32.2.1 Folliculogenesis

Throughout folliculogenesis (Figure 32.1), the follicular somatic cells proliferate, and the oocyte grows in size. Gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)) released from the anterior pituitary and steroid hormones produced from the follicles are the main regulators of this event.

Figure 32.1 Folliculogenesis

(A) A primordial follicle, with the oocyte at the GV stage, is surrounded by a layer of flat granulosa cells and a basal membrane.

(B) A primary follicle. Granulosa cells become cuboidal. Oocyte grows in size.

(C) Secondary follicle. More than two layers of granulosa cells. Theca cells begin to gather around the basal membrane.

(D) Antral follicle. A fluid-filled cavity is formed. Two categories of granulosa cells can be distinguished. Cumulus cells that surround the oocyte and mural granulosa cells that grow closer to the basal membrane of the follicle.

(E) A mature oocyte after its release from a fully grown antral follicle. The oocyte does not have a nucleus anymore. Now it is arrested in Meiosis II and has extruded the first polar body.

Figure 32.1 was created with Biorender.com

Primordial follicles are the most immature stage and are comprised by an oocyte arrested in prophase I (germinal vesicle, GV stage), surrounded by a layer of flattened granulosa cells and a basal membrane. Primordial follicles remain arrested at this stage and several factors are considered to regulate their activation [Reference Williams and Erickson7].

Upon activation, granulosa cells change into a cuboidal shape and the follicle starts expressing receptors for FSH (FSHR). When a full layer of cuboidal granulosa cells is formed, the follicle is named primary. Besides the expression of FSH receptors, at this stage, the follicle growth is responsive but not dependent on gonadotropins. At this stage, also the formation of zona pellucida occurs and an increase in the size of the oocyte is noticed. Gap junctional communications are formed between the granulosa cells and the oocyte for exchange of important factors for oocyte growth.

Granulosa cells start proliferating, forming two layers of granulosa cells. This proliferation leads to the next stage of folliculogenesis, the secondary follicle. The formation of two full layers is accompanied by the recruitment of theca cells around the basal membrane, as well as small blood vessels that supply the growing follicle with gonadotropins and growth factors [Reference Williams and Erickson7].

Following the secondary stage, granulosa cells continue their proliferation and the follicle and oocyte grow in size. This growth is accompanied by the expression of more FSH receptors in the granulosa cells and LH receptors in the theca cells. A fluid-filled cavity called the antrum is formed within the layers of granulosa cells, creating two distinct populations of granulosa cells, the ones surrounding the oocyte (cumulus cells) and the ones that grow closer to the basal membrane (mural granulosa cells) [Reference Hennet and Combelles8].

Follicle and oocyte sizes increase dramatically during folliculogenesis. Follicle diameter ranges from about 30–40 μm in the primordial stage. Primary stage is reached when the follicle grows to 50 μm. Secondary stage is noticed between 100–200 μm after which the signs of antral formation are evident between 180–250 μm. Ovulation occurs when the antral follicle reaches around 20 mm. Oocyte sizes account for 19–30, 40–80, 80–90, and 100–110 μm for each stage respectively [Reference Gougeon9–Reference Telfer11].

32.2.2 Hormonal Cyclicity

When antral follicles reach 2–5 mm in diameter, the cyclic recruitment begins, under the effect of gonadotropins stimulating the beginning of the menstrual cycle and preparing the uterus for implantation (Figure 32.2). In women, the median menstrual cycle lasts 28 days, is regulated by the cyclic release of gonadotropin-releasing hormone (GnRH) and begins with the shedding of the endometrium. GnRH produced by the hypothalamus stimulates the gonadotropin cells of the anterior pituitary to produce FSH and LH. At the beginning of the cycle, the expression of FSH has a positive effect on the proliferation of the granulosa cells and leads to the production of estradiol from the growing follicles. The high expression of estradiol results in negative feedback towards the gonadotropin cells, which now produce less FSH. The low levels of FSH affect the follicular growth of the follicles. Only one follicle will continue to grow, the most sensitive to FSH, while the rest cannot respond to the low stimulus and undergo atresia [Reference Hawkins and Matzuk12]. The selected follicle now produces high levels of estradiol which are responsible for the elevated expression of LH. LH stimulates the production of progesterone, which has positive feedback and is responsible for the mid-cycle increase of FSH. Following 10–12 hours after a peak of LH, ovulation occurs in the middle of the cycle (approximately Day 14) [Reference Reed and Carr13]. A mature oocyte is released from the antral, fully grown follicle, into the fallopian tube and is ready for fertilization. The high levels of estradiol have a proliferative effect on the endometrial cells, while progesterone leads to the decidualization of the endometrial cells, a process important for a successful implantation.

