likely that YAP is also required to mediate trophectoderm lineage segregation in human embryos, as dual suppression of YAP and TAZ strongly suppressed trophectoderm induction in human embryonic stem cells (ESCs). 102 However, in humans, pYAP is localized in the nucleus in both the ICM and trophectoderm during the early blastocyst stage (E5), and the trophectoderm at E5 can regenerate an ICM population. 103 These findings suggest that polarization alone cannot determine p‐YAP nuclear localization and, thus, the first lineage segregation in human embryos. OCT4, an ICM‐specific pluripotency marker, is initially expressed at the eight‐cell stage, similar to mouse embryos, following genome activation and is expressed in all cells until the blastocyst stage at day 6, when OCT4 is restricted to EPI cells. 104 CDX2, a master regulator of the trophectoderm in mouse embryos, is first detected in human embryos at E5, in the trophectoderm of blastocysts following cavitation. 104 In contrast, in mice, Cdx2 initially exhibits mosaic expression at the 16‐cell stage and is eventually uniformly expressed in all trophectoderm cells. 104 Thus, in human embryos at the early stage of blastocyst formation (E5), in contrast to mouse embryos, nuclear expression of YAP is detected in both the ICM and trophectoderm, whereas CDX2 expression overlaps the expression of OCT4 in nearly all trophectoderm cells, similar to the pattern in mouse embryos. 104 In human embryos, the first lineage segregation occurs at ~E5 following cavitation of blastocysts, when CDX2 is first detected. 90 , 92 , 104 Thus, the fluid‐filled lumen of blastocysts, rather than cell polarization, may play a key role in the spatial allocation of trophectoderm and ICM cells in human embryos. Further studies are required to clarify the role of the forces and changes in mechanical properties in fate determination in human embryos. Because of ethical and technical constraints limiting research, little is known about the early human embryo after the polar trophectoderm of day (D) 6 to D7 blastocysts adheres to the uterine wall. 90 , 92 Our knowledge has been limited mainly to the analysis of human embryo specimens from the Carnegie collection, which has been unable to provide insights into the dynamic process of human embryo implantation and what can go wrong. 90 , 105 A very recent study demonstrated that the polar trophectoderm is an evolutionarily conserved regulator of epiblast shape after implantation. 105 Species‐specific remodeling after implantation is likely due to differences in the behavior of the trophectoderm. 105 During the transition from pre‐ to post‐implantation (E4.5 to E5.0), the mouse epiblast transforms from an oval to a cup shape. 89 , 90 , 105 In mouse embryos, implantation and invasion into the maternal endometrium are mediated by the mural trophectoderm. 89 , 90 , 105 Increasing contractility and tension in the polar trophectoderm generate a physical force to push the epiblast into its post‐implantation configuration, which leads to the acquisition of the cup shape. 105 Further apical constriction of the polar trophectoderm results in formation of the post‐implantation egg cylinder. 105 In contrast, implantation and invasion of human embryos into the maternal endometrium are mediated by the polar trophectoderm, 90 , 92 resulting in pulling and stretching of the embryo. 105 The human embryo does not exhibit any sign of horizontal constraint, and polar trophectoderm stretching alone could shape the epiblast into a disk structure. 105 Epiblast shape was analyzed after implantation of human embryos from the Carnegie collection. 105 It was confirmed that human epiblasts initially form an oval shape but then become disk‐like, growing horizontally but not vertically. 105 Rock inhibition of mouse embryos by blebbistatin treatment for 20 h at E4.5 prevented pMyosin‐II‐mediated contractility and forced the epiblasts into a disk‐like shape characteristic of human embryos. 105 Altogether, these findings suggest that physical cues play an essential role in controlling embryonic development in humans but potentially via different mechanisms compared with those in mice. 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 Greater insight into early human embryonic development will be achieved using recent technological advancements, including CRISPR‐Cas9‐mediated genome editing, 106 novel imaging techniques with new computational methods that enable high‐resolution fate maps and maps of tissue morphogenesis at the single‐cell level, 107 a new 3D blastocyst culture system enabling human blastocyst development up to the primitive streak stage (D14), 108 human ESCs that mimic different aspects of embryonic development in vitro, 109 , 110 and development of self‐renewing human trophoblast stem cells 111 and trophoblast organoids 112 , 113 as well as endometrial epithelial organoids (Figure  1 ). 114 , 115 Recent and future developments of innovative tools for mechanobiology. The recent and future developments of novel tools (quantitative force measurements, manipulation of forces, novel in vitro models, computational models) will help us broaden our understanding of the mechanobiology of the female reproductive system. 3D, three dimensional; ECM, extracellular matrix; dECM, decellularized ECM; hESC, human embryonic stem cell; hTSC, human trophoblast stem cell [Colour figure can be viewed at wileyonlinelibrary.com ] The placenta plays a critical role in reproductive success, and it may also be associated with susceptibility to chronic diseases in adults. 116 , 117 Despite the importance of the placenta, little is known about early placental development in humans. 118 The ethical and technical constraints on research, the high divergence of the placenta across different species, and the lack of physiologically relevant in vitro models complicate animal and in vitro experiments. 118 The stiffness of fresh human samples of first‐trimester placental and decidual tissues (6–12 weeks of gestation) and secretory‐phase endometrial tissues (7–10 days after the preovulatory luteinizing hormone surge) was measured using atomic force microscopy. 119 Nonpregnant secretory‐phase endometrial, decidua parietalis, and placental tissues had a stiffness of 102 Pa, whereas the decidua basalis was an order of magnitude stiffer at 103 Pa. 119 They speculated that the increase in stiffness in the decidua basalis is likely due to changes induced by extravillous trophoblast (EVT) invasion; this is because the pre‐decidualized endometrium and fully decidualized decidua parietalis have similar stiffness values, and thus changes in the ECM during decidualization before embryo implantation do not drive the change in tissue stiffness. 119 As EVTs invade, they degrade stromal ECM proteins while simultaneously producing their own distinct ECM networks. 119 , 120 , 121 Implantation involves invasion of the embryonic trophoblast through the endometrial stroma. 86 Decidualization is accompanied by metalloproteinase (MMP)‐dependent ECM degradation. 