Shear wave elastography to assess stiffness of the human ovary and other reproductive tissues across the reproductive lifespan in health and disease

Abstract

The ovary is one of the first organs to show overt signs of aging in the human body, and ovarian aging is associated with a loss of gamete quality and quantity. The age-dependent decline in ovarian function contributes to infertility and an altered endocrine milieu, which has ramifications for overall health. The aging ovarian microenvironment becomes fibro-inflammatory and stiff with age, and this has implications for ovarian physiology and pathology, including follicle growth, gamete quality, ovulation dynamics, and ovarian cancer. Thus, developing a non-invasive tool to measure and monitor the stiffness of the human ovary would represent a major advance for female reproductive health and longevity. Shear wave elastography is a quantitative ultrasound imaging method for evaluation of soft tissue stiffness. Shear wave elastography has been used clinically in assessment of liver fibrosis and characterization of tendinopathies and various neoplasms in thyroid, breast, prostate, and lymph nodes as a non-invasive diagnostic and prognostic tool. In this study, we review the underlying principles of shear wave elastography and its current clinical uses outside the reproductive tract as well as its successful application of shear wave elastography to reproductive tissues, including the uterus and cervix. We also describe an emerging use of this technology in evaluation of human ovarian stiffness via transvaginal ultrasound. Establishing ovarian stiffness as a clinical biomarker of ovarian aging may have implications for predicting the ovarian reserve and outcomes of Assisted Reproductive Technologies as well as for the assessment of the efficacy of emerging therapeutics to extend reproductive longevity. This parameter may also have broad relevance in other conditions where ovarian stiffness and fibrosis may be implicated, such as polycystic ovarian syndrome, late off target effects of chemotherapy and radiation, premature ovarian insufficiency, conditions of differences of sexual development, and ovarian cancer.

Summary sentence: 

Shear Wave Elastography is a non-invasive technique to study human tissue stiffness, and here we review its clinical applications and implications for reproductive health and disease.

Introduction

The female reproductive system is one of the first organ systems to age in the human body with ovarian function decreasing in females beginning in their mid-thirties and ceasing completely at the time of menopause [1–3]. Women are born with a finite pool of oocytes, which are organized into follicles [4]. The majority of these follicles remain quiescent as primordial follicles, forming the ovarian reserve [4]. As women age, the ovarian reserve diminishes in both quantity and quality, leading to diminished fertility. Female reproductive aging is also associated with reduced ovarian hormone production, which can have a wide range of clinical consequences for overall health in women [1–3]. As more women delay childbearing, it is prudent to identify modalities to non-invasively assess ovarian age and, hence, reproductive timeline [5–7]. Although current markers of ovarian age, including anti-Müllerian hormone levels and antral follicle counts early in the follicular phase, help predict oocyte quantity and response to hormonal stimulation in Assisted Reproductive Technologies (ART), these markers do not reflect oocyte quality, chance of conception, or overall ovarian health [8–10]. Development of a non-invasive tool that more comprehensively predicts oocyte quality, fertility, and reproductive longevity has potential to empower patients with information about their reproductive health, overall fertility, and risk for pathologies such as ovarian cancer.

The ovarian microenvironment in mammals undergoes dramatic age-related changes, becoming inflammatory, fibrotic, and stiff [11–23]. With age, mouse ovaries exhibit increased expression of pro-inflammatory molecules as well as the presence of a unique immune cell population associated with chronic inflammation [13, 15, 16, 19]. Moreover, ovaries from reproductively old mice contain high levels of collagen I and III, are enriched in fibro-inflammatory proteins, and have reduced hyaluronan content [11, 13, 24, 25] Many of these phenotypes are conserved in the human ovary as well [26, 27]. This age-dependent increase in ovarian fibrosis is directly associated with increased ovarian stiffness [24]. Stiffness is a biomechanical property measured as the ability of a material to resist deformation in response to a force [28].

Ovarian stiffness has important implications for ovarian function as well as disease. First, the physical environment of the ovary regulates primordial follicle activation. Primordial follicles require a stiffer microenvironment to remain quiescent and a softer, more permissive one to activate and grow [29–38]. Thus, a stiffer microenvironment, due to increased fibrosis, may alter the dynamics of follicle activation and development. Interestingly, growing isolated ovarian follicles in rigid alginate hydrogels that mimic the stiffness of an aging ovary results in decreased follicle growth, estradiol production, and gametes that lack developmental competence relative to follicles grown in softer alginate hydrogels [29–31, 39–42]. Second, ovaries from reproductively old mice exhibit impaired ovulation and subsequent defects in wound healing, which is at least in part due to fibrosis because short-term treatment with antifibrotic agents can restore ovulation [11, 43]. Finally, the age-associated increase in ovarian stiffness may serve as a permissive niche to support ovarian cancer pathogenesis. Ovarian cancer is most commonly diagnosed in postmenopausal women, and ovarian cancer cells prefer to adhere to a collagen-rich and stiffer matrix similar to what occurs in the aging ovary [44–47]. Solid ovarian tumors are known to exhibit increased stiffness compared with non-diseased ovarian tissue [48, 49]. In addition, the stiffer ovarian stroma underlying ovarian cancer is thought to promote tumor progression, metastasis, and chemoresistance [12, 44, 50–53]. There is also evidence that ovaries from women with Brca mutations are associated with increased cancer risk, early menopause, and accelerated ovarian fibrosis compared with ovaries from non-carriers, underscoring the pathologic role of fibrosis in both ovarian aging and cancer risk [12].

