Chemically-induced histopathological progression in the polycystic ovary syndrome (PCOS)- A review

Abstract

Polycystic ovary syndrome (PCOS) is the most common endocrine-gynecological disorder, affecting 4–10% of women of reproductive age. Although a fragment of the involved mechanism behind the PCOS occurrence is discovered, the exact etiology and pathophysiology are not completely understood yet. The pathogenesis of the disease involves several genetic, epigenetic, and environmental factors, as well as poor lifestyle. Chemicals that interact adversely with the endocrine glands are ubiquitous in the ecosystem and are known as endocrine-disrupting chemicals. An exposed population, especially women at a growing age, undergoes many hormonal and physiological changes and is more susceptible to developing endocrinological disorders like PCOS. An effective diagnosis of the disease can be made by observing changes in cells that are associated with the progression of the disease. Histopathological changes provide abundant evidence that is directly or indirectly involved in its progression and help in the evaluation of the complexity of the disease. In case of environmental stress, the body responds via some visible changes in cells, including the position and size of small cysts, changes in atretic follicles and ovarian capsules, thickening of ovarian tunica, and increased intra-ovarian cysts are some of the examples that are involved in etiology of disease. Therefore, in this review, we will try to understand the progression of PCOS by observing morphological changes through the histopathology of associated tissues that can be a plausible predictor of malignant changes in the disease. Our primary aim is to summarize the existing literature of experiments and contribute to a better understanding of the disease and associated problems.

Introduction

Polycystic ovary syndrome (PCOS) was first coined by Stein and Leventhal in the year 1935, therefore, it is also known by the name Stein–Leventhal syndrome [1]. It is the most common, highly prevalent, and multifactorial disorder, which affects around 4–18% of women of childbearing age in the world [2]. However, its prevalence varies among different races and ethnic groups as it is higher among South Asians than Caucasians [3]. As per the World Health Organization (WHO) reports, around 116 million (12%) women were affected by PCOS globally in 2012, and in 2020, this ratio elevated promptly to 26% [1, 3]. However, in India, its dominance is around 11.34% [4]. PCOS is a multifactorial disorder, and the exact mechanism behind its occurrence is unknown. Hyperandrogenism may be the main etiological factor for the occurrence of PCOS along with menstrual cycle disturbance, anovulation, insulin resistance, and obesity [5, 6]. In PCOS condition, the reduced level of follicle-stimulating hormone (FSH) leads to the arrest of follicular maturation in the final stage. The higher level of luteinizing hormone (LH) and testosterone (androgen), along with lower level of FSH disturb the steroidogenesis process. This hormonal imbalance results in a disturbance in the menstrual cycle which makes it difficult for women to conceive [3, 7]. The PCOS ovary gets enlarged as it has to accommodate multiple cysts, generally more than 10. These cysts are generally undeveloped follicles. As the disease advances, the ovary wall gets thickened which prevents the discharge of the ripened follicles, leading to anovulation [1]. Other clinical factors associated with PCOS are dysfunctional uterine bleeding, type II diabetes, ovarian cancer, hypercholesterolemia, imbalance of all the hormones, and cardiovascular disorder [3, 8, 9]. Due to the clinically heterogeneous characteristics of this syndrome, its treatment remains complex with variable responses among PCOS patients [10]. Moreover, it is diagnosed when females have difficulty in conceiving or getting pregnant. In addition to clinical investigations, changes in the morphology and histology of ovaries can also serve as an important biomarkers for PCOS diagnosis because all the changes can be seen clearly.

The PCOS ovary generally shows numerous small antral follicles, hyperplasia, hypertrophy of theca cells, and stromal and cortical thickenings. The increase in the number of small antral follicles (2–9 mm) is attributed to the higher proportion of follicles from the primordial phase which are destined to become mature follicles (primary). But when the follicle maturation arrest occurs, then these small antral follicles which have prolonged lifespans, hamper the development of dominant follicles. According to Jonard and Dewailly [11], all these happen due to androgen excess. Several investigations relate excess androgens to exposure to xenobiotics and other environmental toxicants. In the environment several toxicants including endocrine disrupting chemicals (EDCs), pesticides, heavy metals, pharmaceutics, etc. are present which either directly or indirectly upon exposure to humans result in hormonal imbalance. Among them, EDCs are major drivers involved in the excess production of androgens and the imbalance of hormones, consequently leading to anovulation, infertility, disruptive ovarian functions, polycystic ovaries, and other histopathological alterations [3, 12, 13]. Experiments with different animal models demonstrated that several xenobiotics, industrial chemicals, drugs, pharmaceuticals, and exogenous androgen sources contribute to PCOS pathogenesis [13–16] as they may affect the functioning of the hypothalamus pituitary gonadal axis. Therefore, it becomes necessary and significant to map out the histopathological changes in the progression of PCOS. Hence, in this review, there is a thorough assessment of the occurrence of histopathological progression in polycystic ovaries after exposure to several agents.