122 A potential underlying mechanism of the increase in stiffness in the decidua basalis as a result of EVT invasion may be partly explained by epithelial to mesenchymal transition (EMT), which occurs during physiological EVT differentiation. 123 , 124 Mechanical cues, including matrix stiffness, tissue geometry, and intracellular forces, could act in concert with biochemical signals to regulate EMT. 125 , 126 , 127 , 128 EMT has been extensively studied in numerous cancer cells, and increased matrix stiffness in the tumor microenvironment induces tumor cell migration and invasion through the EMT. 125 , 126 , 127 , 128 It remains to be clarified when and how the tumor ECM increases matrix stiffness. However, studies have suggested that tumor stiffening is mediated by tumor cells themselves. 125 Cells undergoing EMT feed forward to enhance matrix remodeling via a positive feedback loop. 125 Tumor cells undergoing EMT can in turn modulate the mechanical properties of the ECM by increasing cell contractile forces, inducing extensive deposition of ECM proteins, and inducing cancer‐associated fibroblasts (CAF)‐mediated ECM remodeling to further stiffen the ECM. 125 These findings suggest that EVT invasion via EMT itself may increase the stiffness of the decidua basalis. YAP/TAZ, the key transcriptional coactivator in the Hippo pathway, is regulated by various mechanical cues, including matrix stiffness, cell‐cell contact, and cell shape. 129 TAZ was found to be weakly expressed in the villous cytotrophoblasts (vCTBs) and cell column trophoblasts (CCTs) of early placental tissues. 130 TAZ promotes EMT in various cancer cells, 131 , 132 , 133 in turn inducing EVT invasion, which may increase the stiffness of the decidua basalis. In contrast to TAZ, YAP was strongly expressed in the nuclei and cytoplasm of vCTBs and CCTs; however, low levels were detected in EVTs. 130 YAP expression is primarily associated with trophoblast stemness and proliferation. 130 YAP is a cofactor for TEAD4, and both are specifically expressed in CTB progenitors in the first‐trimester human placenta. 134 The TEAD4/YAP1 complex is essential for trophoblast progenitor self‐renewal. 134 TEAD4 expression in vCTBs and CCTs is strongly reduced in patients experiencing idiopathic recurrent pregnancy loss (RPL). 134 Human trophoblast stem cells from RPL patients showed a strong reduction in proliferation and a prominent defect in placental villus formation. 134 Rescue of TEAD4 expression restored the self‐renewal ability of trophoblast stem cells from RPL patients. 134 Impairment of the Hippo signaling pathway could be a molecular cause of early human pregnancy loss. YAP/TAZ is regulated by various mechanical cues. 16 Further studies are required to determine whether and how mechanical cues play a role in early human placental development, and whether mechanical perturbation results in early pregnancy loss. The recent development of novel tools, such as self‐renewing human trophoblast stem cells 111 and trophoblast organoids, 112 , 113 may further improve our understanding of early human placental development (Figure  1 ).

Here, I provide a brief introduction on mechanical signaling and mechanotransduction. Please refer to excellent previous review articles for more details. 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 Biochemical signals generated in response to hormones or other soluble factors have been investigated extensively, whereas much less is known about mechanical signaling, which refers to intracellular signaling events triggered by a mechanical force, in the female reproductive system. 4 Living cells generate and sense forces. 1 , 2 , 3 Intrinsic or intracellularly generated forces, including elasticity, stiffness, viscoelasticity, and adhesion, are transmitted to other cells either directly via cell‐cell junctions, such as cadherin‐based adhesions, or indirectly via cell‐extracellular matrix (ECM) interactions, such as integrin‐based adhesions. 13 , 14 , 15 , 16 , 17 , 18 , 19 Extrinsic forces comprise solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal and interstitial) applied externally on cells. 13 , 14 , 15 , 16 , 17 , 18 , 19 Mechanical signaling differs from biochemical signaling in signaling characteristics, mode of transmission, and directionality. 11 , 12 Mechanical signaling can transmit information over long distances more rapidly and efficiently compared with biochemical signaling. 11 , 12 Biochemical signaling is diffusion‐based, and the strength of the signal decreases with distance, whereas mechanical forces can act at a distance to induce mechanochemical conversion in the nucleus and alter gene activities, and the strength of the signal does not decrease with increasing distance or time. 11 , 12 Mechanotransduction is the overall process by which cells sense and respond to externally applied or internally generated mechanical forces and convert them into an intracellular response. Mechanoreceptors such as integrins and cadherins, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates critical nuclear events. 13 , 14 , 15 , 16 , 17 , 18 , 19 Forces are transmitted from the cell surface and cytoskeleton across the nuclear envelope to the interior of the nucleus, triggering changes in chromatin organization and gene expression. 14 , 15 , 16 , 17 Furthermore, findings suggest that the nucleus can act as a cellular mechanosensor and respond directly to mechanical forces. 13 , 18 , 19 Biomechanical and biochemical morphogenetic processes in embryonic development were long considered to be disconnected. 20 , 21 , 22 However, recent data indicate robust coordination between biochemical and biomechanical morphogenesis during embryonic development. 20 , 21 , 22 There are feedback loops between mechanical and biochemical signals, also termed mechanochemical feedback loops. 20 , 21 , 22 These bidirectional interactions between mechanical and biochemical signals play key roles in morphogenesis and pattern formation in both development and disease. 20 , 21 , 22 To determine how mechanical cues are translated into function, we need to understand the interplay between mechanical and biochemical signals in the complex environment of a living organism.

The present review provides the current knowledge regarding the mechanobiology of the female reproductive system. Because we do not yet fully understand the mechanobiology of female reproductive system physiology, whether and how mechanical perturbations are involved in reproductive system disorders are unclear. Recent and tremendous technological advancements in mechanobiology research will help us understand the role of mechanical forces in reproductive system disorders, such as embryo implantation failure and early pregnancy loss. Today, significant efforts are still needed to fully understand the mechanobiology of the female reproductive system. However, in the near future, I hope we can determine how biochemical, genetic, and mechanical factors are integrated to regulate the female reproductive system, and how perturbations in these factors result in related disorders.

