4.1 Introduction

Infertility is a common problem with significant psychological, economic, and medical implications. It is estimated to affect one in eight women [1]. There are approximately 70 million infertile couples worldwide, most of whom reside in developing countries [2]. In the USA, the CDC estimated that of married women ages 15–44, 1.5 million (6%) are infertile, and of all women ages 15–44 years old, 7.4 million have used infertility services [3]. Two of the most common mechanisms of infertility are failure of fertilization and failure of implantation. Fertilization is the union of a sperm and an oocyte, while implantation is the attachment of a developed blastocyst to the uterine endometrium. Although the majority of our knowledge on this subject relies on other mammalian fertilization models, with advancing technology and a deeper understanding of gamete biology, transport, and implantation, our knowledge and available treatment options and medical care for infertile couples have significantly improved.

In this chapter, we will summarize the current evidence on molecular and cellular interactions that are essential for successful fertilization and implantation and their relevance to current clinical reproductive medicine.

Clinical Case

A 27–year-old woman is undergoing in vitro fertilization (IVF) for idiopathic infertility. Her investigation revealed no abnormality. Her partner has normal sperm parameters. She undergoes in vitro fertilization (IVF). Eleven oocytes are obtained with fertilization resulting in 9 embryos. Seven embryos develop to the blastocyst stage. One is transferred but no pregnancy occurred. She has 2 subsequent frozen thawed cycles with no pregnancy. She has 2 remaining embryos. She consults you for an explanation and plan.

4.2 Sperm Transport in the Male Reproductive Tract

A mature and healthy spermatozoon is essential for reproductive success. The male gonads, commonly referred to as testes, have two important functions for reproduction. The first is to produce a constant supply of spermatogenic stem cells and mature them through meiosis into spermatozoa , a process called spermatogenesis . The second is to produce sex hormones , which have diverse metabolic and reproductive functions essential for spermatogenesis and male endocrinology. Even though these functions are accomplished in two distinct anatomical compartments of the testes, the interstitial compartment and seminiferous tubules, respectively, they are very reliant on each other.

Seminiferous tubules are the most abundant anatomical component of the testicle. Each testicle contains approximately 360 m of seminiferous tubules. This convoluted tubular structure is lined by Sertoli cells, which act to nourish and aid the process of spermatogenesis . In between the Sertoli cells, germline stem cells undergo differentiation into spermatogonia and ultimately mature into spermatozoa . In order to protect spermatogenesis , Sertoli cells form tight junctions between each other called the blood-testis barrier. This barrier helps regulate the entry of hormones and nutrients, and most importantly, prevents an immune response to the developing spermatozoon .

The interstitial compartment of the testes contains Leydig cells, blood vessels, myofibroblastic cells, and nerves, which all contribute to spermatogenesis via hormone production and control of the local environment. Leydig cells produce the majority of the androgens needed for spermatogenesis and male reproductive function.

It is well known that ultimate control of spermatogenesis and production of gonad-derived steroid hormones comes from the anterior pituitary gonadotropins LH and FSH . Luteinizing hormone (LH) , secreted by the anterior pituitary gland, stimulates Leydig cells to produce androgens. Intra-testicular testosterone exerts its effect through binding to testosterone receptors found in Sertoli, Leydig, and peritubular cells.

The other gonadotropin, follicle-stimulating hormone (FSH ) , stimulates Sertoli cells to produce androgen-binding protein, which binds androgen and optimizes local androgen levels that are essential for spermatogenesis . Other secretory functions of the Sertoli cell include, but are not limited to, production of aromatase, which converts androgens to estrogens, and release of inhibin/activin, which regulate the release of FSH.