Pathology and etiology of PCOS

PCOS is a multifactorial disorder hence the exact cause is unknown. However, several factors implicated by researchers in the PCOS pathophysiology are broadly classified into environmental and genetic factors. In addition, nutritional factors have also been implicated in the development of PCOS. These are presented in Fig. 1.

Fig. 1.

Illustrates the PCOS pathology and etiology with environmental, nutritional, and genetic factors

Environmental and nutritional factors

According to several investigators, the major factors which contribute to PCOS are poor diet and an imbalanced lifestyle [3, 4, 17]. An increased amount of androgens prevents the discharge of the ovum from the follicles. Thus, an unhealthy and poor diet and stressful lifestyle could worsen the symptoms of PCOS. Besides, exposure to several toxicants, including EDCs, androgens, etc. is responsible for PCOS pathogenesis.

Genetic factors

These factors provide additional insight to determine the epidemiology, prevalence, and presence of PCOS. Based on these factors, PCOS is divided into four different types.

1. Insulin resistance PCOS: A high level of insulin is the common and highly prevalent reason for PCOS [18].

2. Adrenal PCOS: Stimulation of adrenal secretions during early puberty causes adrenal PCOS. Patients with adrenal PCOS generally experience more stress due to excess DHEAS (dehydroepiandrosterone sulfate, an androgen of adrenal glands) [19].

3. Inflammatory PCOS: Chronic low-grade inflammation is generally found in PCOS patients [20].

4. Post-pill PCOS: Caused by contraceptive pills and hormonal disturbances [21].

5. Increased insulin level and insulin resistance also contribute to the pathogenesis of PCOS.

Method of histological slide preparation

The healthy female Wistar rats (n = 5; 6 weeks old) with a mean weight of 130 ± 4 g obtained from the Central Animal House Facility, Jamia Hamdard, the work approved by Institutional Animal Ethics Committee (IAEC, project # 1952). The ovaries were fixed in 10% formalin. The tissues were dehydrated in xylene and different grades of alcohol by following the standard protocol, and blocks were made by embedding them in paraffin wax. Tissue sections of 5 µm thickness were incised from the blocks and mounted onto the slide. Subsequently, they were rehydrated slowly with grades of alcohol and cleared in xylene, stained with haematoxylin (H) and eosin (E), and observed under the light microscope. The histological images of the ovarian tissue provided in the manuscript are the authors’ original unpublished data.

Morphology and histology of follicles in the healthy ovary

Generally, the normal and healthy rodent ovary are surrounded by a single layer of peritoneal mesothelium - the ovarian surface epithelium (OSE, Fig. 2B), which is in continuation with mesovarium (towards inside) and supports the ovary. The cells of OSE can be squamous or cuboidal or columnar or pseudostratified columnar, based on the changes in underlying parenchyma during the estrous cycle. The body of the ovary is formed by a stroma consisting of spindle-shaped fibroblast-like cells and collagen fibers. Hence, the stroma below the OSE is dense and fibrous. It forms the narrow and distinct zone called the tunica albuginea (TA). The stroma below the TA is divided into the peripheral cortex and central medulla (Fig. 2A). However, the medulla is not always visible in histological sections. Besides, rete ovarii is also present, which arises from the anastomosing of several tubules embedded within the stroma and lined with cuboidal and columnar epithelium. In sexually mature rats, five types of follicles are present which include primordial follicles, preantral follicles, small antral follicles, large ovulatory antral follicles, and corpus luteum [22]. According to the presence or absence of an antral cavity, the follicles are named preantral or antral follicles. The primordial, primary, and secondary follicles come under the preantral category whereas early tertiary follicles are described as small antral and mature tertiary follicles as antral follicles. At the antral phase, most follicles undergo atretic degeneration as part of a normal cycle. However, only a few of them reach the preovulatory phase under the influence of gonadotropins [23].

Fig. 2.

A Cortex and medulla regions of rat ovary and B ovarian surface epithelium which covers the entire ovary. Staining was done with H and E. Scale bar 100 µm

The types of follicles present in the ovary are classified as follows:

1. Primordial follicles: This is the earliest stage of follicle development and is present within the peripheral cortex just below TA. It contains an oocyte surrounded by a complete or partial layer of squamous follicular epithelium (granulosa) cells (Fig. 3A).