A major challenge in mechanobiology is investigation of the cellular and molecular mechanisms linking mechanical forces to biological responses and understanding how these cellular and molecular events contribute to development, physiology, and disease. 135 Progress in the field of mechanobiology has been limited by the lack of appropriate experimental tools for (1) quantitative force measurement and (2) force manipulation. 135 Traditional animal models and standard two‐dimensional (2D) in vitro cell culture are not appropriate due to poor control of applied force and highly artificial cell culture environments, which do not recapitulate the in vivo situation. 134 Recent technological advancements have contributed to our understanding of the role of mechanical cues in both physiology and diseases. 135 , 136 , 137 , 138 , 139 , 140 Measurements of forces have been critical in enabling the growing field of mechanobiology (Table  1 ). 134 , 135 , 136 , 137 , 138 , 139 There are two types of tools for force measurements: one measures the forces actively generated by cells without applying any external force, and the other measures cellular responses to mechanical forces or mechanical quantities such as cell stiffness. 135 , 136 , 137 , 138 , 139 , 140 Over the past 20 years, a wide repertoire of techniques has been developed to measure forces. 135 , 136 , 137 , 138 , 139 , 140 These techniques, based mostly on ex vivo and in vitro conditions, have been extensively reviewed in. 135 , 136 , 137 , 138 , 139 , 140 However, these techniques are still far from becoming routine biological laboratory tools because multidisciplinary expertise is required to conduct and interpret the measurements. 135 , 136 , 137 , 138 , 139 , 140 Furthermore, quantitative comparisons of the mechanical properties measured by different methods are limited. 140 Wu et al. evaluated and compared the mechanical properties of MCF‐7 human breast cancer cells cultured under the same environmental conditions in vitro using well‐established techniques (atomic force microscopy, magnetic twisting cytometry, particle‐tracking microrheology, parallel‐plate rheometry, cell monolayer rheology, and optical stretching). 141 They showed that the mechanical properties of MCF‐7 human breast cancer cells can vary by orders of magnitude, depending on the length scale at which cell viscoelasticity is probed (eg, from the diameter of an actin fiber to the size of a whole cell). 141 Those authors clearly showed that cell mechanics depend on the level of mechanical stress and rate of deformation to which the cell is subjected, the geometry of the mechanical probe used in the experiments, the probe‐cell contact area, the probed location in the cell, and the extracellular context. 141 Janmey et al. 142 speculated that these differences in mechanical properties probably reflect the heterogeneous structure of the cell and noncontinuous nature of the cytoskeleton more than the inherent uncertainty in measurements by any single method. Thus, it may also be true that quantitative comparison among different cells even when using the same methods may be limited. Measurement of the stiffness of a tissue includes the cellular, nuclear, and ECM stiffnesses as a whole. However, in general, the composition of the ECM determines the stiffness of a tissue. 143 , 144 Dynamic cellular interactions and remodeling processes in the ECM result in heterogeneity in ECM mechanical properties. 145 Thus, when measuring the stiffness of different tissues (eg, diseased versus their counterpart normal tissues) using the same methods, caution in interpreting the results may be required. Furthermore, the measurement of cell and tissue mechanical properties in vivo is not completely feasible and extremely challenging. 135 In vivo, cells are connected to a host of materials and other cells, all of which contribute to the generation and propagation of cellular forces. 135 , 137 Very recently, a 3D magnetic device was developed to quantify the spatial stiffness distribution in living mouse embryos; it can map the absolute stiffness and viscosity of bulk tissues that are not influenced by morphogenetic cell behaviors. 146 Currently, in the clinical setting, elastography techniques based on ultrasonography, optical coherence tomography, and MRI are widely used for noninvasive measurement of in vivo mechanical properties in patients. 147 , 148 Elastography techniques are very useful to measure the in vivo mechanical properties of bulk tissue and provide valuable information for diagnosis and therapy. 147 , 148 However, they lack the resolution to dissect dynamic cell‐cell and cell‐ECM interactions, spatiotemporal changes in the ECM, or internal or external mechanical forces. 136 Microfluidics represents the engineered manipulation of fluids, usually in the range of microliters (10 −6 ) to picoliters (10 −12 ), in networks of channels with dimensions of tens to hundreds of micrometers to provide precise control of fluids at microliter volumes (Table  2 ). 149 , 150 In the human reproductive system, microfluidics was proposed in the 1990s as a new approach to minimize the specialized requirements required for successful IVF. 151 , 152 , 153 Several groups have developed microfluidic technology platforms to evaluate the female reproductive system. 151 , 152 , 153 Gnecco et al. 154 developed an organ‐on‐a‐chip microfluidic model of the human endometrial perivascular stroma to examine the crosstalk between stromal and endothelial cells. Primary human uterine microvascular endothelial cells were seeded on the top chamber and primary human ESs on the bottom chamber of the chip. 154 The microfluidic device provided continuous laminar shear stress (perfusion) to the endothelial cells to mimic hemodynamic forces derived from blood flow. 154 The work of Gnecco et al. 154 demonstrated that hemodynamic forces induce secretion of specific endothelial cell‐derived prostanoids that enhance endometrial perivascular decidualization via a paracrine mechanism. Several groups have developed microfluidic technology platforms that mimic mechanical stimuli similar to those in the oviduct in vivo to investigate the effects of mechanical cues on embryonic development. 57 , 58 , 59 , 151 , 152 , 153 Furthermore, a microfluidic oviduct‐on‐a‐chip platform was developed to maintain the morphological and functional structure of oviduct epithelial cells, similar to the in vivo oviduct. 60 For more details, please see the above section regarding the effect of mechanical cues in the oviduct on embryonic development. A hydrogel is a water‐swollen, physically or chemically crosslinked 3D network of hydrophilic polymer chains. 155 , 156 As naïve‐ECM mimics, both natural hydrogels, formed from natural polymers, and synthetic hydrogels, formed from synthetic polymers, have been widely used to investigate the effects of ECM mechanical properties on cell function. 155 , 156 , 157 In the field of female reproduction, alginate hydrogels have been used to investigate secondary follicle growth in mice. 42 The growth of mouse secondary follicles was investigated on 3D alginate hydrogels with varying shear elastic modulus values: high (3% alginate), intermediate (1.5% alginate), and low (0.7%, oxidized and irradiated alginate). 42 Follicles cultured on the 0.7% (low) alginate hydrogels showed increased follicle growth, higher rates of antral and thecal layer formation, and higher oocyte quality. 42 In another study, mouse embryos cultured on 3D type I collagen gels (1 kPa stiffness) had increased two‐cell, blastocyst, and hatching frequencies and TE cell numbers compared with embryos cultured on polystyrene petri dishes. 158 Furthermore, fetuses derived from embryos cultured on collagen in vitro had a greater placental weight at E12.5. 158 There are many advantages to using natural hydrogels, such as they are biocompatible and bioactive, and the abundant endogenous factors promote the functions of many cell types. 155 , 156 , 157 However, there are some disadvantages, including low stiffness, limited long‐term stability, and batch‐to‐batch variability. 155 , 156 , 157 Particularly, for mechanobiology studies, fine tuning the mechanical properties of natural hydrogels is difficult. 155 , 156 , 157 Tunable synthetic hydrogels may be more suitable as an experimental tool to finely control hydrogel stiffness. 155 , 156 , 157 The most common tool used for culturing cells on substrates of variable stiffness is polyacrylamide hydrogels, which can be easily fabricated using standard chemical techniques, and stiffness can be precisely adjusted by mixing various acrylamide (monomer) and bis‐acrylamide (cross‐linker) concentrations. 155 , 156 , 157 One major limitation is that polyacrylamide hydrogels cannot be used to encapsulate cells three‐dimensionally due to toxicity of the hydrogel precursors. 