Figure 32.2 The menstrual cycle.

Day 1 of the cycle begins with menstruation. Under the effect of follicle stimulating hormone (FSH) a number of small antral follicles are recruited. A rise in the oestradiol levels leads to a decrease in FSH. Only one follicle, the most sensitive to FSH will continue to grow, producing high levels of oestradiol. High levels of oestradiol lead to a peak in luteinizing hormone (LH) levels. On Day 14 of the cycle and 10–12 hours following the LH peak, ovulation occurs. The oocyte is released from the follicle into the fallopian tube. The remaining follicle becomes the corpus luteum, producing high levels of oestradiol and progesterone that have a proliferative action on the endometrium. Corpus luteum has a lifespan of about 14 days. If fertilization does not occur, decreased levels of progesterone and oestradiol can no longer support the endometrium, which sheds, and a new cycle begins.

Figure 32.2 was created with Biorender.com

The remaining follicle will transform now into a corpus luteum, which under the effect of FSH and LH, will continue the production of progesterone, estradiol, and inhibin, and inhibit the growth of other follicles. A corpus luteum has a lifetime of about 14 days. If fertilization does not occur, the levels of steroid hormones progressively decrease, leading to rising levels of gonadotropins. As the levels of FSH start increasing, recruitment of another group of antral follicles is initiated with the new menstrual cycle [Reference Hawkins and Matzuk12].

32.2.3 The Role of Androgens

Androgens have a very important role in the cyclicity of the cycle and normal folliculogenesis, as they are involved in the biosynthetic pathway of estrogens. In women, dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), and dihydrotestosterone (DHT) are available in the serum. DHEAS and DHEA are predominantly derived from the adrenal glands, whilst A4, T, and DHT are equally derived from both the ovaries and the adrenal glands. T and DHT are the only active androgens which can directly exert their action through the androgen receptor (AR), while DHEAS, DHEA and A4, need to be converted to T or DHT to promote androgenic effects. Estrogens are produced through a multistep biosynthetic pathway, and T is the last step before their formation. Estrogens are produced making use of the two cells–two gonadotropins system (Figure 32.3). Theca cells, which express LH receptors, use cholesterol as a base to produce progesterone and pregnenolone, in response to LH. They are converted to DHEA and then to A4. Theca cells use the 17βHSD enzyme to convert A4 to T. The enzyme for the conversion of T to estradiol is missing in the theca cells, and so, T defuses to the granulosa cells, where it gets converted to estradiol by aromatase. Since biosynthesis of estradiol needs androgens and estradiol is important for folliculogenesis and the cyclicity of the cycle, androgens play an important role in the normal physiology of the ovary [Reference Walters and Handelsman14].

Figure 32.3 Two cells–two gonadotropins system.

Under the binding of LH through their receptors, theca cells synthesize androstenedione using cholesterol, with many intermediate enzymatic steps. Androstenedione is then converted to testosterone, with the 17βHSD enzyme. Testosterone diffuses then to the granulosa cells, where aromatase is present. Aromatase activity is induced with the binding of FSH on its receptor on the granulosa cells. Aromatase converts testosterone to oestradiol.

Figure 32.3 was created with Biorender.com

Androgens can mediate their effects indirectly through T conversion to estradiol, or directly, through their binding to the AR. AR is present in the brain, granulosa, and theca cells of human follicles, stromal cells, and corpus luteum. It has been proposed that androgens have a stimulatory role in the follicles to express the FSH receptor, making them more sensitive to FSH [Reference Zhang, Zhang and Shu15].