2. Primary follicles: Squamous epithelium surrounding the primordial follicle differentiates into a single layer of cuboidal granulosa cells to form primary follicles. Sometimes, intermediate follicles are also present between primordial and primary follicles, consisting of both squamous and cuboidal cells, if cuboidal cells predominate, then they are classified as primary follicles (Fig. 3B).

3. Secondary follicles: When a single layer of cuboidal granulosa cells proliferates into several layers (more than one layer) to form a zona granulosa surrounding the oocyte, then it is called a secondary follicle (Fig. 3C). It does not contain any visible antrum. Between the oocyte and the zona granulosa, there is a thick layer of glycoprotein and proteoglycan, the zona pellucida. With further growth of secondary follicles, some fluid-filled spaces (vesicles) may develop in the zona granulosa, this stage is called the vesicular follicle. Furthermore, the stromal cells surrounding the developing secondary follicles are arranged concentrically to form theca folliculi. The basement membrane separates the zona granulosa from theca folliculi.

4. Tertiary follicles: In these follicles’ large central cavity, the follicular antrum appears which is fluid-filled and surrounded by zona granulosa (Fig. 3D and 4A). The primary oocyte is eccentric in position and present in the mass of granulosa cells known as cumulus oophorus. The granulosa cells which are in the immediate surroundings of the oocyte are called corona radiata. The theca cells form two zones theca interna and theca externa. The theca interna comprises polygonal cells with vacuolated cytoplasm and displays the ultrastructural properties of steroid-producing cells, such as numerous mitochondria, lipid droplets, and smooth endoplasmic reticulum. Thus, serves as the principal site of synthesis of androstenedione. The theca externa consists of spindle-shaped cells and does not perform any endocrine functions.

5. Preovulatory/graafian follicle: Not all but a few tertiary follicles enter the preovulatory phase. They undergo further changes in their morphology. The antral cavity enlarges causing a reduction of zona granulosa, and cells of cumulus oophorus degenerate, resulting in the detachment of the primary oocyte, due to which it floats freely within the antrum.

Fig. 3.

Different types of follicles with oocyte in rat ovary. A primordial follicle, B primary follicle, C secondary follicle and D tertiary follicle with distinct antral cavity and oocyte. Staining was done with H and E. Scale bar 50 µm

Fig. 4.

A Tertiary follicle with eccentrically placed oocyte and B corpus luteum within the ovary. Staining was done with H and E. Scale bar 50 µm. All figures are created by the authors. No permission was required

The primary oocyte completes its first meiotic division just before ovulation and forms the secondary oocyte. After ovulation of the secondary oocyte, the granulosa and theca cells of the remnant follicle undergo hypertrophy and hyperplasia, and the process is called luteinization under the influence of LH and prolactin. Along with luteinization, the basement membrane degenerates, due to which theca interna and zona granulosa get separated and infiltration of the postovulatory follicle occurs from theca interna (Fig. 4B). The resulting mature corpus luteum (eosinophilic structure) may bulge out from the ovarian surface. However, before luteinization, the corpora lutea appears basophilic. The maturation of the corpus luteum (yellow body) occurs during the estrous cycle in which it is formed before regressing over the course of several subsequent cycles. Therefore, at least three sets of corpora lutea are present within the ovaries of normally cycling rats. The corpus luteum degenerates, so its size shrinks and is characterized by high amounts of fibrous tissue and yellow–brown lipofuscin pigments. Due to the high amounts of fibers during the regression phase of the corpus luteum, it is termed corpus albicans (white body). In the case of rats, this structure gets regressed completely and no remnants of fibrous tissue are left within the ovary.

It is to be noted that only a few primordial follicles progress via folliculogenesis to form graafian follicles and ovulate. The remaining tissue undergoes follicular degeneration or atresia during the follicular maturation process.

Identification of atretic and cystic follicles in PCOS condition

The morphology of normal and healthy follicles has already been described. However, the atretic and cystic follicles both are different morphologically as well as functionally. Few researchers described the signs of an atretic follicle as the one whose oocyte nucleus is degenerating, wrinkled or shrunk nuclear membrane, with presence of vacuoles in the oocyte, degeneration in granulosa cells like karyorrhexis, pyknosis, and cell shrinkage [24–26]. Features of cystic follicles were described by Shi et al. [26] and Merlo et al. [27] as they were lined by one to four or five-cell thick layers of flat to round granulosa cells, showed dilation, and were filled with follicular fluid.