Methodology to non-invasively quantify ovarian stiffness could provide a novel clinical biomarker for the ovarian reserve, follicle dynamics, gamete quality, and ovarian cancer risk which would have impacts for both clinicians and patients. In this review, we provide an overview of the non-invasive quantitative imaging technique shear wave elastography (SWE) and its potential applications for exploration of reproductive health and disease.

SWE is a safe and effective tool for non-invasive assessment of tissue stiffness

Stiffness of human organs and soft tissues is dynamic and can change drastically both with physiologic aging and pathologic conditions such as cancer and chronic liver disease (CLD) (Table 1). Thus, the ability to non-invasively assess tissue stiffness represents a significant advance with direct clinical applications for assessing organ function, health, and disease progression. Several techniques have been developed to estimate tissue stiffness using non-invasive imaging modalities, including strain elastography, SWE, and magnetic resonance elastography (MRE) [73, 106–108]. Strain elastography approximates tissue stiffness based on the speed at which ultrasound waves return to the probe after the tissue of interest is manually deformed by the ultrasound probe, but this measurement is only semi-quantitative and can vary widely between operators [74, 106–108]. In contrast, SWE is an ultrasound imaging tool to quantify soft tissue stiffness with less operator dependence [106]. It uses a push pulse, often referred to as acoustic radiation force, from the ultrasound probe to generate shear waves in target soft tissues [54, 106, 107]. Shear waves propagate perpendicular to the direction of the push pulse and travel faster through stiffer tissue (Figure 1A) [54, 73, 106]. Ultrafast imaging from the same ultrasound probe tracks shear wave propagation, which directly correlates with tissue stiffness [54, 106, 107]. In other words, shear wave speed can be converted into the Young modulus in kPa, a measure of tissue elasticity, from which a color-coded 2-dimensional elasticity image is created and overlaid on the grayscale ultrasound image in the region of interest (ROI) to spatially map out local variation in tissue stiffness (Figure 1A) [106]. The Young modulus can be estimated by a simple mathematical equation: E = 3ρc² in the isotropic media, where E is Young modulus in units of pressure, normally kilopascals; ρ is the material density; and c is the shear wave velocity [73, 108]. Shear wave speed is directly measured by the ultrasound probe, and tissue density is estimated based on the tissue of interest or is assumed to be 1 g/mL for soft tissues [73]. For the ovary in particular, ovarian density has been estimated at ~1 g/mL [109]. MRE has recently been used in quantifying liver stiffness, in which shear waves are generated in the soft tissues using a low-frequency (50–60 Hz) external vibration source that is secured onto the abdominal wall over the liver [110]. In the past two decades, SWE has been extensively studied in liver fibrosis and in thyroid and breast neoplasms. More recently, the applications of SWE have been extended to assess prostate gland, lymph nodes, and musculoskeletal conditions (Figure 1B) [54–57, 62, 63, 65, 66, 73–76, 111, 112].

Table 1.

Current clinical and research applications of SWE (non-reproductive)

Condition	Justification	Outcome	Citations
Chronic liver disease (CLD)	CLD results in increased collagen deposition and fibrosis, which is clinically relevant for staging and monitoring disease progression.	Increased stiffness predicts fibrosis in CLD and can be used for disease monitoring and staging.	[54–61]
Thyroid cancer	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[62]
Prostate cancer	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[63, 64]
Breast cancer	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[65–69]
Lymph node tumors	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[70–72]
Tendinopathies	Diseased tendons weaken and have disordered ECM prior to rupture.	Decreased stiffness predicts tendinopathy and can be used to predict risk of rupture and track healing after tendon repair.	[73–76]
Uterine fibroids	Fibroids are stiff and have a high collagen content.	Fibroids are stiffer than non-diseased myometrium, which can be used to track growth of fibroids and identify new lesions before they become symptomatic. Decreases in stiffness following treatment (ex. uterine artery embolization) may be useful for monitoring treatment efficacy.	[77–85]
Uterine adenomyosis	Adenomyosis lesions contain a higher content of ECM components.	Adenomyosis lesions are stiffer than non-diseased myometrium, which can be used to track growth of lesions and identify new lesions before they become symptomatic. Decreases in stiffness following treatment (e.g. treatment with GnRHa) may be useful for monitoring treatment efficacy.	[77, 80–83, 86–88]
Endometrial cancer	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[89–93]
Cervical cancer	Malignant tissue is stiffer than healthy tissue.	Stiffer lesions are more likely to be malignant.	[94–101]
Preterm labor risk	Remodeling and softening of the cervix is essential for labor and may occur early in cases of preterm labor.	Decreased stiffness of the internal cervical os in the second trimester predicts risk of preterm labor.	[102–105]

Figure 1.