154 , 155 , 156 Our group used polyacrylamide hydrogels to investigate the effects of matrix stiffness on human endometrial epithelial cells (EEs). 159 Endometrial epithelial cells were grown on polyacrylamide hydrogels with varying levels of stiffness (Young's modulus, 2, 4, 8, 16, or 30 kPa) or on plastic. 159 The cells grown under 2 kPa retained their epithelial‐related phenotype, whereas those grown under 30 kPa became elongated and showed F‐actin‐positive stress‐fiber‐like structures. 159 Among the cells grown on plastic, only those located in the center retained their epithelial phenotype. 159 Endometrial epithelial cells stimulated with TGF‐β1 underwent a partial EMT‐like process even on a soft matrix (2 kPa Young's modulus). 159 A recent study investigated the effects of controlled substrate stiffness, mimicking human tissue mechanics, using polyacrylamide hydrogels, on human pluripotent stem cells (hPSC) lineage specification. 160 RNA sequencing analysis showed that a substrate with a stiffness similar to that of the liver (3 kPa) triggered the expression of endoderm‐specific genes ( EOMES , SOX17 , and FOXA2 ), whereas harder substrates (similar to bone or tissue culture polystyrene) did not. 160 The lncRNA LINC00458 was identified as a critical regulator of endodermal lineage commitment in response to ECM stiffness. 158 Primary vCTBs isolated from patients at the time of natural vaginal delivery or BeWo cells (human placental choriocarcinoma cells) were cultured on substrates with different substrate stiffness levels: sub‐physiological (shear elastic modulus, 0.1 kPa), normal (1.3 kPa), pathological (7 kPa; preeclamptic), and extreme pathological (17.4 kPa; preeclamptic). 161 The fusion ratio was nearly two‐fold higher for vCTBs cultured on a substrate of normal stiffness (1.3 kPa) compared with pathological stiffness (7 kPa). 159 Furthermore, that study showed that when BeWo cells were grown on a composite hydrogel substrate consisting of parallel soft (1.3 kPa) and stiff (17.4 kPa) strips, the fusion ratio was significantly lower for both the soft and stiff regions compared with uniformly soft substrates, suggesting that fusion of trophoblasts grown on tissues of normal stiffness could be affected by adjacent pathologically stiff regions. 161 Matrix stiffness may have an indirect effect from a distance by being transmitted through another structure. 161 This is a good example of how mechanical cues can be transmitted through another structure. 12 Although computational models of the female reproductive system are not very new, until now, very limited studies have developed computational mechanobiology models to investigate the female reproductive system, 162 partly because of the lack of high‐quality experimental data on mechanobiology. 163 More than 50 years ago, computational models were used to reveal theoretical ovum transport mechanisms in the oviduct. 164 , 165 In 1980, Verdugo et al. 164 used a stochastic model to show that both ciliary forces and random tubal contractions are involved in ovum transport in the ampulla of the rabbit oviduct. Later in 1983, Blake et al. 165 demonstrated steady transport of the ovum, whereas muscular activity resulted in highly oscillatory motion, using a simple fluid dynamic model of ovum transport in the ampulla and isthmus of the human oviduct. A sequence of contractions in the uterine direction is required for ovum transport. 165 Eytan et al. 166 developed computational models to investigate the effects of uterine contractions on the site of embryo implantation. Their computational model of uterine fluid motion simulated by a finite, uniform 2D channel indicated that the magnitude of uterine contractions may be key in determining the success and site of implantation. 166 Then, using the same model, they showed that an increased pressure gradient within the fallopian tube in hydrosalpinx generates reflux currents that can push the embryo away from the implantation site. 167 To simulate the sagittal cross section of the uterus more accurately, those authors expanded their model to a 2D finite nonuniform channel and showed that transport phenomena were maximal when wall displacements were symmetric and the inclination angle small. 167 The intrauterine flow induced by relatively symmetric uterine contractions and the small angle between uterine walls may accelerate transport of the embryo away the implantation site. 167 Normal placental development is critical for reproductive success. 118 However, how the human placenta develops has not been clarified. 118 In vivo experiments of early human pregnancy are prohibited for ethical reasons. 118 Furthermore, there is great variation in placental types across mammals. 118 A computational model may be promising for investigating the development of the human placenta. Rejniaka et al. 168 developed a computational model of the growth mechanics of the trophoblast bilayer (inner CTBs and the outer syncytiotrophoblasts) in chorionic villi. This model revealed different viscoelastic properties of the three main components of the trophoblast tissue (basement membrane, CTBs, and syncytium), of which the syncytium layer is the most flexible and the basement membrane most rigid. 168 The model also captured the mechanics of the viscous, incompressible fluid in which the tissue is immersed. 168 Three critical cell processes for whole placental development, division, growth, and fusion, were incorporated. 168 The model showed that the stiffness of these two layers of trophoblasts affects the shape of CTBs and the whole trophoblast. 168

The field of mechanobiology focuses on how the responses of cells, tissues, and organs to mechanical cues, resulting from both intracellularly generated and externally generated forces, contribute to development, differentiation, physiology and disease via the integration of medicine, biology, engineering, and physics. 1 , 2 , 3 How living cells can sense their environment and adequately respond in terms of morphology, migration, proliferation, differentiation, and survival requires understanding at multiple scales, from molecules to single cells, tissues, and organs. 1 , 2 , 3 More than a century ago, mechanical forces were proposed to drive embryogenesis and bone structure. 1 , 2 , 3 The importance of mechanical forces for biological regulation was recognized in the field of developmental biology at the beginning of the twentieth century. 1 , 2 , 3 However, there were no experimental tools available to directly test the role of mechanical forces in biological regulation. 1 , 2 , 3 The greatest challenge in mechanobiology is quantifying how biological systems sense, transduce, respond to and apply mechanical signals. 1 , 2 , 3 With the recent advent of biophysical and molecular technologies, today mechanobiology is an emerging multidisciplinary field ranging from cell and developmental biology to bioengineering, material science, and biophysics. 1 , 2 , 3 The impact of biochemical and genetic factors in biology has been studied extensively in the 20th century. Professor Ingber from the Wyss Institute, who has made major contributions to the mechanobiology field, stated, “The time has come to recognize that all of these factors—biochemical, genetic and mechanical—are equally important for biological control, and that to truly understand living systems, we must break through our silos of knowledge and create a fully integrated explanation of how our bodies work. 3 ” The female reproductive system is a complex multi‐organ system with multiple closely regulated functional processes. It is clear that we need to understand how biochemical, genetic, and mechanical factors act together to control the physiology of the system and how perturbation of these factors results in pathological consequences, to truly understand the physiology, disorders and diseases of the female reproductive system. However, mechanobiology in the field of human female reproduction has been extremely challenging technically and ethically. More studies are needed to understand the roles of forces and changes in the mechanical properties of female reproductive system physiology, and little is known about how mechanical perturbations result in the related disorders. 4 , 5 , 6 , 7 , 8 , 9 , 10 However, given the importance of mechanobiology in human biology and development, 2 the present review aimed to provide the current knowledge on mechanobiology of the female reproductive system. I focus on the early phases of reproduction from oocyte development to early embryonic development, with an emphasis on current progress. Because of the limited knowledge in humans, I include evidence from animal studies, both in vivo and in vitro. I also provide a perspective on the critical challenges in the field of mechanobiology of the female reproductive system.