Role of histology in the diagnosis and pathogenesis of PCOS

PCOS condition cannot be diagnosed with blood tests, biochemical measurements, and cultures. There are no specific tests available for it. The past medical history, changes in body weight, and indicators of insulin resistance could be helpful to some extent whereas, examination of the pelvis, transvaginal ultrasound, and assessing the hormone levels are the most commonly used methods [13]. According to the National Health Service (NHS), the irregular menstrual cycle, elevated amounts of androgens, and scans representing polycystic ovaries are among the designated criteria for PCOS [28]. According to the Rotterdam criteria, if the ultrasound investigation shows two of the following parameters that are oligo/anovulation, the presence of polycystic ovaries, and clinical or biochemical hyperandrogenism could indicate PCOS condition [29]. Rotterdam criteria also clarified that not every PCOS woman has a polycystic ovary; likewise, every ovarian cyst may not be an indicator of PCOS. However, these frequently used diagnostic methods cannot give an exact and clear picture of pathogenesis in the polycystic ovary. Samples of polycystic ovaries from humans are also limited because the availability of wedge resection is not recommended in PCOS patients. Therefore, further studies are needed to be done on polycystic ovaries in model organisms. Histopathological tools can easily detect changes in the morphology of the tissue/cell in several disease types like cancer. Therefore, histopathological progression in the polycystic ovary can set a benchmark in the diagnosis of this disorder.

Pache et al. [30] asserted that although hyperplasia in the theca cell layer has not been a specific criterion for PCOS, it remained an acceptable histological basis for the diagnosis of the disorder on exposure to androgen. Canning et al. [31] studied differences in follicle numbers in the ovaries of five different strains of mice (neonates and 42 days old), namely DBA/2, 129/Sv, FVB, C57BL/6, and AKR/J. In neonates, minimum follicles were found in the ovaries of AKR/J females whereas the maximum was in 129/Sv females. While in 42 days old female, follicle numbers were comparable. However, strain-dependent differences were observed in the rate of primordial follicle growth and follicular atresia. Tilly [32] also found upto tenfold differences in the early follicles of mouse ovaries. Therefore, follicle counts become important to differentiate polycystic ovaries from healthy ovaries. Several other researchers also reported enhanced follicle recruitment in the PCOS conditions [11, 25, 33–35]. The appearance of atretic and cystic follicles, along with an abnormal reproductive cycle, are common features of PCOS in human and rodent models [26, 36–39]. Abbott et al. [40] observed several enlarged and sclerotic follicles along with many cystic follicles, hypertrophy in the whole ovary, thickening of the capsule (> 100 μm), increase in subcapsular follicular cysts, premature theca cell luteinization, decrease in corpora lutea and albicantia, and fibrosis and hyperplasia in the stroma. Caldwell et al. [36] found an abnormal estrous cycle along with numerous multicystic follicles in mice treated with dihydrotestosterone (DHT). There are no known serum markers by which follicle numbers of normal, atretic, and cystic formations can be counted accurately in the ovaries at the different developmental stages. Hence, histomorphological evaluation can be employed to solve this issue.

The counting of different follicle types can impart important information about ovarian function, particularly the relationship between folliculogenesis and the growth factors that regulate it. This may also be important to ascertain the relative functions of gonadotropins, steroid hormones, and an intraovarian environment containing growth factors regulating the survival and maturation of follicles at any stage of their development. Therefore, the identification of ovarian follicles and associated cells is important in the diagnosis of the diseased condition.

Histological alterations in the female reproductive tract- ovary, uterus, and vagina

Although PCOS shows many morphological alterations that are directly or indirectly involved in the etiology of the disease such as the irregular menstrual cycle [16], increased body weight index, ovarian weight [41], hormonal fluctuations [42], increase in blood glucose and insulin level [41] it also shows numerous histological signs that could possibly be the hallmark for the disease like few of which are described here from existing literature (Table 1).

Table 1.

