2. Anatomy and Physiology of Ovarian Follicle

Marija Dundović¹, Lada Zibar² and Mariaelena Malvasi³

(1)

Department of Human Reproduction and Assisted Reproductive Techniques, Clinical Hospital Center Osijek, Osijek, Croatia

(2)

Clinical Hospital Merkur, Zagreb, Croatia

(3)

Department of Anatomical Medical and Histological Science of the Locomotor System, Sapienza University, Rome, Italy

Marija Dundović (Corresponding author)

Lada Zibar

2.1 Follicle Growth

2.2 Relevance of Hormones

2.3 Growth Factors Influencing Ovarian Follicles

2.4 Applications in Assisted Reproduction Techniques (ART)

References

Keywords

FolliculogenesisOocyte maturationGrowth factorsOocyte qualityFollicle recruitmentOocyte morphology

Female reproductive system is a place of origin of a new human life. It produces female gametes, gives a supportive environment for fertilization and embryo development, ultimately it nurtures a growing fetus for 40 weeks of gestation. Women are born with two ovaries placed on either sides of the uterus in the abdomen. They have a complex role on regulating menstrual cycle, producing hormones and monthly giving a single mature oocyte that is ready for fertilization, subsequently giving a couple a chance for pregnancy. It is the intention of this chapter to describe a structure of a single follicle that is a place of human oocyte origin, its development through various stages of woman’s life and menstrual cycle. Also, we will explain the effect of hormones on ovarian tissue and follicle development and how they affect oocyte maturation, what happens to the follicle after the rupture, and what is the significance of various growth factors and chemical signals in physiology of the follicle.

Gametogenesis, oogenesis in this particular case, represents developmental stages of oocytes.

Oogenesis starts before birth when primordial germ cells migrate do gonadal ridge and start their differentiation to oogonia. Oogonia divide by mitosis and reach a number of 7,000,000 by the fifth month of gestation and at the same time some of them differentiate into primary oocytes. By the seventh month of gestation, most of them become atretic and primary oocytes that do survive and enter in prophase of the first meiotic division and become dormant in that form all the way to puberty [1]. Primary oocyte together with its layer of follicular cells makes a primordial follicle. Also, it is well known that a female newborn carries approximately 1–2 million of primary oocytes and that number decreases to 400,000 at puberty and about 400 hundred reach maturation and ovulate. It is interesting to see what happens to all the oocytes that become atretic. Vaskivuo et al. [2] published a paper in 2001 that explained the survival of human ovarian follicles from fetal to adult life and the mechanism of apoptosis in human ovaries. Their results show that a large amount of oocytes degenerate during fetal life through apoptosis, and it is already evident in the 13th week of gestation. Their findings also showed that the rate of apoptosis in the adult ovary increased with growing follicular size and only slightly affected early growing follicles. It is a mechanism for eliminating recruited follicles that do not reach dominant follicle stage. It is also important to notice that during fetal stage apoptosis is a mechanism for eliminating oocytes, but in adult life apoptosis is located in granulosa cells and they play a major role in follicular demise.

Ovaries are specific by their structure since at a monthly basis they go through extreme changes and reorganization. Follicles that contain oocytes go through various stages of development that include primordial, primary, secondary, preantral and antral follicles that ultimately give rise to a large follicle called Graafian follicle (Fig. 2.1). This process is controlled by various hormones and growth factors [3].

../images/462633_1_En_2_Chapter/462633_1_En_2_Fig1_HTML.jpg — Fig. 2.1

Stages of development of human ovarian follicles

Environment in which oocyte matures has been proven to influence the quality of the oocyte and subsequently entire embryo and its implantation potential. This is especially interesting to observe from assisted reproductive techniques perspective where a mature oocyte with excellent metabolic activity plays a crucial role in embryo development and its morphology (Fig. 2.2).

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Oocyte maturity: (a) germinal vesicle, (b) MI—immature oocyte, (c) MII—mature oocyte (author: MarijaDundović)

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Oocyte maturity: (a) germinal vesicle, (b) MI—immature oocyte, (c) MII—mature oocyte (author: MarijaDundović)

Dumesic et al. (2015) [4] have investigated a relationship between follicular fluid and cumulus cells and oocyte health. They found that signaling between oocytes and somatic cells changed intrafollicular environment that controlled follicle growth and which antral follicle was to be selected to ovulate a healthy oocyte. Cellular metabolism is key to a normal meiotic resumption. Maternal ageing or metabolic disease perturb cellular mechanisms within the oocyte, alter macromolecules, and induce mitochondrial mutations which hurts the oocyte.

It is clear that the environment that brings an oocyte to its maturity plays a crucial role and not just in maturity but also in oocye shape and quality (Figs. 2.3 and 2.4). Therefore, there will be a great emphasis on folliculogenesis and how the actual follicles change and what structural changes give rise to a mature oocyte.