The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Any changes in the extracellular environment, cell structure, the cellular mechanosensing process itself, or the involved downstream signaling pathways can result in several disease conditions. 12 , 169 , 170 To understand how mechanical factors are involved in disorders of the female reproductive system, it is first critical to determine how they are involved in the physiology of the female reproductive system. Studies have shown that well‐controlled mechanical cues are important in the early development of the embryo in mouse models. 88 , 91 , 93 , 101 , 105 New insights gained from previous studies may have translational applications. Recent mouse experiments showed that the external geometrical constraints imposed by the uterine environment are an important determinant of early mouse embryonic development. 88 , 91 It is speculated that mechanical imbalances among the embryo, decidua, and uterine muscle contractions may adversely affect early embryogenesis in utero and thereby result in spontaneous abortion at an early stage of mammalian pregnancy. 88 , 91 Peristaltic contractions of the human uterus throughout the menstrual cycle have been investigated extensively. 61 , 66 , 67 , 68 , 69 However, to date, no clinical study has focused on the role of uterine peristaltic contractions in human embryonic development after embryo implantation. Early pregnancy loss is a major clinical problem in reproductive health. 171 Furthermore, diseases associated with disturbed mechanotransduction signaling include developmental defects, such as congenital heart diseases. 170 Although extremely challenging, further investigation into whether and how mechanical imbalances among the embryo, decidua, and uterine muscle contractions affect early human embryonic development and induce early human pregnancy loss and/or developmental defects will provide insights on early pregnancy loss and developmental defects. To address these questions, mouse models are very useful, and eventually, we may be able to address these questions in women. Today, we can investigate whether and how uterine contractions occur after embryo implantation in women by MRI‐based motion tracking, as used in one study to evaluate uterine contractions in 46 healthy pregnant women during the second trimester. 172 In that study, uterine contractions were detected on 18.6% of the MRI scans (11/59) at 14–18 weeks of gestation, increasing to 26.5% (14/53) at 19–24 weeks of gestation. 172 Previous studies have suggested that the biomechanical properties of oocytes/embryos are potentially novel biomarkers for selecting high‐quality oocytes/embryos, leading to better IVF/ICSI outcomes. 45 , 46 , 47 , 152 , 153 However, before use of these biomarkers can become routine in clinical practice, there are many technical challenges that we must overcome. 152 , 153 First, we need to validate whether the biomechanical properties of bulk embryos are appropriate biomarkers. 45 It is very likely that a stiff zona pellucida and soft cytoplasm have distinct impacts on embryonic developmental, and thus a new method may be required to measure the mechanical properties of each structure individually. 45 Consequently, no invasive automated assessment of oocyte/embryo mechanics would be required. 152 , 153 Microfluidic technology may have great potential application in such assessments, although it is still far from routine use even in basic research. 152 , 153 Second, well‐controlled prospective clinical studies are required to determine whether noninvasive, automated assessment of oocyte/embryo mechanics in terms of morphology is superior to conventional selection. 45 , 46 , 47 , 152 , 153 It is important to determine whether the assessment of mechanical properties can provide a clinical benefit for IVF patients. 152 , 153 Recent studies clearly showed high regulative flexibility of human preimplantation embryos, which may partly explain why successful live births can result from transfer of embryos of poor quality in terms of morphology. 102 , 103 It is clear that new biomarkers are required to select high‐quality oocytes/embryos 173 ; however, it remains unclear whether the biomechanical properties of oocytes/embryos are potential biomarkers. 152 , 153 To simulate mechanical stimuli (shear stress, compression, and friction force) similar to those in the fallopian tube, 49 different culture systems have been developed. 49 , 50 , 51 , 52 , 53 , 54 , 55 , 57 , 58 , 59 , 60 In the early 2000s, studies showed that excess shear forces were harmful to mouse embryonic developments. 49 , 53 , 54 , 55 Subsequently, several groups showed that transient mechanical stimuli may benefit embryonic development. 57 , 58 , 59 Hydrosalpinges have a deleterious effect on embryonic development, and hydrosalpinx induces an increased pressure gradient within the fallopian tube. 174 Higher and continuous shear stress in hydrosalpinx may partly explain the deleterious effect of hydrosalpinges on embryonic development. 49 , 53 , 54 , 55 , 60 , 174 However, to date, there is no robust evidence that integration of physiological, mechanical stimuli mimicking those of the Fallopian tube has any clinical benefit on human oocytes/embryos during IVF procedures. In this review, I focused on the early phases of reproduction from oocyte development to early embryonic development. However, perturbations in mechanical cues could be involved in many disorders/diseases of the female reproductive tract, such as preterm birth, cervical incompetence, intrauterine growth restriction, endometriosis, adenomyosis, uterine fibroma, and genital prolapse. 5 , 6 , 7 , 9 , 10 , 169 , 170 Efforts to gain new insights into the mechanobiology of the female reproductive system will help us broaden our understanding of, and develop new strategies for, the treatment and/or prevention of these clinically important disorders/diseases in women (Figure  2 ). 5 , 6 , 7 , 9 , 10 , 168 , 169 Potential involvement of perturbations in mechanical cues in disorders/diseases of the female reproductive system. Little is known about how mechanical perturbations result in the related disorders/diseases of the female reproductive system. Efforts to gain new insights into the mechanobiology of the female reproductive system will help us broaden our understanding of, and develop new strategies for, the treatment and/or prevention of these clinically important disorders/diseases in women [Colour figure can be viewed at wileyonlinelibrary.com ] Immune cells migrate within varying environments via the blood and lymphatic circulation to perform immunosurveillance and effector functions. 175 Therefore, it is no surprise that immune cells experience a wide range of mechanical forces, from piconewtons at the nanoscale level to several orders of magnitude higher at the tissue level. 175 , 176 , 177 , 178 , 179 Thanks to recent technical advances in imaging modalities and biophysical tools, growing evidence indicates critical roles of mechanical forces in immune cell functions. 175 , 176 , 177 , 178 , 179 Endogenous mechanical forces from the ECM or neighboring cells, as well as exogenous mechanical forces, act at the cellular level in immune cells, similar to other cells described above. 