Histological abnormalities observed in the reproductive tract of rats

S. No	Tissue	Histological abnormalities observed	References
1	Ovary	Multiple variably sized cysts and cystic follicles, arrest of follicular growth, and cortex region shows many cystic follicles.	[16]
2	Uterus	Accumulation of endometrial glands, infiltration of eosinophil in the stromal region, thickening of uterine wall and lumen, and vacuolation.	[41]
3	Vagina	No significant changes were observed most frequently. Vaginal smear observations were made for changes in estrous cycle.	[42]

Environmental agents (xenobiotics) contributing to PCOS

Recently, investigations in animal models showed that the etiology of PCOS is closely linked to the excess of androgens, which would interfere with the function of the hypothalamic-pituitary–gonadal axis and thus lead to reproductive dysfunction in adults. The EDCs could be one of the reasons for excessive androgens [43–46]. The EDCs are found in everything we use in daily life [47]. The United States Environmental Protection Agency (USEPA) characterizes an EDC as “an exogenous substance which interferes with the synthesis, secretion, and transport of hormones in the body which are responsible for maintaining homeostasis, development, and reproduction [48]. As far as their structure is concerned, they contain phenols or halogens which impart them the imitating power to the steroid hormone actions therefore, they act as hormones’ antagonists or agonists in binding to their receptors [47, 49]. Many researchers have reported higher serum EDC concentrations in PCOS patients [4, 47, 49]. Chronic exposure to EDCs from the antenatal to adolescent stage causes susceptibility to PCOS [47, 50]. For instance, bisphenol A (BPA) is a synthetic compound widely utilized in epoxy resins, polycarbonate plastics, baby bottles, food packages, and polyvinyl chloride (PVC) [51, 52]. It interacts with estrogen receptors (ER) and non-classical membrane ER and thus directly affects oogenesis [47, 52, 53]. Besides EDCs, there are some heavy metals and their salts like mercury, cadmium, cadmium chloride, lead, and lead acetate, which are found to cause PCOS-like pathologies in the ovaries [27, 35, 54–57]. Overall, several reports emphasize the fact that PCOS can be induced by exposure to a variety of toxicants, exogenous androgens, xenobiotics, etc.

Effects of environmental agents and androgens on histopathological alterations in the ovary and uterus

There are several studies which linked environmental exposure with the PCOS. For instance, Aldeli et al. [58] reported the reprotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in BALB/c mice for two generations (F₁ and F₂). TCDD (25 μg/kg) impaired hormone synthesis and induces cystic and atretic follicles in F₁ generation. However, in the F₂ generation, abnormal hormone levels, along with necrosis of granulosa cells, dissolution of oocytes, and blood vessel congestion, were observed. Javed et al. [59] highlighted that a low dose of BPA (50 μg/kg) caused disturbed levels of sex hormones and androgens and also observed cystic and atretic follicles, and necrosis in the ovary of Wistar rats. The endometrium and perimetrium of the uterus were also found to be degenerated. Zhan et al. [60] found a strong positive correlation between levels of BPA and its analogs and PCOS in a hospital-based case–control study. Experimental studies investigating the effects of exposure to major chemicals, such as heavy metals and endocrine disrupters on the reproductive system of female rats that could play a plausible role in the etiology of PCOS are summarized in Table 2. Besides, the imbalance as well as excess of androgens and hormones can also result in the development of PCOS (Table 3).

Table 2.

Effects of environmental chemical exposure on histological alterations indicating the risk of PCOS