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(a) Very irregular shaped germinal vesicle, (b) zona free—no oocyte retrieved within zona pellucida (author: MarijaDundović)

../images/462633_1_En_2_Chapter/462633_1_En_2_Fig4_HTML.jpg — Fig. 2.4

Irregularities in oocyte structure, mainly cytoplasm, seen when performing ICSI (intracytoplasmic injection of sperm) (a and b) (author: Marija Dundović)

2.1 Follicle Growth

Changes in structure and size of a follicle that lead to a mature oocyte happen in a process of folliculogenesis. It is a process that takes approximately 1 year in women, and it includes growth of a recruited primordial follicle that develops into a specialized Graafian follicle which will either ovulate to give a mature oocyte or die by atresia.

Mechanisms that regulate folliculogenesis are under control of changing concentrations of hormones and growth factors, at endocrine level they are regulated by the central nervous system, anterior pituitary, and ovary cascade system [5]. It is important to mention the synergy of these two control systems where growth factors can enhance or reduce the action of certain hormones locally—autocrine or paracrine system of control. Interestingly, very similar mechanisms control early embryogenesis and blastocyst implantation. Atwood and Meethal (2016) [6] examine spatiotemporal regulation of mentioned signals and confirm that hypothalamic-pituitary-gonadal hormones regulate folliculogenesis, follicular quiescence, ovulation, follicular atresia, and corpus luteal functions. After conception and in early embryo development, autocrine and paracrine signaling becomes increasingly important, and these signals are crucial for synthesis of human chorionic gonadotropin which is a proof of embryo existing in female reproductive system. This hormone ultimately has an effect, upon blastocyst arrival in the uterus, on tissue remodeling and supports controlled invasion of the blastocyst in the endometrium.

Regarding the structural changes, human folliculogenesis can be divided into four main steps that include initiation of follicular growth, early follicular growth, selection of one follicle from a pool of selectable follicles, and maturation of preovulatory follicle. In the work of Gougeon (2010) [7], we can see that primordial, transitory, and small primary follicles constitute ovarian reserve, and initiation of follicular growth starts when oocyte nucleus reaches a critical diameter of 19 μm.

After entering the growth phase, granulosa cells proliferate and the oocyte grows at the quickest rate so follicles become secondary follicles with multiple layers of granulosa cells and are around 2 mm in diameter. Selectable follicles measure from 2 to 5 mm and their number ranges from 3 to 11 in an ovary of women 24–33 years of age. Research has shown that the selected follicle is the one that is the healthiest, grows faster than the other ones, and is the most sensitive to FSH (follicle-stimulating hormone). From the time of selection to ovulation, the selected follicle rapidly changes in size from approximately 6 mm up to 18 mm and more, and it is important to point out that in the mid-follicular phase the preovulatory follicle becomes highly vascularized through theca. Theca cells have a very important role in folliculogenesis (Fig. 2.5)—they synthesize androgens, connect granulosa cells and oocyte during development, and provide support for a growing follicle. They are, to simplify, an outer layer of the follicle and have their origin in stromal tissue surrounding the primordial follicle. They are specialized cells that are recruited to surround an activated follicle and provide structural support and acquire a capillary network [8].

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Development of theca cells

Regarding the recruitment of follicles into later stages of development, literature shows the two types: initial and cyclic recruitment. Initial recruitment involves primordial follicles that aren’t under influence of hormones, they remain dormant and this happens continuously throughout life after follicle formation and oocytes have just started to grow. Cyclic recruitment involves antral follicles in growth phase that are under FSH influence, start their recruitment after onset of puberty with grown oocytes, and can undergo apoptosis as a mechanism of cellular death if not selected to reach maturity and ovulate (Fig. 2.6 and Table 2.1) [9].

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History of ovarian follicles

Table 2.1

Differences between initial and cyclic recruitment of ovarian follicles

Stages	Initial recruitment (initiation of growth)	Cyclic recruitment (escape from atresia)
	Primordial	Antral (human: 2–5 mm in diameter: rodents: 0.2–0.4 mm in diameter)
Hormones involved	Not determined	FSH
Default pathway	Remain dormant	Apoptosis
Timing	Continuous throughout life, begins after follicle formation	Cyclic (human: 28 days, rodents: 4–5 days), starts after puberty onset
Oocyte status	Starting to grow, not capable of undergoing germinal vesicle breakdown	Completed growth, competent to undergo germinal vesicle breakdown