Spermatogenesis takes approximately 72 days. After production in the seminiferous tubules, spermatozoa are transported to the rete testis, efferent ducts, caput epididymis, corpus epididymis, and finally to the cauda epididymis, where they are stored until ejaculation [4]. The epididymis is not merely a storage site for spermatozoa; in fact, it is the site where spermatozoa undergo physiological modifications that result in the acquisition of progressive motility and the ability to undergo capacitation [5]. Spermatozoa in the epididymis contain free sulfhydryl groups rather than disulfide bonds, and the oxidation of those free groups helps stabilize the sperm structures [6]. Spermatozoa are able to become motile in the epididymis, but their motility is suppressed by acidification of the epididymal lumen [7]. During the transport in epididymis the sperm interacts with the seminal fluid and enriched with cholesterol, glycophospholipids. This process decreases the sperm membranes fluidity to prevent premature acrosomal reaction [8]. Activation of cannabinoid receptors on the sperm surface also helps to keep spermatozoa in an immotile state [9]. Additionally, various secretory proteins in the epididymal lumen may contribute to sperm maturation [10]. Extracellular vesicular proteins containing MIF and aldose reductase can be transported from the apical surface of the epididymal cells to spermatozoa thereby allowing spermatozoa to acquire new surface proteins that are controlled by androgen involved in further maturation of the spermatozoa [11].

Human spermatozoa seem to rely on glycolysis for ATP production and the activity of glycolytic enzymes is modified during epididymal maturation [6, 12].

With ejaculation , the spermatozoa are rapidly transported from the epididymis through the ductus deferens where they are mixed with seminal vesicle and prostatic secretions. In fact, only 5% of the ejaculate volume is composed of spermatozoa. The bulk of the ejaculate is composed of secretions from the seminal vesicle (70%) and prostatic secretions (25%). Less than 1% of the ejaculate volume comes from the bulbourethral glands.

Seminal vesicle secretions have an alkaline nature and contain rich nutritional substances, which serve as an initial energy source for spermatozoa , and proteins responsible for the “coagulum” formation, which is important to stabilize the deposited sperm in the female reproductive tract. An important component of prostatic secretions is a prostate-derived serine protease, prostate-specific antigen (PSA), responsible for liquefaction of the coagulum so that the sperm can swim freely once in the vaginal vault.

Components of ejaculate differ among individuals, as well as within a single individual. Initially, prostatic, non-coagulable secretions are followed by the sperm-rich non-coagulable component. Subsequently, seminal vesicle secretions predominate, which results in coagulation of the ejaculate. The initial non-coagulable spermatozoa have the advantage of entering the female reproductive tract earlier than the spermatozoa that are trapped in the coagulum. An average ejaculate contains 200–500 million spermatozoa, most of which are mature and motile [13].

In addition to spermatogenesis , penile anatomy is also important for reproductive success. Various anatomical abnormalities like penile curvature and uncorrected urethral openings can cause male factor infertility .

4.3 Sperm Transport in the Female Reproductive Tract

After ejaculation , mature spermatozoa are deposited near the external cervical os, or in the anterior vaginal fornix. The ejaculate coagulates within a minute and forms a loose gel in humans rather than a compact gel that is observed in rodents. The principal proteins involved in coagulation are semenogelin I and semenogelin II, which are secreted from seminal vesicles [14]. The gel formation minimizes the back-flow of deposited sperm into the vagina and protects spermatozoa against the harsh vaginal environment, yet a median of 35% of spermatozoa are still lost through retrograde flow down and out of the vagina [15]. The coagulate is then enzymatically digested in about 30–60 min. PSA is the main enzyme that is involved in this digestion. Although the alkaline nature of the seminal plasma protects the spermatozoa from the acidic vaginal environment, this protection is only transient and sperm is protected for 2 h. Alkaline environment of the ejaculate increases the pH of the sperm cytoplasm and sperm become mobile [16]. Spermatozoa have to leave the coagulant quickly to escape from inactivation or immune attack; they are only able to remain motile in the vagina for a few hours. Within a few minutes of vaginal deposition, human sperm begins to leave the seminal pool and enter the cervical canal [17]. Cervix and the uterotubal junction are two mechanical barriers that spermatozoa need to breach. The amount of sperm that transverse the cervix depends on multiple factors, including sperm concentration, morphology, motility, molecular characteristics of the sperm surface, as well as genetic factors of the spermatozoa. Only the highest quality spermatozoa can breach these barriers, which is an evolutionary protective mechanism against polyspermy [18]. Majority of the remaining spermatozoa that do not enter the female reproductive tract are either inactivated by the acidic environment or phagocytized.