S. No	Chemical name	Species	Dose	Route & exposure duration	Findings	References
1	BPA	Sprague Dawley rat	50 µg/kg/d	Subcutaneous injection (50 µg/50 uL PND1 to PND10)	Tremendous increase in the number of cysts, lesser number of corpora lutea, high number of atretic follicles (cystic), and lower antral follicles.	[34]
2	Lead acetate	BALB/c mice	30 mg/kg/d	Oral gavage (60 days)	Reduced primary follicular count, arrest in the growth and development of follicles.	[55]
3	Lead acetate	Adult Wistar rat	30,100, & 300 ppm	Drinking water (28 days)	Endometrium thickening, vacuolar degeneration in epithelial cells, narrow lumen, and degeneration of goblet cells.	[54]
4	Di (2-ethyl hexyl) phthalate (DEHP)	Mouse (CD-1) (transgenerational study)	Prenatal exposure of DEHP (0.05 mg/kg/d, 5 mg/kg/d) from gestational day 0.5-PND21 pups	Diet (chow)	Less number of primordial follicles (DEHP 0.05 mg/kg/d, 5 mg/kg/d) in the F1–F3 generations-more number of preantral follicles (DEHP 0.05 mg/kg/d, 5 mg/kg/d) in the F1–F3 generations. Less number of antral follicles in the F1 and F2 generations (DEHP 0.05 mg/kg/d). Decreased number of antral follicles (DEHP 5 mg/kg/d) in the F1 generation.	[61]
5	DEHP	CD-1 mice	20 & 200 µg/kg/d & 500 & 750 mg/kg/d	Oral (from gestational day 11 till birth)	In the F1 generation prenatal exposure causes a reduction in follicle numbers while in F2 and F3 generations rise in follicle numbers and increase in ovarian cysts.	[62]
6	Mercury	Wistar albino rat	1st dose 4.6 μg/kg/d & subsequent dose 0.07 μg/kg/d	Intramuscular injection (30 days)	Thickening of tunica albuginea in the ovaries, fibrils increased with connective tissues, congested capillaries and blood vessels. Oocyte and follicle borders were irregular. Thickening in uterine endometrium and myometrium areas.	[27]
7	Tributyltin and BPA	Sprague Dawley rat	TBT (10 & 100 ng/kg/d) BPA (50 μg/kg/d)	Subcutaneous injection daily from PND1-PND16	Decreased number of corpora lutea and antral follicles. Increased number of atretic follicles and presence of cysts.	[63]
8	Cadmium chloride (CdCl2)	Wistar rat	0.09 & 4.5 mg/kg	Oral gavage (90 days)	Cycle length prolongation, degenerated corpus luteum, and reduced number of oocytes.	[56]
9	Cd	Adult Wistar rat	CdCl2 (100 mg/L)	Oral with drinking water (30 days)	Reduced number of preantral follicles, antral follicles, and reduced granulose thickness.	[57]
10	CdCl2	Sprague Dawley rat	5 mg/kg	Oral gavage (6 weeks)	Higher number of antral and atretic follicles.	[35]
11	Polystyrene microplastics	Wistar rat	0.015 mg/kg/d, 0.15 mg/kg/d	Drinking water (90 days)	Thinning of granulose layer of secondary follicles and decreased number of growing follicles.	[64]
12	BPA	Wistar rat	50 & 500 µg/kg/d	Subcutaneous injection (21 days)	Development of cyst in ovarian follicles, loose attachment of theca cells, and necrosis.	[59]

Table 3.

Induction of PCOS by androgens and hormones and associated histological alterations

S. No	Chemical name	Species	Dose	Route & exposure duration		Findings	References
1	Estradiol valerate	Wistar Kyoto rat (adult virgin)	4 mg	Intramuscular injection (single dose)		Many atretic follicles and regressed corpora lutea.	[65]
2	Cortisone	B6A mice (neonatal)	20 μg	Subcutaneous injection (3–6 days)		Ovaries lacked corpora lutea and multiple follicular cysts were present.	[66]
3	Estrogen	Mice	20 μg	Subcutaneous injection (15 days)		Follicular cysts formed and caused anovulation. Corpora luteum was absent.	[67]
4	Dihydrot-estostero-ne (DHT)	Mice	250 μg	Subcutaneous injection (16–18 days of gestation)		Large number of small antral follicles in ovary, thin granulosa cell layer within follicle and thick theca cell layer.	[43]
5	Testoster-one + high fat diet	Wistar rat	1 mg/ 100 g b.wt	Subcutaneous injection (28 days)		Many preantral, atretic, and cystic follicles along with enlargement of stroma.	[68]
6	Testoster-one	Sprague Dawley rat	2.5 mg/d	Subcutaneous injection		Increase in cystic, atretic, and preantral follicles. Decrease in corpora lutea.	[69]
7	DHT	mice	10 mg	Subcutaneous implant (90 days)		Numerous multicystic follicles.	[36]
8	DHEA + High-fat diet	C57BL/6 Mice	6 mg/ 100 g b.wt + 60% high-fat diet	Subcutaneous injection (20 days) + oral diet		More cystic and antral follicles and reduced corpora lutea.	[37]
9	Mifeprist-one (Antiprog-esterone) (RU486)	Wistar rat	5 mg, 7.5 mg, & 10 mg	Subcutaneous Injection (9 days)		Enlarged ovaries with arrested follicular development.  Increased atretic follicular cysts and hypertrophy of granulosa cells.	[70]
10	Letrozole	C57BL/6N female mice	50 μg/d	Subcutaneous injection (5 weeks)		Irregular estrous cycle.  Many cysts in ovaries were observed.	[44]
11	Letrozole	Sprague Dawley rats	1 mg/ kg/d	Oral (15 days)		Increase in number of cystic and atretic follicles. Decreased corpus luteum and increased thickness of peripheral theca layer.	[71]
12	Dehydroe-piandrost-erone (DHEA)	Naval Medical Research Institute (NMRI) rat	6 mg/kg.b.wt	Intraperitoneal injection (20 days)		Increased number of cystic follicles, decreased number of antral follicles and corpus luteum, and anovulation.	[72]
13	Insulin + Human chorionic gonadotr-opin (hCG)	Sprague Dawley rat (70 days old)	Insulin 0.5 to 6 IU/d + hCG3 IU/d	Subcutaneous injection (twice daily for 23 days)		Decrease in luminal epithelial cell numbers and layers, glands and gland conglomerates, and decrease in endometrium and myometrium.	[73]
14	DHEA	C57BL/6 mice	6 mg/100 g.b.wt	Subcutaneous injection (20 days)		Reduction in healthy antral follicles, oocyte, and corpora lutea. Also, granulosa layer of follicles appears thin and degenerated.	[74]
15	DHEA	Sprague Dawley rat	6 mg/100 g.b.wt	Subcutaneous injection (4 weeks)		Increased number of multiple follicular cysts and increased weight of ovary.	[45]
16	Human chorionic gonadotr-opin + insulin	Sprague Dawley rat	3 IU/d (hCG) & 0.5–3 U/d insulin	Subcutaneous injection (22 days)		Numerous cystic follicles, degenerated granulosa cell layer, and reduced corpora lutea.	[75]
17	DHT	C57Bl/6 J female mice	10 mg DHT + diets varied in protein, carbs, and fat content	1 cm SILASTIC brand DHT implant (10 weeks)		Decrease and lack of corpora lutea.	[76]
18	Testoster-one	Zebra fish	0.25, 2, 10, & 100 ng/mL	Dissolved in swimming water (3 days)		Arrest in follicular growth, oligo-ovulation, and ovarian enlargement.	[77]
19	DHEA	Sprague Dawley	7 mg/ kg. b.wt	Subcutaneous injection (90 days)		Higher number of cystic and atretic follicles, hyperthecosis of internal cell layer, and thinning of the ganulosa layer.	[78]
20	Estradiol valerate	Wistar rats	4 mg	subcutaneous injection (single administration)		Reduction in size of ovary, increased size of cysts, degenerative secondary follicles were present, and Corpus lutea absent.	[46]
21	DHT	Sprague Dawley	83 µg/d/kg.b.wt	Subcutaneous injection (90 days)		Larger antral follicles, thickened theca interna, multiple cystic follicles lacking antrum and corpus lutea, and reduction in endometrium thickness.	[78]