175 , 176 , 177 , 178 , 179 Furthermore, immune receptors themselves respond to mechanical stimuli during antigen recognition, and thus lymphocyte activation is a mechanosensitive process. 175 , 176 , 177 , 178 , 179 The immune system drives a range of reproductive processes and normal physiological interactions between the female reproductive and immune systems, which are critical in female reproductive health. 180 , 181 , 182 , 183 The field of mechanoimmunology is still in its infancy. 175 , 176 , 177 , 178 , 179 Furthermore, reproductive immunology has been incredibly difficult to understand for both clinicians and scientists. 180 , 181 , 182 , 183 However, because of the important roles of the immune system and mechanical cues in the female reproductive system, the field of “reproductive” mechanoimmunology will contribute to the future directions of basic research and clinical applications. This will be an immense challenge. At the very least, as Jain et al. clearly stated “We should keep in mind that any experimental data, without carefully consideration of the importance of physical forces and factors, may lead to the wrong assumptions 176 . The most essential and fundamental questions to be addressed first are whether mechanical properties differ between the two major compartments of the ovary (cortex and medulla) in normally ovulating human ovaries, and whether the difference is involved in dormancy and activation of primordial follicles, followed by folliculogenesis. Without this knowledge, we cannot understand the true mechanisms of human folliculogenesis and related disorders such as PCOS and primary ovarian insufficiency. In such disorders, the mechanical properties of the different compartments may differ from those of normally ovulating ovaries. 8 , 24 Because of ethical limitations in human investigations, and because the laboratory mouse is not entirely suitable for investigating the mechanobiology of human ovaries, 28 large animal models, particularly cows, horses, and sheep, which have been validated for evaluation of ovarian function in humans, will be appropriate for addressing these questions. 29 Colloidal probe atomic force microscopy will be useful to map and quantify the mechanical microenvironment across different compartments as well as that surrounding follicles at different stages, in large animal models, as a very recent study demonstrated in mouse ovaries. 27 A new multipole magnetic device was recently developed for 3D mapping of the absolute stiffness and viscosity of tissue in a nontoxic manner. 146 This device will also be useful, although some modifications may be required, for quantification of the spatial stiffness distribution in the ovaries of large animals. The mechanisms underlying follicle rupture during ovulation remain to be clarified. Human preovulatory follicles grow to approximately 20 mm, approximately 600 times larger than the initial activated primordial follicle. 184 Thus, it is very likely that volumetric expansion of pre‐ovulatory follicles is a potential mechanism of follicle rupture before ovulation. A recent mathematical model of the blastocyst as a thin‐walled pressure vessel demonstrated that volumetric expansion, rather than hydraulic pressure generated inside blastocysts, induces zona pellucida rupture and embryo hatching. 48 The model showed that embryos do not spontaneously hatch after reaching the peak pressure but rather require a further decrease in zona pellucida thickness to break, similar to a balloon bursting after being inflated beyond the critical pressure. 48 Very interestingly, more than 50 years ago, studies showed no significant increase in intrafollicular pressure before ovulation, and artificially increasing intrafollicular pressure by fluid or oil injection did not induce follicle rupture in rabbits 34 , 35 and pigs. 36 This mathematical model of a thin‐walled pressure vessel 48 will be useful for investigating the roles of intrafollicular pressure and follicular wall thickness in follicle rupture during ovulation. Furthermore, this mathematical model 48 will be used to investigate the impact of mechanical forces from fluid‐filled growing follicles on folliculogenesis. Given the important roles of the hydraulic pressures inside blastocysts as well as volumetric expansion in the embryonic development, 98 , 100 cyclically well‐controlled mechanical forces, such as stretching and compression, from fluid‐filled growing follicles and a sudden decrease in mechanical forces after ovulation may play important roles in folliculogenesis in normally ovulating ovaries. Further studies are required to investigate whether and how such cyclical changes in follicular hydraulic pressure and/or volumetric expansion affect the surrounding cells, including somatic and germinal cells. Furthermore, using a similar mathematical model, 48 we may also investigate the impacts of mechanical forces induced by hydraulic intracystic pressure and volumetric expansion in endometriomas on ovarian function. Persistent stretching of adjacent ovarian tissue by endometriomas has been hypothesized to diminish the ovarian reserve. 185 However, no studies have investigated the mechanical forces in surrounding normal ovarian tissues/cells generated by endometrioma, the biochemical signaling pathways induced by mechanical forces, or the effects of mechanical, and biochemical cues generated by endometriomas on normal ovarian function. 3D models of in vitro ovarian folliculogenesis in large non‐rodent animals will be very powerful tools to investigate ovarian mechanobiology. 186 However, there are many challenges to overcome before establishing such models. 186 The duration of folliculogenesis is much longer and the size of follicles much larger than those of mice, complicating the creation of 3D models. 186 To generate optimal microenvironments that can recapitulate in vivo folliculogenesis, precise spatial mapping of biochemical and mechanical properties in whole ovaries in vivo is required. 187 Next, we need to determine which signals are required to maintain dormancy versus activate primordial follicles. 187 New culture methods as well as 3D scaffolds will be required to establish long‐term culture of 3D ovarian folliculogenesis models in large non‐rodent animals. 187 The recent development of 3D printed bioprosthetic ovaries in mice showed 3D bio‐printed scaffolds as promising tools for the development of 3D ovarian folliculogenesis models. 188 Many cell types have been observed to migrate toward stiffer regions of mechanical gradients in a process termed durotaxis. 189 , 190 , 191 Durotaxis is the tendency of most cells to move toward stiffer substrates when migrating on a compliant gradient; this cellular behavior is based on the mechanical, rather than biochemical, properties of the microenvironment. 189 , 190 , 191 Cells spread and migrate more easily on stiff substrates than on compliant substrates. 189 , 190 , 191 Because many migratory cells express MMPs and ADAMs, which degrade and soften the ECM, the cells can degrade the ECM locally, thereby generating a stiffness gradient along the cells track. 191 Local softening can also be achieved independently of MMPs. 192 , 193 Durotaxis has been observed in various cell types in vitro and has been implicated in embryonic development, wound healing and fibrosis, and cancer metastasis. 