32.3.1 Ovarian Physiology and Anatomy

Testosterone treatment during gender-affirming procedures in persons suffering from gender dysphoria has an effect on the normal physiology of the ovary. Testosterone treatment may occur with many different formulations and supplements, several doses and frequencies depending on the supplement and route of administration. Increased testosterone levels alter the secretion of gonadotropins and ovarian hormones, important for folliculogenesis and cyclicity of the menstrual cycle. Testosterone supplementation also leads to cessation of the menstrual cycle, which is one of the most wanted effects for transmen. The time to amenorrhea may vary depending on the individual and the type of the treatment, but usually occurs within 6 months, and most patients experience cessation of cyclicity within 1 year. Frequently, induction of complete amenorrhea is only achieved by additional prescription of a progestin, preferentially lynestrenol due to its strong affinity to the endometrium. Effects are largely reversible if the treatment is discontinued and the menstrual cycle may recur, as long as oophorectomy has not taken place. Most of the time, testosterone treatment causes anovulation by the negative effect on the hypothalamic-pituitary axis but not always, and spontaneous pregnancies may occur in transmen that have intercourse with cismen or transgender women (pre-GRS) [Reference De Roo, Tilleman, T’Sjoen and De Sutter2]. Thus, testosterone treatment should not be regarded as a contraceptive [Reference Light, Obedin-Maliver, Sevelius and Kerns16,Reference Krempasky, Harris, Abern and Grimstad17]. This is of great importance, as testosterone is known to have potential teratogenic effects and its use should be avoided during pregnancy.

Cessation of testosterone can restore the normal cyclicity of the menstrual cycle following an average of 4 months [Reference Irwig and Legato18] and pregnancies have been achieved in transmen wishing to have their own biological children. For adult transmen that wish to preserve their fertility, controlled ovarian hyperstimulation and consecutive oocyte collection and cryopreservation is proposed either prior to the start of testosterone treatment or after discontinuation of the treatment. Discontinuation of testosterone supplementation has been proposed for at least 3 months before ovarian stimulation [Reference De Roo, Tilleman, T’Sjoen and De Sutter2]. Transmen respond well to stimulation, as the ovarian reserve is not affected. A study by Amir et al. compared the antral follicle count (AFC), oestradiol peaks, oocyte yield, and oocyte maturation between transmen with previous testosterone treatment and testosterone native transmen with fertile ciswomen that served as oocyte donors. No statistically significant difference was found between the different populations in the above-mentioned characteristics. The discontinuation of the treatment ranged between 5 and 21 months, while the length of the treatment seemed to affect the number of good-quality blastocysts frozen [Reference Amir, Yaish and Samara19]. The same trend was noticed in the study of Adeleye et al., as the patient with the longer testosterone exposure retrieved the lowest number of good-quality embryos [Reference Adeleye, Cedars, Smith and Mok-Lin20]. These results propose a possible inferior quality of the retrieved oocytes and embryos when the treatment length is longer but yet, no clear guidelines have been established on the correct dosing and optimal length of the treatment to avoid potential negative effects on the fertility outcome.

The effect of testosterone has also been explored in ovarian tissue after surgical removal in the course of gender-affirming surgery. Ovaries from 40 transgender patients under continuous testosterone treatment were removed and prepared for cryopreservation. Immature oocytes were collected during the process of tissue cryopreservation and underwent in vitro maturation. Maturation rate was 38.1% showing potential to resume meiosis II, a prerequisite event for fertilization. Following freezing and thawing, the oocytes displayed a normal spindle formation and proper chromosomal alignment, both markers of good oocyte quality [Reference Lierman, Tilleman and Braeckmans21]. In a similar study, conducted by De Roo et al., patient characteristics, type and length of testosterone treatment did not affect the maturation efficiency of GV oocytes collected during tissue cryopreservation, but AMH levels seemed to be positively correlated to the maturation rate [Reference De Roo, Lierman and Tilleman22]. Nevertheless, the fertilization of these oocytes remains an important next step to further establish this technique, as exposure to continuous supraphysiological testosterone supplementation may have deleterious effects on the fertilization process. A recent study by Lierman et al., explored the ability of these oocytes to be fertilized and monitored subsequent embryo development. In total, only 23.8% of 1903 retrieved Cumulus oocyte complexes (COCs) matured. Following vitrification and subsequent thawing, 139 oocytes were injected with a single sperm. Interestingly, 34.5% of the injected oocytes were normally fertilized but aberrant cleavage patterns were observed in 45.8% of the zygotes (including no extrusion of second polar body, direct cleavage and reverse cleavage). Early embryo arrest occurred in 91.7% of the zygotes, resulting only in one blastocyst on Day 5 [Reference Lierman, Tolpe and De Croo23]. However, data from cancer patients undergoing these procedures to maintain fertility are promising, as pregnancies and live birth rates have been reported after maturation of the GV oocytes and subsequent fertilization [Reference Lierman, Tolpe and De Croo23–Reference Prasath, Chan and Wong26].