As the spermatozoa enter the cervical canal, they encounter the cervical mucus. At the time of ovulation, under the influence of estrogen, the cervical mucus is highly hydrated and the cervical pH becomes alkaline, which are optimal for spermatozoa transport and activation [19]. Failure of these physiological changes are of clinical importance since hyperandrogenic women have acidic cervical pH likely contributing to infertility [20]. Additionally, coitus on the day of maximal mucus hydration is more closely related with pregnancy success than using other indicators such as basal body temperature [21]. If the conception does not occur during this period, then, under the effect of progesterone, the cervical mucus gets thicker and creates an unfavorable environment for passage of spermatozoa.

Cervical mucus acts as a selective gate for sperm transport. Cervical mucus also acts as a barrier to abnormal sperm transport, selecting for the more vigorous and motile sperm [19, 22]. Additionally, due to flow of uterine secretions, cervical mucus is aligned to form a microarchitecture in cervical mucosal grooves. The microarchitecture is thought to guide spermatozoa to the uterus [23]. Spermatozoa carrying fragmented DNA are filtered in the cervical mucus, likely as a result of their inadequate membrane surface characteristics and motility. This selection helps to prevent sperm with poor DNA quality to reach to oocyte that would otherwise result in poor quality embryos [24].

Like the vagina, the cervix also contains immunologic barriers such as immunoglobulins, the complement system, and neutrophils, that together act to combat entry of microorganisms. However, with the aid of the seminal plasma proteins which coat spermatozoa against immune attack, highly motile spermatozoa can escape this barrier without difficulty.

As sperm enter the uterus, they are able to quickly transverse the cavity. Uterine smooth muscle contractions, which are directed caudally, increase in intensity during the late follicular phase [25]. The smooth muscle contractions appear to be limited to the layer of myometrium directly beneath the endometrial layer [26]. Thus, it appears that both active flagellar beating and uterine contractions aid in transportation through the uterine cavity. It was also suggested that these contractions could draw watery cervical mucus into the uterus. As the uterine lumen is small in volume and cervical mucus is plentiful during the peri-ovulatory phase, this would easily drag the spermatozoa through the uterine lumen [27].

The final part of the sperm transport is the passage through the uterotubal junction. In most mammals, it is narrow and may be filled with mucus. Although mucus has been shown in the uterotubal junction in humans, this does not appear to be a rate-limiting factor for sperm transport [17]. Only a few spermatozoa traverse the oviduct at any given time and move towards the ampulla, the most common site of fertilization. Movement is facilitated by oviduct contractions and fluid flow.

4.4 Sperm Capacitation

Although mature, spermatozoa are not able to fertilize an oocyte immediately after spermatogenesis . A series of molecular and physiological events that begin in the cervix and occur in the female reproductive tract give the spermatozoa the ability to fertilize the oocyte; this process is known as capacitation [28]. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, membrane hyperpolarization, cholesterol loss, and changes in the phosphorylation state of many proteins [29]. Capacitation normally takes place within the female reproductive tract; however, it can be mimicked in the laboratory by incubation of sperm in a defined medium containing bicarbonate, a cholesterol acceptor like albumin, calcium, and an energy source such as pyruvate, glucose, or lactate [24].

Spermatozoa have two principal structural compartments, the head and the tail, which behave relatively independent from each other during capacitation. However, some studies suggested that they are functionally related, and as capacitation starts in the tail (hyperactivation), it subsequently triggers capacitation in the other compartment, the sperm head (acrosomal reaction) [24].