In addition, Chang et al. (1998) [10] have studied neutrophil—interleukin-8 system in human folliculogenesis and have come to a conclusion that neutrophils and endogenous interleukin-8 are expressed in the theca vasculature during folliculogenesis in normal ovulatory women, being particularly abundant in cohort follicles undergoing atresia. This led them to propose that there could be a system involving these factors that could function as a mechanism for follicle atresia. Many other studies have shown that granulosa cells that surround the oocyte are essential in determining the survival of follicles. Matsuda et al. (2012) [11] described the regulation of follicular growth and atresia by granulosa cells, and the key factor was the appearance of apoptotic granulosa cells in atretic follicles which led to completely apoptotic granulosa in progressed atretic follicles with a disruptive granulosa layer. The deprivation of key survival-promoting factors or stimulation by death ligands caused apoptosis. Cumulus cells that directly surround the oocyte originate from granulosa cells and have a great role in oocyte maturation and growth (Fig. 2.7).

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(a) Cumulus oocyte complex (COC)—the oocyte is visible and surrounded with cumulus cells, (b) oocyte derived from COC in a

Transcriptome analysis (mRNA expression) in cumulus cell can indicate quality of the environment the oocyte was exposed to while maturing and give rise to some biomarkers that can be an indicator of oocyte and later embryo fitness resulting in healthy pregnancies [12].

We can say that in terms of structural changes, follicular antrum or cavity certainly has very expansive growth and makes the majority of volume of preovulatory Graafian follicle (Figs. 2.8, 2.9, and 2.10). Rodgers and Irvine-Rodgers (2010) [13] have described the process of follicular antrum and fluid formation in a way that granulosa cells produce hyaluronan and proteoglycan versican generates osmotic gradient that draws fluid from thecal vasculature (Fig. 2.11). Aquaporins from granulosa cell could be involved in water transport into the follicle. Follicular fluid is of complex composition that has a significant relevance for developing oocytes. Ambekar et al. (2013) [14] have carried series of tests in order to characterize proteome of human follicular fluid. They report identification of 480 proteins, 320 of which have never been described before in the follicular fluid. The identified proteins include growth factors, hormones, signaling receptors, enzymes, antibodies, and complement. This will help in understanding the process of follicular maturation and in case of IVF patients, some proteins can be used as biomarkers for oocyte quality and eventually IVF success.

../images/462633_1_En_2_Chapter/462633_1_En_2_Fig8_HTML.png — Fig. 2.8

Structure of Graafian follicle

../images/462633_1_En_2_Chapter/462633_1_En_2_Fig9_HTML.png — Fig. 2.9

Structure of cells that surround an oocyte in a Graafian follicle

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Follicle with oocyte

../images/462633_1_En_2_Chapter/462633_1_En_2_Fig11_HTML.png — Fig. 2.11

Ovarian follicle recruitment

Jozwik et al. (2006) [15] aimed to determine amino acids, ammonia, and urea concentrations in human ovarian follicular fluid and compare it to concentrations found in plasma. They found threefold increase of glutamate concentration (Table 2.2) in follicular fluid, higher concentrations of ammonia in follicular fluid, and no significant difference in urea concentration. These findings may reflect utilization of amino acids and transport characteristics of the follicular cells which can be an important factor in embryo development.

Table 2.2

Comparison of concentrations of ammonia and urea in preovulatory fluid to concentrations of ammonia in whole blood and urea in plasma

Substance	Concentration in blood or plasma	Concentration in follicular fluid	P-value	Difference between blood or plasma and follicular fluid	Blood or plasma-to-follicular fluid ratio
Ammonia (μΜ)	22.11 ± 1.96 (whole blood)	38.87 ± 2.23	<0.001	−16.77 ± 2.42	0.58 ± 0.05
Urea (mM)	3.37 ± 0.18 (plasma)	3.36 ± 0.22	0.911	0.015 ± 0.063	1.01 ± 0.02

When speaking of follicular fluid composition and general follicle as a microenvironment for a developing oocyte, it is important to note the existence of oxidative stress in the form of reactive oxygen species (ROS) as products of metabolism within the follicle. Melatonin, generally known as a pineal gland secrete that regulates circadian rhythm and reproduction, is found to be an excellent free radical scavenger in the ovarian follicle. Tamura et al. (2013) [16] summarized new findings related to beneficial effects of melatonin which enters the follicular fluid via blood. It acts as a potent antioxidant (Fig. 2.12) and contributes to oocyte maturation and embryo development.

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Roles of melatonin

This chapter also demonstrated usage of melatonin as an infertility treatment since research has shown that it elevates fertilization and pregnancy rates.

With the usage of scanning and transmission electron microscope (SEM and TEM) Makabe, Naguro, and Stallone (2006) [17] were able to demonstrate the fine relationship existing between oocyte and follicular cells during various stages of follicular development (Figs. 2.13, 2.14, 2.15, 2.16, and 2.17).