SWE is a quantitative technique that has been used broadly to study tissue stiffness. (A) Softer tissues have slower shear wave propagation and lower shear wave velocity while stiffer tissues have faster propagation and higher shear wave velocity. Shear wave velocity measurements can be used to estimate the Young modulus of the ROI. (B) SWE has been used clinically in a variety of non-reproductive tissues. Preliminary studies have been completed in several reproductive tissues and pathologies.

One of the most widely adopted clinical uses of SWE is in the context of liver stiffness change associated with underlying CLD [54, 55, 57–59, 112]. Prior to use of elastography technologies, the only option for assessing liver fibrosis was biopsy and histologic evaluation [57, 59, 112]. Transabdominal SWE is a non-invasive, low cost, and low risk tool used to closely monitor disease onset and progression, becoming an important complementary tool for detection, staging and management of CLD caused by viral hepatitis, alcohol-associated fatty liver disease, nonalcoholic fatty liver disease, and autoimmune liver disease (Table 1) [54, 55, 57, 59, 60]. Studies show that diseased liver has measurable increases in shear wave velocity and hence stiffness even at early fibrotic stages [54]. The stiffness increases further with late-stage fibrotic disease and cirrhosis compared with early-stage and non-diseased tissue [54]. Stiffness change measured by SWE over time in patients with liver disease is valuable at stratifying the risk of mortality and may be useful for identifying patients that need more comprehensive care [61]. In addition to measuring general stiffness globally in the liver, SWE technologies are being piloted to assess focal lesions in the liver and portal hypertension by measuring increases in spleen stiffness [54, 112].

SWE has also been used in the oncology setting in the thyroid, breast, prostate, and lymph nodes to differentiate tumors, especially malignant solid tumors, from adjacent normal tissues based on the assumption that malignancies have a higher cell density than surrounding normal tissues and/or fibrous collagen accumulation and crosslinking resulting in increased local fibrosis (Figure 1B; Table 1) [62, 63, 66, 70, 71, 112]. Although the definitive diagnosis for cancerous lesions in each of these conditions relies on biopsy, SWE has proven to be an effective predictive tool for determining whether a concerning finding on a physical exam is likely to be malignant [112]. For example, the Tsukuba score, a metric derived from measurements of stiffness in breast lesions that are correlated to risk of malignancy, was shown to have a sensitivity and specificity for malignancy of greater than 80% in several clinical trials [67–69, 112]. In suspected malignancies of the thyroid, prostate, and lymph nodes there was similarly a sensitivity and specificity of greater than 80% for diagnosing malignancy [64, 72, 113]. In each of these evaluations, measurements were taken by placing the ultrasound probe directly on the soft tissue overlying the ROI and making measurements at several locations within and outside of the target lesion [112].

There is emerging evidence for the utility of SWE in assessment of the uterus, endometrium, and cervix (Table 1). SWE has had clinical utility in the context of diagnosis of uterine pathologies, including fibroids, adenomyosis, and cancer which all have increased shear wave velocity and stiffness compared with non-diseased tissue [77–83, 86, 87, 89, 94]. As such, SWE may be a valuable tool to quantify regional differences in tissue stiffness for diagnosis, staging, and monitoring of these uterine pathologies. SWE has also been applied to endometrial and cervical cancer, where cancerous lesions in both pathologies exhibit increased stiffness relative to healthy tissue [84, 89–93, 95–101]. In these studies, SWE was able to identify malignant lesions and measure invasion of endometrial cancer into the myometrium and cervical cancer into the uterus, and vaginal fornix [93, 99, 100]. Beyond diagnosis, SWE has also been briefly studied in the evaluation of potential therapeutics that target uterine lesions. Gonadotropin hormone-releasing hormone agonists (GnRHa) are used to treat adenomyosis and may improve fertility outcomes. One prospective study using SWE found that stiffness of adenomyosis lesions significantly decreased after 6 months of GnRHa treatment [88]. In the case of fibroids, uterine artery embolization is often used as a treatment alternative to hysterectomy and myomectomy to provide symptomatic relief to patients [85]. One study of SWE in patients before and 1.5 months after uterine artery embolization found that fibroids were significantly less stiff after treatment [114].

SWE has also been used in assessment of the risk of preterm labor in pregnant patients. The cervix undergoes biomechanical changes throughout pregnancy, culminating in a localized softening of the cervix at the time of parturition. Women at risk of preterm labor may have altered cervical stiffness at an earlier point in pregnancy, so cervical stiffness measured by non-invasive SWE may serve as a useful biomarker for assessing risk of preterm labor [102]. A meta-analysis that included 7 studies and 1488 pregnant women found that SWE had a sensitivity and specificity of 84 and 82%, respectively, at predicting preterm labor risk, and showed increased predictive power compared with cervical length measurements [103]. In these studies, the stiffness of the internal and external os of the cervix was measured during the second and third trimesters [103–105]. Overall, these studies demonstrate that SWE is a safe tool with the potential to characterize the physiology and pathophysiology of reproductive tissues and directly inform reproductive health interventions. However more research is needed to assess its effectiveness, accuracy, and reliability in these contexts.