Epidemiological studies related to PCOS due to environmental exposure

To date, very limited studies have been performed in humans regarding PCOS due to environmental exposure. BPA, phthalates, phthalic acid esters (PEAs), octylphenol (OP), perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), monobenzyl phthalate (mBz), triclosan (TCS), and organic UV filter -octocrylene are some of the EDCs on which epidemiological studies were formed related to PCOS (Table 4). In current clinical practice, ovarian tissue biopsies are rarely performed solely to diagnose PCOS; therefore, no histological study was found. Instead, the transvaginal ultrasound, a noninvasive technique, is commonly used for the diagnosis of cystic and atretic follicles in humans. However, the conventional and transvaginal ultrasound does not present the overall morphology of the PCOS ovary and may complicate the follicle counting, resulting in misdiagnosis. Therefore, there is a need for the development of noninvasive and more efficient diagnostic methods.

Table 4.

Epidemiological studies on chemically-induced PCOS in humans

S No	Chemical name	Biological material	Findings	References
1	Phthalic acid esters (PEAs), BPA, octylphenol (OP)	Serum	PEA and BPA showed no significant differences in PCOS patients and the control group. However, OP caused insulin resistance in the PCOS group.	[79]
2	BPA	Serum	Significantly higher level (1.2 ng/mL) in serum of PCOS women than nonhyperandrogenic peers.	[80]
3	Perfluorooctanoate (PFOA), perfluor- ooctane sulfonate (PFOS), monoben- zyl phthalate (mBzP)	Serum and urine	PCOS case patients have higher mean PFOA (4.1 μg/L) and PFOS (8.2 μg/L) in serum and lower urine mBzP (7.5 μg/g creatinine).	[81]
4	BPA	Serum	Serum BPA level (1.1 ng/mL) which was higher in adolescent PCOS girls than healthy girls. Ultrasound investigation reveals polycystic ovaries.	[82]
5	BPA	Urine	Higher concentration of BPA (3.34 ng/mL) in urine samples of PCOS women than the non-PCOS women.	[83]
6	BPA	Serum	Serum concentration was 0.202 ng/mL in PCOS women, which correlates positively with testosterone levels and free androgen index.	[84]
7	Di(2-ethylhexyl) phthalate (DEHP)	Follic-ular fluid	Women with PCOS have mean 1.68 ng/mL DEHP in follicular fluid than healthy ones.	[85]
8	Triclosan (TCS)	Urine	PCOS group had higher level of TCS 1.49 μg/g creatinine which positively correlated with LH and LH/FSH ratio.	[86]
9	Organic UV filter -octocrylene	Urine	Positively associated with PCOS in women.	[87]