147 , 189 , 190 , 191 , 194 , 195 However, whether durotaxis occurs in vivo remains elusive, and addressing this will require the development of new tools. 190 First, we need appropriate tools to map tissue stiffness in bulk tissues three‐dimensionally. 190 For this purpose, a multipole magnetic device was recently developed to map the absolute stiffness and viscosity of a living mouse embryo three‐dimensionally. 146 Using this method, correlations between stiffness gradients and mesodermal cell migration patterns were found, raising the possibility that durotaxis guides cell movement in vivo. 146 However, other tools that precisely modify stiffness in vivo are required to functionally probe the role of stiffness gradients. 146 , 190 Because all directional migration cannot be explained solely by chemotaxis in living tissues, and mechanical cues play an essential role in biological control, it is very likely that durotaxis occurs in vivo. 190 In the female reproductive system, durotaxis may potentially be involved in several processes in addition to embryonic development. The mechanisms underlying folliculogenesis remain to be clarified. 196 However, it has been suggested that after activation of primordial follicles, growing follicles migrate from the stiff cortex to soft medulla. 24 However, if the medulla is stiffer than the cortex in human ovaries, as recent studies have shown in bovine 30 and mouse ovaries, 27 durotaxis might be involved in human folliculogenesis. The ovarian wound healing process after ovulation is not clear. However, during skin wound healing, MMP‐1 expression (MMP‐1 breaks down interstitial collagen types I, II, and III) increases rapidly and peaks in migrating basal keratinocytes at the wound edge on day, 1 followed by a gradual decrease. 197 Furthermore, fragmentation of the ovarian cortex facilitates the conversion of G‐actin into F‐actin, leading to disruption of the Hippo signaling pathway to allow secondary follicle growth. 198 , 199 , 200 These findings raise the hypothesis that in normally ovulating ovaries, ovarian tissue injuries from ovulation induce ECM degradation and matrix softening, which in turn trigger activation of dormant primordial follicles. Growing follicles may migrate into the medulla, which is stiffer than the cortex, via durotaxis. In PCOS ovaries, durotaxis may be impaired due to hardening of the cortex and/or loss of the stiffness gradient within the ovarian components, resulting in follicular arrest. Surgical treatment for PCOS may induce MMP‐1, which degrades the ECM and induces matrix softening, resulting in a softer cortex before surgery. Consequently, follicles can migrate and grow in the medulla via durotaxis. Durotaxis may also be involved in initial embryo implantation. During initial embryo implantation, embryos invade the underlying stromal cells. 86 In mice, Lox, which enhances ECM stiffness, is first detected in the subluminal stroma surrounding the implanted blastocyst on day‐5 of pregnancy. 83 Then, from days 6 to 8, Lox expression is strongly detected in the decidual cells surrounding the embryos. 83 Blastocyst invasion and stromal cell migration are impeded by β‐aminopropionitrile, an inhibitor of Lox activity. 83 Spatiotemporal changes in endometrial ECM stiffness may induce blastocyst invasion and stromal cell migration via durotaxis in mice. Further experiments will be required to confirm this in mouse models. An in vitro 2D co‐culture model of human embryos with decidualized primary human ESCs showed that the motility of the stromal cells increases at implantation sites. 86 The migration of human ESCs away from the implantation site may facilitate trophoblast invasion and implantation of the embryo into the stromal compartment. 86 This increased motility of stromal cells at implantation sites was correlated with a localized increase in Rac1 activation and a reciprocal decrease in the level of RacGAP1, which inactivates Rac1. 86 cdGAP, an adhesion‐localized Rac1 and Cdc42‐specific GTPase‐activating protein, is required for durotaxis in U2OS osteosarcoma cells. 201 The stiffness of human ESCs was decreased during in vitro decidualization. 78 The stiffness of patient‐derived tumor cells and cancer cell lines is inversely correlated with migration and invasion through 3D basement membranes. 87 These findings raise the questions of whether durotaxis is involved in stromal cell migration and embryo invasion, and whether the decreased stiffness of decidualized ESCs facilitates stromal cell migration, during initial human embryo implantation. Durotaxis may also play a role in EVT migration. EVTs migrate from softer endometrium to stiffer myometrium. 202 In normal pregnancies, the interstitial EVTs stop migrating once they reach the inner third of the myometrium. 202 It was shown that durotaxis requires individual focal adhesions to sense local ECM stiffness to guide durotaxis, and that FAK/phosphopaxillin/vinculin signaling defines the rigidity range over which this dynamic sensing process operates. 203 , 204 FAK is a key kinase involved in early trophoblast cell differentiation, and it plays a role in regulating cell proliferation and motility during early placental development. 205 Activated FAK is localized in proliferating EVTs between 4 and 8 weeks of human gestation and is decreased markedly after 10 to 12 weeks of gestation. 205 Furthermore, the strongest durotactic migratory response was observed on the softest regions of a stiffness gradient (2–7 kPa), with decreased responsiveness on the stiffer regions of the gradients, in all cancer cell lines tested (U87‐MG, T98G, MDA‐MB‐231, and HT1080), as well as in noninvasive human fibroblasts (BJ‐5ta normal fibroblasts). 189 EVTs may not migrate beyond the inner third of the myometrium due to a stiff endometrium along with a marked decrease in the level of activated FAK. In vitro experiments to investigate whether durotaxis is involved in the female reproductive system, such as in migration of growing ovarian follicles, embryo implantation, and/or EVT migration, will be required to test these speculations. Huse clearly stated, “We can only study what we can measure, and as the field of mechanobiology moves forwards, it will be advisable to keep this obvious dictum in mind (Figure  1 ). 175 Progress in mechanobiology is dependent on development of technologies that enable precise physical measurements in living cells”. 135 To fully understand the mechanobiology of the female reproductive system in humans, in which the dynamic, complex architectural multiscale organization of tissues is elaborate, noninvasive techniques that provide precise high‐resolution mechanical properties at multiple scales, from molecules, to single cells, tissues, and organs, are essential and indispensable. 136 Today, microfluidic technologies are still far from routine biological experiments, even in basic research, partly because of the technical limitations of most biological laboratories. 149 , 150 , 151 , 152 , 153 However, in the future, microfluidic technologies will be an indispensable tool for investigating the cellular microenvironment in development and disease for the following reasons. The cellular microenvironment is defined by physical and chemical signals that can influence cellular behavior, either directly or indirectly. 3 Recent advances in microfluidic technologies may enable precise control of the mechanical and chemical microenvironments of cultured cells. 