SWE is a clinically relevant tool for assessment of ovarian stiffness

Because SWE is an important tool for non-invasively assessing tissue stiffness, it may serve as an effective tool for assessing fibrosis in the ovary with advanced reproductive age. For this technology to be effective, it is essential that measurements can be made in a range of ovarian compartments given the heterogeneous nature of ovarian tissue which consists of stroma, vasculature, and follicles including large fluid-filled antral stage follicles. To assess feasibility of the regional quantification of stiffness in ovarian tissue using SWE, a bovine model was used to establish proof-of-concept measurements [115]. This preclinical study with SWE using bovine ovaries ex vivo demonstrated a quantifiable difference in tissue stiffness between anatomical regions [115]. In this bovine model, the inner medullary region of the ovary was quantifiably stiffer than the outer cortical region [115]. Notably, these findings differ from the prevailing paradigm that the ovarian cortex is stiffer than the medulla. This paradigm was initially defined by histologic studies that use collagen density as a surrogate for tissue stiffness [29, 34, 116, 117]. In addition, ovarian collagen density directly correlates to increased tissue stiffness [24]. Increased cortical stiffness has also been demonstrated in an ex vivo bovine model [118]. However, an additional study of regional tissue stiffness using a mouse model, where the ovary is not distinctly compartmentalized into the cortex and medulla, found that the exterior region of the ovary was softer than the interior, as measured by atomic force microscopy [119]. These contradictory findings may represent differences between ex vivo measurements and in vivo physiology or different criteria that each of these studies use to distinguish between ovarian compartments, differences across animal models, or differences between cycle stages. More studies are needed to clarify and define the regional stiffness of ovarian compartments, particularly as it relates to defined differences in their stromal composition. Ultimately, this proof-of-concept study demonstrated that it is feasible to use SWE to quantify ovarian stiffness and distinguish between regional differences in stiffness in the ovary despite tissue heterogeneity [115].

SWE can be used to measure ovarian changes associated with reproductive aging

Feasibility and considerations for SWE measurements of the ovary

Given the potential broad relevance of this technology to the study of ovarian function and disease, we have developed a preliminary general protocol to perform SWE on human ovaries. In brief, transvaginal ultrasound images of the uterus, ovaries, and adnexa were obtained per routine clinical protocol with a GE Logic Fortis HDU ultrasound device (SN LFO301952, GE HealthCare). SWE was performed on the ovaries using a 2D transvaginal probe (IC5-9-D; SN 1278929WX3) at 7-9 MHz frequency to acquire necessary images. The least possible compression was applied to the intravaginal region most adjacent to the ovary, placing the ovary within 2 cm of depth from the transvaginal ultrasound probe (Figure 2A). Individuals were asked to stop breathing for a few seconds to minimize any motion artifacts. The target area including the ovary was determined through the identification of ovarian landmarks, and the SWE function was activated. Twelve SWE frames were acquired per ovary, and the SWE confidence map was utilized to define the areas with highest confidence for stiffness assessment (Figure 2A—three example frames are shown). Regions of high confidence are determined by several factors that include but are not limited to a normal shear-wave propagation through the region, a high amplitude of the shear waves, and distance from acoustic radiation force impulse pulse and its impact on the displacement curves [120]. The ultrasound scanner we used integrates these factors and provides a confidence map to improve the accuracy of the stiffness measurements (Figure 2A—three example frames are shown). This confidence map is displayed as a color-coded overlay on the grayscale image. The white/bright yellow color on this map indicates that the acquisition in that area has the highest confidence in which to make measurements. Typically, areas devoid of blood vessels and antral follicles represented high-confidence regions. Within each frame, a 5 mm ROI was placed in a high-confidence area and SWE measurements were made. Overall, 12 stiffness measurements were taken per ovary, and either ROI-specific or average stiffness values can be reported (Figure 2A—three frames are shown, Figure 2B). This same procedure was repeated for the contralateral ovary.

Figure 2.

Human ovarian stiffness assessment using SWE. (A) The least possible compression is applied with the ultrasound probe and SWE is performed only for the ovary (arrow) within 2 cm distance from the ultrasound probe (0.76 cm on the image indicates distance from the ovary to the ultrasound probe). F, antral follicle; S, ovarian stroma. SWE confidence map defines the areas with the highest confidence for ovarian stiffness assessment. Confidence ranges from 0% (dark red) to 100% (bright yellow). Ovarian stiffness measurements are performed in non-overlapping areas (ROI) across the ovary in high-confidence areas. The values are averaged to calculate the average ovarian stiffness. Stiffness measurements heatmap shows stiffness range (increasing stiffness from blue to red). (B) Stiffness measurements taken in individual ovaries are heterogenous. A total of 12 measurements from each ovary of a 31-year-old patient are shown in (B). The patient images shown in (A) are not from the same patient as the measurements displayed in (B).