Ultrasound elastography (UE)

It is one of the emerging techniques used to take images of the tissues with different grades of elasticity and deformation when external force is applied [88]. It supplemented the conventional ultrasound method and diagnosed the tissue morphology, stiffness, and blood flow features. This technique uses shear wave elastography (SWE), which generates transverse waves with the help of acoustic radiation pulses that create vibrations in the tissue. For measuring elasticity in real time, different color codes have been used along with Young’s modulus calculation. Their higher values indicate more stiffness [89]. This technique has successfully been utilized in obstetrics and gynecology [90], and for the detection of thyroid nodes [91], hepatic fibrosis [92], and breast lesions [93]. It has also been applied in clinical diagnosis as well as on rat models of PCOS and correlated positively with the histological data, hormone assay, protein and gene expression data, transcriptomics and proteomics data [94].

Histopathological changes observed in alternative models used in PCOS-related research

Danio rerio (zebra fish) is an aquarium fish and serves as a popular model for research in scientific fields like neurobiology, evolutionary sciences, genetics, and developmental biology and toxicology [78, 95–98]. It also shows the close degree of similarity with the reproductive regulation system of humans and the presence of similar hormones and responses, anatomy, physiology, gene functions, and expressions [98–100]. Ovarian development can be effectively studied using zebra fish model [101]. Therefore, it could serve as the ideal model for investigating embryonic development as well as fertility. Thus, due to its high versatility, it is now frequently used as an alternative model to observe the progression of PCOS by any chemical or drug screening for mechanistic evaluation [77, 102]. Liu et al. [77] reported arrest in follicular growth, oligo ovulation, ovarian enlargement, and reduction in reproduction capacity in zebra fish, characterizing it as PCOS fish. Sudhakaran et al. [102] investigated enlarged ovaries and cysts along with other hormonal changes in zebra fish and described it as the best non-mammalian PCOS model.

Recent advances in histopathology

With the increase in the advancement of technology and microscopy, the use of artificial intelligence (AI) and machine learning (ML) in histology will revolutionize the examination of tissues and cells in clinical observations. The laborious process of tissue-sectioning and processing can be a lengthy process that requires a lot of attention and care. Advances in digital world image processing can be revolutionary in these steps and enhance rapid diagnosis in a limited time with more accuracy. The new technique involves simple new features and advent histology as proposed earlier in one of the studies by a group of researchers, which shows drenching a whole tissue sample with dyes having the fluorescent property that eliminates the requirement of the tissue-mounting step on microscopic slides. They used the ultraviolet surface excitation phenomenon in microscopy and with image processing software to create a virtual hematoxylin (H) and eosin (E) environment [103]. Similarly, many studies have been performed using a more advanced fluorescence microscope [103–106].

Histopathology has already made a significant position in the field of biomedical research in its traditional methods in such a way that it would take years for the advanced techniques to replace it. The techniques will be required to be efficient, practical and accessible to each and everyone just like the simple H and E staining which is highly economical and simple.

The present review demonstrates that androgens, hormones, and environmental toxicant exposure result in reproductive abnormalities that favor PCOS and PCOS-like phenotypes. Moreover, ovarian histopathology in animal models is widely studied while that of the uterus and vagina it is scarcely reported. The morphology of follicles and their counts can serve as important parameters for the prediction of PCOS. Therefore, among the different identification criteria of PCOS, histopathology can be added as one of the identifying features of pathogenesis. However, in humans, wedge resection of ovarian tissue is very scarcely done to diagnose PCOS. Therefore, ultrasound elastography, a newly developed non-invasive technique, is currently in use for PCOS diagnosis. It correlates the histological and molecular data accurately in the disease diagnosis.

Funding

This work was supported by a research grant from the Indian Council of Medical Research (ICMR), New Delhi (Project no. 5/4–5 Ad-hoc/Neuro/231/2020-NCD-1).

Declarations

Conflict of interest

Mehjbeen Javed, Suramya, Shahzad Ahmad, and Sheikh Raisuddin declare that they have no conflict of interest.

Ethical approval and consent to participate

Not applicable.