There is an ongoing debate on whether testosterone treatment can induce polycystic ovary syndrome (PCOS) in the ovary [Reference Moravek27]. Ovaries collected following supraphysiological supplementation of testosterone display in some cases the phenotype of PCOS patients. PCOS is the most common metabolic syndrome in cisgender women of reproductive age. The syndrome is characterized by anovulation, hirsutism, and menstrual disturbances, but not all of them are always present and the pathophysiology of this disease is not clearly determined. Typical endocrine abnormalities involve excess expression of LH and androgens in the serum whilst concentrations of FSH appear normal or reduced [Reference Franks, Stark and Hardy28]. Increased concentration of serum LH leads to overstimulation of the theca cells in the follicles, to produce androgens. Nevertheless, as the FSH levels remain low, the granulosa cells are not stimulated enough to make use of the aromatase and convert testosterone to estradiol. As a result, the environment of the follicle remains highly androgenic and there is no selection of a dominant follicle, leading to atresia and anovulation in many cases. Nevertheless, anovulation has been noted in women with no hyperandrogenism or high LH levels in the serum [Reference Trikudanathan29]. A distinct characteristic of PCOS is the formation of many (>12) small follicles (2–9 mm) in the ovaries, as seen by transvaginal ultrasonography and histopathology [Reference Azziz30]. Despite as yet being inconclusive, it seems that androgenic environment is linked to PCOS. Since transmen that undergo testosterone treatment are exposed to high levels of androgens, some studies have evaluated the effect of testosterone on the morphology of the ovarian tissue in transmen undergoing oophorectomy. Pache et al. reported an altered morphology following histopathology in the ovaries of transmen undergoing testosterone treatment compared with control, non-stimulated patients. The changes involved enlarged ovaries, collagenization of ovarian cortex, theca interna luteinization, and stromal hyperplasia [Reference Pache, Chadha and Gooren31]. De Roo et al. described follicular distribution in the ovaries of transmen, which resembled that of cis women. Patients were treated with testosterone for more than a year and ovaries were collected while they underwent ovariectomy for gender-affirming surgery and ovarian tissue cryopreservation. During ovarian tissue cryopreservation, cumulus oocyte complexes (COCs) were collected following medulla manipulation and underwent in vitro maturation. These COCs, which probably escape antral follicles during tissue dissection, contain quite a high number, totalling about 27 as a mean in each patient, which is higher compared to that noticed in cancer patients (a mean of 8.1 COCs). This high number could have been induced by the FSH and LH downregulation, resulting in anovulation and subsequent accumulation of antral follicles [Reference De Roo, Lierman and Tilleman22]. Another study by Grynberg et al. included 112 transmen that underwent at least 6 months of testosterone treatment and evaluated the histology of the ovaries following oophorectomy. Ovaries of 89 out of 112 transgender patients (79%) displayed PCOS phenotype. Histopathologic criteria for PCOS ovaries were observed again and included collagenization of the cortex, luteinization and hyperplasia of stroma cells, follicular atresia, and more than 12 antral follicles in the ovary. Furthermore, patients with PCOS phenotype also displayed enlarged ovaries [Reference Grynberg, Fanchin and Dubost32]. Nevertheless, this high incidence of PCO morphology is not reported by all studies [Reference Grimstad, Fowler and New33,Reference Ikeda, Baba and Noguchi34], while transvaginal ultrasound data from transmen showed no significant difference in the occurrence of PCO morphology in adults exposed to high androgen levels and control patients [Reference Caanen, Schouten and Kuijper35]. However, textural profile analysis in cortical pieces from transmen and oncological patients by De Roo et al. demonstrated a stiffer ovarian cortex originating from transgender patients. These results confirm altered physical properties in the outer part of the cortex due to testosterone supplementation. This could be explained by the presence of androgen receptors in the epithelium, which can stimulate cell proliferation. This is an important observation, as the cortical environment can have a great effect on the activation, growth, and ovulation of the follicles [Reference De Roo, Tilleman and Vercruysse36]. Altered extracellular matrix has also been described in PCOS patients, with a more rigid cortex, causing an aberrant follicular development, as PCOS patients are characterized by a high number of small antral follicles and no pre-ovulatory follicles [Reference De Roo, Tilleman and Vercruysse36].

Fallopian tube function might also be affected by ovarian physiology, as paracrine and endocrine factors from the ovary have been found in high concentrations in the fallopian tube following ovulation. Androgen receptors have been found in the fallopian tube and high levels of testosterone may result in alteration of cilia genes expression and cilia movement [Reference Jackson-Bey, Colina and Isenberg37].