We then applied this protocol to assess stiffness of the ovarian parenchyma in various contexts, such as across an age spectrum (Figure 3A and B), adjacent to cyst structures (Figure 3C and D), and within endometriomas (Figure 3E). Of note, we observed heterogeneity in measurements, including higher values in the cases of advanced reproductive age and endometrioma. In the case of cystic structures, non-viscous fluids do not support shear wave propagation. Therefore, a signal void is typically seen in the center of a large simple cyst. Depending on the complexity and viscosity of a cystic structure, low stiffness measurements may be obtained in the cystic structure. We observed low stiffness in regions adjacent to the cysts. When an ROI is relatively small, the adjacent cystic structure may affect the stiffness measurement in the ROI. In addition, these results are only from one patient, so further analyses will need to be done to better understand the impact of ovarian cysts on the biomechanical properties of the adjacent ovarian stroma. Whether these patterns hold true in a larger population, if the high numbers of follicles in the young age group may cause heterogenous measurements of stiffness, and what the clinical significance of the physical ovarian microenvironment is are areas of active investigation.

Figure 3.

SWE based assessment of ovarian stiffness is feasible across age groups and pathological conditions. Ovarian stiffness measurements (A) in a 31 year old patient with 15 antral follicles per ovary, (B) in a 41 years old patient with 2 antral follicles per ovary, (C) adjacent to a large simple cyst, (D) adjacent to a hemorrhagic cyst, (E) adjacent to an endometrioma. F, antral follicle; S, ovarian stroma. For each patient, the image on the left is a representative image that best highlights the pathology of interest and the image on the right shows the regions where SWE measurements were made.

Both general ultrasound and SWE specific limitations can cause variability in soft tissue stiffness estimation. These limitations include poor acoustic windows, depth and limited penetration, the size and echotexture of the target soft tissue, and hormonal effects on the ovaries and uterus [121]. The current depth limit for SWE is 8 cm. Therefore, transabdominal SWE measurement may not be reliable when performed on obese patients [122]. Overlying body fat and fatty tissues, such as fatty liver, have higher acoustic attenuations which reduce the reliability of SWE. Small size or heterogenous target tissue can also affect the robustness of stiffness measurements. Variability in SWE imaging may also be operator-dependent and is associated with a learning curve. However, the use of the confidence map may help reduce operator bias.

Reproductive aging and interventions to extend reproductive longevity

As fibrosis is a key hallmark of reproductive aging, SWE may be an ideal tool for monitoring changes to ovarian stiffness as a predictor of fertility and progression toward menopause. In addition, interventions that prevent or clear fibrotic tissue from the ovary may be able to extend the reproductive lifespan [43]. Currently the only way to assess the impact of these medications on ovarian stiffness is through animal models or analysis of limited human ovarian tissue that can be obtained for research purposes [26, 43]. In human studies, postmenopausal women on metformin, a first-line medication to treat insulin resistance, had ovarian collagen levels and organization that were similar to the ovaries of nondiabetic pre-menopausal women (13). This antifibrotic mechanism of metformin is further exemplified in the mouse ovary where metformin prevented age-related fibrosis by modulating the stromal population of fibroblasts, myofibroblasts, and immune cells [16]. Other agents such as Etanercept (an anti-inflammatory target of TNF- α/β), Pirfenidone (an antifibrotic target of collagen I/III), and nintedanib (a small molecule tyrosine kinase inhibitor) also reduce collagen content in mouse ovaries compared with untreated controls [24, 43]. Thus, these antifibrotic agents could be groundbreaking to extend reproductive longevity, but larger-scale human studies are essential prior to broad clinical application. The major barrier to development of these trials is the invasive nature of ovarian tissue assessment to monitor drug response. Using SWE to non-invasively assess ovarian tissue stiffness would allow for close monitoring and a quantitative output of the efficacy of these drugs on ovarian stiffness that could be directly compared with measured clinical outcomes (Table 2).

Table 2.

Proposed research applications of SWE (reproductive aging)

Condition	Justification	Utility	Citations
Ovarian aging	Ovaries become fibrotic with advanced reproductive age.	Increased ovarian stiffness may serve as a biomarker for reproductive aging and could assess the efficacy of interventions to slow or reverse reproductive aging (e.g. metformin, antifibrotic agents).	[11–13, 24, 26, 27, 29–34, 43]
Ovarian cancer	Malignant tissue is stiffer than healthy tissue.	Identifying regions of elevated stiffness could be used for early ovarian cancer screening. It could also be useful for monitoring for fertility and ovarian cancer risk in patients with Brca mutations.	[12, 14, 44–53, 123–125]
Premature ovarian insufficiency (POI)	Some causes of POI are associated with ovarian fibrosis and accelerated reproductive aging.	Increased ovarian stiffness may be useful for monitoring fertility and diagnosing POI.	[116, 126–137]
Assisted reproductive technologies (ART)	Repeated ovarian stimulation during ART may increase inflammation in the ovary, possibly leading to increased fibrosis.	Assessing changes to ovarian stiffness in patients undergoing ART could lead to modulations in ART treatment plans and help patients make informed decisions about undergoing IVF. Pairing SWE with ART outcomes will also provide important information about the impact of ovarian stiffness on fertility.	[12, 138, 139]

Ovarian cancer

SWE already has demonstrated utility in distinguishing between benign and malignant lesions in many different organs. In the ovary SWE could play a role in predicting malignancy, assessing cancer risk based on the ovarian microenvironment, and monitoring women with genetic mutations that predispose them to ovarian cancer (Table 2). SWE-based monitoring of ovarian stiffness may be particularly relevant for patients with genetic mutations that predispose them to the development of ovarian cancer, including Brca mutations. For many of these women, the choice of if or when they should have prophylactic oophorectomies is complex [123–125]. A tool to non-invasively profile ovarian stiffness may give these women an indication of the progression of their ovarian fibrosis, which may help them be more informed as they make these difficult decisions. In addition, as ovarian cancer is often diagnosed at later stages with worse prognosis, SWE may be a valuable screening tool for early detection and risk assessment. The utility of SWE for ovarian cancer is a crucial area of study to see if the technology may provide beneficial insights as to the risk and prognosis of ovarian cancer.