149 , 150 Microfluidics can be used to apply both solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal and interstitial) to cells. 149 , 150 Furthermore, advances in fabrication methods will enable integration of solid and fluid forces within a single device to recapitulate the tissue microenvironment more accurately. 149 , 150 Both mechanical and biochemical signals play important roles in development and disease. 3 Furthermore, there are feedback loops between mechanical and biochemical signals, termed mechanochemical feedback loops, and these bidirectional interactions play key roles in morphogenesis and pattern formation in both development and disease. 22 Furthermore, given the importance of cell‐ECM interactions, a major technological challenge is to develop in vitro ECM tools that mimic both normal and pathological ECM in the female, reproductive system in vivo. 187 , 206 , 207 Previous studies showed that synthetic hydrogels are a useful tool to investigate the effects of ECM stiffness on cell function. 155 , 156 , 157 However, naïve ECMs are viscoelastic and thus exhibit stress relaxation. 208 , 209 , 210 , 211 Both stiffness and substrate stress relaxation are fundamental physical properties that can affect cell behavior and function. 208 , 209 , 210 , 211 Furthermore, substrate topography and stiffness interact to regulate the mechanical properties of cells. 212 , 213 A significant interaction between substrate stiffness and topography was found in the elongation and orientation of cardiac fibroblasts and in the viscoelastic behavior and smooth muscle gene expression of mesenchymal stem cells. 212 Recently, a microfabrication method was developed to independently control the stiffness (3–145 kPa) and topographical structures (5–10 μm in size) of polyacrylamide hydrogels. 213 The design of well‐defined micro‐ and nanostructural characteristics of ECM topography, including the architecture, geometry, size, and organization of the ECM network, is required to simulate the native ECM. 214 Decellularization techniques support the creation of biocompatible ECM hydrogels, providing tissue‐specific environments for both in vitro cell culture and in vivo tissue regeneration. 215 , 216 However, a major limitation of mechanobiology experiments is that the mechanical strength of decellularized ECM hydrogels is lower than that of native tissue. 215 , 216 Decellularized ECM hydrogels lose essential mechanical and structural stability during decellularization and digestion processes. 215 , 216 Tuning the mechanical properties of decellularized ECM hydrogels to mimic native tissue in vivo without compromising biocompatibility is required. 215 , 216 Genetic engineering and hybrid scaffolds offer potential solutions. 217 A recent study showed that using MDA‐MB‐231 cells (human invasive breast cancer cell line) with varying LOX expression levels by genetic engineering, decellularized ECM scaffolds with different stiffness levels can be generated. 217 Another study developed tunable printable hydrogels composed of decellularized human heart ECM using either methacrylated gelatin (GelMA) or GelMA‐methacrylated hyaluronic acid (MeHA) hydrogels, which were dual crosslinked using UV and microbial transglutaminase, to create cardiac tissue‐like constructs. 218 By altering the concentrations of GelMA and MeHA individually and mixing them at different ratios, the Young's modulus values can be adjusted over a wide range. 218 Recent developments in human organoid technology including endometrial epithelial cell organoids 114 , 115 and trophoblast organoids 112 , 113 have provided a deeper understanding of human biology and diseases. However, despite tremendous advances in organoid technology, there are still many limitations in traditional organoid systems. 219 , 220 , 221 A major limitation is that they lack key specialized cell types (vasculature, stromal, and immune cells) and fail to recapitulate the complexity of native organs. 219 , 220 , 221 To understand the underlying mechanisms of female reproductive system physiology and related disorders such as embryo implantation and early embryonic development, molecular and functional interactions of embryos or trophoblasts with decidua including epithelial, stromal, and immune cells should be investigated. 212 , 223 , 224 , 225 Complex organotypic models involving cancer organoids and endometrial organoids co‐cultured with stromal and/or immune cellular components have been developed. 222 , 226 Interestingly, a recent study developed an air‐liquid interface (ALI) method to establish patient‐derived organoids (PDOs) preserving diverse endogenous immune cells and other non‐epithelial cell types. 227 The cancer tissues were physically (not enzymatically) minced into fragments, and ALI PDOs cultures were established from 100 individual patient tumors representing 19 distinct tissue sites and 28 unique disease subtypes. 227 However, it remains unclear whether the ALI method can also be applied to female reproductive tissues. The organoid‐on‐a‐chip model, which synergistically combines the best features of organ‐on‐a‐chip and organoid models, can help address the major technical challenges in organoid research, including microenvironmental control of organoids (biophysical and biochemical microenvironments, nutrient supply), more controllable and more conducive environments for co‐culture of different cell, tissue and organ types in organoid systems, and minimizing variability. 228 Organoid‐on‐a‐chip systems will provide a promising platform to investigate the physiology and related disorders of the human female reproductive system. 228 Computational models have become a standard tool for testing hypotheses in biological settings. 163 , 229 , 230 , 231 They cannot prove whether a proposed mechanism is true for an observed phenomenon, but they can demonstrate whether the mechanism is sufficient to produce the observed results. 163 , 229 , 230 , 231 Because computational models allow testing of numerous hypotheses via simulated experiments and identify the most probable/correct hypotheses to prove via laboratory experiments, these models are useful before conducting laboratory experiments, especially when investigating complex relationships among biochemical and mechanical pathways at multiple levels. 163 , 229 , 230 , 231 Thus, a computational model is a promising tool to identify the most probable/correct hypotheses in the field of mechanobiology. 163 , 229 , 230 , 231 However, to prove a hypothesis, high‐quality experimental data are indispensable. 163 , 229 , 230 , 231 Furthermore, to generalize the findings to the clinic, “the verification, analytical validation and clinical validation” process is essential. 232 However, in the study of the human female reproductive system, even the first step (verification) is challenging due to a lack of high‐quality multiscale experimental data. Nevertheless, in the future, multiscale computational models that can recapitulate multiple complex feedback loops between mechanical and biochemical signals will be a powerful tool to help enable true understanding of the physiology and related disorders of the female reproductive system. A close collaboration with computational biologists will be required. 163 , 229 , 230 , 231