Premature ovarian insufficiency

SWE could also have utility in premature ovarian insufficiency (POI), which is a heterogenous set of iatrogenic, environmental, genetic, and idiopathic conditions associated with an early decline in the ovarian reserve [126, 127]. In both human ovarian tissue and preclinical animal models, there is some evidence that ovaries in individuals with POI have increased fibrosis which may contribute to the early decline in the ovarian reserve [128–132]. Chemotherapy and radiation cause accelerated follicle depletion which is associated with iatrogenic POI and early menopause. Interestingly, fibrosis and increased ovarian stiffness are late effects of these treatments which may further accelerate ovarian aging [116, 129, 133–137]. The rate of fertility decline in each case of POI is heterogenous and difficult to assess [132]. SWE could be used to non-invasively track progression of disease, provide a quantitative metric for fertility, and assess the efficacy of possible clinical interventions on maintenance of the ovarian reserve (Table 2). SWE may be useful for tracking the extent of ovarian fibrosis and assessing the efficacy of interventions that may preserve ovarian function and prevent fibrosis during cancer treatments.

Assisted Reproductive Technologies

There has been an exponential rise in the use of assisted reproductive technologies (ART) and in vitro fertilization (IVF) over the last 30 years, accounting for nearly 8 million live births worldwide [138]. Despite widespread use of ART, little is known about the impact of the ovarian microenvironment on outcomes of fertility interventions. This is particularly relevant for patients undergoing ART for age-related infertility. Using SWE in parallel with routine ultrasound scans during IVF treatments could provide a wealth of information about how ovarian stiffness corresponds to hormone levels, egg quality and quantity, embryo quality, and pregnancy outcomes. In addition, little is known about the long-term effects of gonadotropin stimulation and oocyte retrieval on ovarian stiffness. IVF stimulation involves the use of exogenous gonadotropins to coordinate mass follicle recruitment until ovulation, where the oocytes are retrieved through ultrasound guided needle aspiration. How ovarian stimulation and oocyte retrieval affect the ovarian microenvironment in the short and long-term and their effects on the lifelong endocrinological function of the ovary remain to be investigated. Moreover, ovarian exposure to a wide range of medications during IVF could have varying degrees of effect on fibrosis formation. For example, ovarian stimulation in mice with Cetrotide (a common GnRH antagonist used in IVF) reduces the degree of ovarian fibrosis, thereby improving ovarian function [139]. Given patients undergoing IVF stimulation require frequent transvaginal ultrasounds to monitor follicle development, SWE could provide key insight on ovarian microenvironmental changes throughout stimulation and following repeated stimulations (Table 2). Additionally, SWE could help compare ovarian fibrosis risk between various stimulation regimens which could guide treatment plans. Understanding if there is a relationship between IVF and ovarian fibrosis may impact elective fertility preservation decisions.

SWE may have additional reproductive applications beyond aging

Polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is a complex endocrine disorder that affects 6–10% of reproductively aged women and is associated with anovulatory infertility and metabolic dysfunction [140]. Ovaries from women with PCOS have increased collagen and fibrosis which may contribute to ovarian dysfunction [141–143]. In addition, metformin has documented antifibrotic effects on the ovary and can be used clinically to increase ovulation rates in patients with PCOS [144–146]. Ovarian stiffness, therefore, may be a valuable biomarker for PCOS diagnosis and progression (Table 3). The diagnostic utility of SWE in PCOS has been explored in a few studies. In one early trial with 37 PCOS patients and 16 control patients, no detectable difference was found in ovarian stiffness measured transabdominally with SWE between patients with PCOS and controls [147]. In contrast, all subsequent studies have shown a consistent increase in ovarian stiffness of patients with PCOS when measured via transvaginal ultrasound [148–150]. In each of these studies, the ultrasound probe was placed transvaginally in a supine patient and measurements were taken in a vein-and cyst-free region while the patient held their breath to minimize motion artifacts. Beyond diagnosis, SWE could also be used to assess disease progression, serve as a biomarker for fertility outcomes, and assess response to novel treatments in PCOS patients.

Table 3.

Active areas of research for clinical applications of SWE (reproductive)

Condition	Justification	Outcome	Citations
Polycystic ovarian syndrome (PCOS)	PCOS is associated with hyperthecosis and increased accumulation of fibrotic tissue within the ovary.	Ovaries of patients with PCOS are stiffer, which may serve as an early diagnostic tool. Increased ovarian stiffness may be useful for earlier PCOS diagnosis, monitoring of disease progression, and assessing efficacy of treatments for PCOS.	[140–143, 147–150]
Endometriomas	Endometriomas contain stiffer tissue than other cysts in the ovary.	Endometriomas are stiffer than hemorrhagic cysts, which can be used to non-invasively differentiate between the two conditions. Increased stiffness associated with endometriomas may provide insights on disease progression, treatment effect, and impact on fertility.	[151–155]
Hormonal contraceptives	Hormonal contraceptives prevent ovulation, which decreases ovarian remodeling and accumulation of fibrosis.	Assessing changes to ovarian structure with hormonal contraceptive use could quantify utility of contraceptives for reducing cancer risk and preserving fertility.	[156]
Gender-affirming hormone therapy	Ovarian exposure to testosterone leads to increased fibrosis compared with untreated ovaries.	Assessing changes to ovarian structure with testosterone use could quantify impact of hormones on reproductive aging and function.	[157, 158]
Obesity	Obesity is associated with systemic inflammation, widespread tissue fibrosis, and impaired ovarian function.	Monitoring changes in stiffness with increased adiposity could provide insights into the impact obesity has on fertility and the impact of weight loss interventions on ovarian health.	[159–182]
Connective tissue disorders	Connective tissue diseases cause widespread dysfunction and alterations to tissue structure, which may impact the reproductive tract.	Quantifying stiffness of the female reproductive tract in patients with connective tissue disorders may help identify and treat those at risk of infertility and obstetric complications.	[183–191]
Differences of sex development (DSD)	DSD in individuals with ovaries is associated with ovarian dysgenesis and hormonal alterations that may lead to stromal hyperplasia, increased rigidity, and loss of the ovarian reserve.	Measuring stiffness in ovaries of DSD patients could give insights into the reproductive function and fertility of these patients.	[192–196]

Endometriomas

Endometriomas are cystic lesions that arise in women with endometriosis when ectopic endometrial tissue invades into the ovary [151]. SWE has demonstrated utility to differentiate endometriomas from hemorrhagic cysts in the ovary, which may reduce the need for surgery and biopsy of endometriotic lesions (Table 3). Endometriomas had significantly increased stiffness compared with hemorrhagic cysts and had a sensitivity and specificity of 82.1 and 79.2%, respectively, for differentiating between the two diagnoses as confirmed with histopathology and follow-up ultrasonography [152]. These findings are consistent with our measurements (Figure 3C–E). Further studies could use SWE to monitor for progression and recurrence of endometriomas. Importantly, SWE could also be used to assess the impact of endometriomas on ovarian fibrosis, as endometriomas are associated with increased ovarian fibrosis and accelerated decline of the ovarian reserve [151, 153–155]. SWE could serve as a biomarker of ovarian function in patients with endometriosis and may detect changes in the ovarian stroma even before endometriomas are symptomatic and detectable.

Hormone-based medications

Hormone-based contraceptives and medications for gender-affirming therapy for transgender patients are taken throughout the reproductive lifespan. Oral contraceptive use has been associated with decreased ovarian cancer risk, possibly due to fewer cumulative ovulation events that contribute to inflammation and fibrosis [12, 156]. Other hormone-based therapeutics may increase tissue stiffness. For example, some preliminary studies on ovaries exposed to testosterone as part of gender-affirming therapy exhibit increased tissue stiffness compared with untreated ovaries [157, 158]. In both cases, more information is needed to conclusively assess the impact of these hormone-based treatments on the ovarian microenvironment. As the rate of utilization of hormone therapy as part of gender-affirming care continues to increase, there is a pressing need now more than ever to develop non-invasive tools that can monitor long-term effects of these treatments on the structure and function of the reproductive tract [197]. SWE could be used to non-invasively quantify how these medications impact ovarian fibrosis, subsequent fertility outcomes, and risk of developing cancer (Table 3).

Obesity

Obesity is associated with systemic inflammation, which may alter ovarian structure and function. In rodent models, obesity is associated with depletion of the primordial follicle pool with concurrent increases in antral follicle counts, and increased follicle atresia [159–162]. Oocytes from obese rodents exhibit decreased rates of meiotic progression, diminished communication between oocytes and cumulus cells, and decreased blastocyst formation after fertilization [163–167]. Clinically, this translates to elevated rates of infertility, anovulation, and poorer response to fertility treatments [168]. In women undergoing fertility treatment for male-factor infertility, the rate of conception for normal weight women based on BMI was 21–30% higher than that of obese women [168–170]. Women with a BMI of >25 kg/m² had an odds ratio of 1.67 for early miscarriage compared with non-obese women, possibly due to diminished oocyte quality and endometrial receptivity [171, 172]. There is also a connection between obesity and some subtypes of ovarian cancer. Obese women have an odds ratio of 1.1 for development of low-grade serous ovarian carcinoma compared with non-obese women [173]. In addition, obesity is associated with increased collagen deposition and fibrosis in ovarian cancer lesions and increased chemoresistance [174]. Although none of these studies looked specifically at ovarian fibrosis or stiffness, obesity is known to cause fibrosis in a broad range of tissues including cardiac, liver, lung, and adipose tissues [175–178]. In each of these tissues, obesity-induced fibrosis is driven by an inflammatory cytokine milieu that includes tumor necrosis factor alpha (TNF- α), transforming growth factor beta (TGF- β), and a variety of adipokines [175–178]. Short-term treatment of mice with leptin impairs ovulation, suggesting that adipokines may regulate ovarian function [179, 180]. In the ovary, TGF- β overexpression is associated with fibrosis in both PCOS and endometriomas [181, 182]. SWE could be used to answer important questions about the exact link between obesity and reproductive function (Table 3). In the context of obesity, SWE could also be used to evaluate the effect of weight loss on reproductive tract biomechanics and function.

Connective tissue disorders

Ovaries contain a broad array of extracellular matrix proteins, including collagens and elastins, that are crucial for maintaining the proper physiologic environment for ovarian function and folliculogenesis [183, 184]. Many extracellular matrix components are implicated in human diseases when mutated. This includes Ehlers–Danlos syndrome (variable collagen mutations), Marfan syndrome (fibrillin 1 mutations), and muscular dystrophies (variable mutations including in dystrophin, laminin, and collagens) [185]. Whether or not ovarian function is altered in these diseases is an active area of investigation. This is particularly relevant as many of these patients rely on ART and pre-implantation genetic screening to prevent disease inheritance [186]. A survey of more than 1200 patients with Ehlers–Danlos syndrome found that 41.1% of surveyed patients reported infertility, 67.2% reported abnormal menstrual cycles, and 57.2% of respondents who had at least one confirmed pregnancy reported a spontaneous abortion [187]. Pregnant patients with Marfan syndrome have a higher risk of premature rupture of membranes and premature birth [188]. Mutations in fibrillin-1 and fibrillin-3 have a documented association with ovarian cancer tumorigenesis and PCOS, both of which are associated with ovarian tissue fibrosis [189, 190]. Case reports of patients with mutations in lamin A/C demonstrate an association with ovarian failure and hypogonadism [191]. SWE could be used to better understand the reproductive implications of these heterogenous disorders on ovarian, uterine, and cervical function (Table 3). This tool could also be used to non-invasively assess fertility or potential for obstetric complications in these high-risk patients.

Differences of sex development

Differences of sexual development (DSD) encompass a spectrum of congenital conditions associated with the abnormal embryologic development of the internal and external genital structures. While the use of SWE in DSD is underexplored, it has potential to provide insight into ovarian composition and reserve in these patients (Table 3). DSD in individuals with ovaries can be classified due to changes in sex chromosomes, such as in Turner syndrome (complete 45, XO) or mosaic Turner syndrome (45,XO/46XX). Given that ovarian dysgenesis is a hallmark of Turner syndrome, detecting the onset of premature ovarian failure is prudent as it can occur as early as in childhood [192]. Histopathologic studies of fetal ovaries with Turner syndrome have underscored that there is an early presence of stromal fibrosis and accelerated germ cell loss [192, 193]. Another classification of DSD in XX individuals involves the presence of ambiguous genitalia, which is secondary to excess androgen exposure from disorders in steroidogenesis (such as congenital adrenal hyperplasia) versus in utero exogenous exposure [194]. The biochemical alterations to ovarian tissue throughout the reproductive lifespan in patients with DSD are still poorly understood, but likely lead to alterations in ovarian stromal composition [195]. For example, the excess production of potent adrenal androgens in congenital adrenal hyperplasia not only directly inhibits ovarian follicular growth, but also may stimulate production of extracellular matrix leading to ovarian stromal hyperplasia and rigidity [195, 196]. Lastly, ovarian dysgenesis in DSD can also involve loss of function mutations in various genes (such as Foxl2), which leads to an accelerated loss of primordial follicles resulting in premature ovarian failure. Given prior studies in patients with POI have identified an inverse relationship between stiffness and ovarian reserve, SWE could serve as a noninvasive tool to characterize and screen for this rapid ovarian aging in patients with DSD [128–132].

Conclusion

Dynamic changes to the structure and function of the female reproductive tract occur with aging and a variety of pathologic conditions. The ability to non-invasively quantify changes in tissue stiffness of reproductive organs and track them dynamically over time would have significant diagnostic and prognostic value for patients. SWE has been tested and used clinically in the liver, musculoskeletal system, and multiple different organs in the case of malignancies. Establishing a standard operating procedure for measuring ovarian stiffness in patients across the reproductive lifespan and ovarian pathologies is an essential first step in assessing the value of SWE for reproductive health. Preliminary data of SWE in the reproductive tract have already been generated in the context of PCOS, endometriomas, and several uterine and endometrial pathologies. However, the potential value extends well beyond these conditions. SWE may be an ideal non-invasive technique for understanding how ovarian tissue stiffness is modulated with disease and medications, which will consequently provide insights into the relevance of fibrosis to women’s reproductive health and will better inform clinical interventions to improve reproductive longevity.

Authors contributions

EJZ wrote the manuscript. MZ, LH, LLF, TA, and AT assisted with the writing and editing of the manuscript. EB and FED conceived the original ideas and contributed to the editing of this paper. All authors participated in discussions regarding this review and approved its final submission.

Conflict of interest: The authors have declared that no conflict of interest exists.

Data availability

Not applicable.

Ethics Statement

This project is IRB approved (Northwestern University IRB STU00218882).