Hyperactivation, which occurs in the tail (or flagellum), increases the speed, velocity, and rate of flagellum beating when compared to spermatozoa prior to capacitation. The spermatozoa exhibit asymmetrical flagellar beats with increased amplitude of principal flagellar bend and a typical high velocity figure-eight pattern of movement. This pattern generates enough propulsive power to allow spermatozoa to navigate through the viscous oviduct fluid and penetrate the outer layers of the oocyte, namely, the cumulus oophorus and corona radiata [30]. Hyperactivation is triggered by an alkaline environment and subsequently increased intracytoplasmic calcium levels. Calcium enters into the spermatozoa from both the external milieu by sperm ion channels and release from intracellular stores [16, 31]. In addition to calcium alterations, changes in membrane permeability to potassium, sodium, protons, bicarbonate, and chloride contribute to sperm capacitation [6]. Mutations of these ion channels were found to be associated with male subfertility [32].

The spermatozoon head, where the “acrosomal reaction” occurs, is further divided into two parts: the acrosomal region and nucleus. The acrosomal region contains various enzymes that play a critical role in penetrating the zona pellucida and fusion with the oocyte, whereas the nucleus carries the paternal genetic code. The acrosomal reaction is the last step of capacitation and occurs when the spermatozoon approaches the oocyte, normally in the ampulla of the oviduct. It is described as acrosomal exocytosis and is a prerequisite for fertilization because it allows the sperm to penetrate the oocyte zona pellucida. The acrosome contains various hydrolytic enzymes like proteases, arylsulfatases, phosphatases, phospholipases, hyaluronidase, and acrosin. Calcium is obligatory for acrosomal exocytosis. There are various theories on the source of calcium increase needed for acrosomal exocytosis. One theory is that the depletion of calcium from the acrosome activates Ca++ channels, which allows the entry of Ca++ from the surrounding medium [33]. Other theories suggest that the exposure of the sperm head to the zona pellucida proteins or progesterone releases calcium stores, which are found in the redundant nuclear envelope at the posterior end of the sperm head. The increase in calcium starts from the head-tail junction and the calcium wave propagates towards the head [31]. An increase in calcium levels leads to a concomitant increase in cAMP levels, with the release of the vesicular fusion proteins; the acrosome completely discharges its enzymatic contents to penetrate the zona pellucida and fuses with the oocyte plasma membrane. The fertilization process will be discussed further, after a discussion of the oocyte.

An understanding of the capacitation process has useful clinical applications in some couples with infertility. Since the fertilizable life of a sperm is decreased once it has been capacitated, with evaluation of acrosomal status and in vitro capacitation, the timing of conception can be precisely controlled and aid in the treatment infertility.

4.5 Oocyte Development

4.5.1 Early Follicular Development

During early embryonic development, at the seventh week of gestation, gonadal stem cells derived from the yolk sac endoderm migrate to the gonadal ridges. After this migration, primordial germ cells undergo mitosis and substantially increase in number becoming “oogonium.” During embryonic development, oogonium form nests and are not initially surrounded with somatic cells. These oogonial cells are then individually surrounded with flat pre-granulosa cells, forming primordial follicles. The process of forming oogonium from primordial germ cells continues until around the third trimester. Concomitantly, around the 11th week of gestation, oogonia begin to enter their first meiotic division to become primary oocytes, but this division gets arrested at the dictyotene stage. Primary oocytes will stay arrested at the dictyate stage of prophase I until postnatal life when the female enters menarche. With each menses/ovulation, only a few primary oocytes will continue to develop while the others remain arrested. The number of primary oocytes is highest in the 20th gestational week [34], estimated to be around 7 million, and steadily decreases thereafter. It was previously believed that no oocytes were produced in reproductive-age women; however, recent data suggest the possibility of oogonial stem cells, which can give rise to new oocyte-like structures in the adult [35,36,37,38]. Further studies are needed to understand their biology and contribution to the “oocyte pool.” This pool of available oocytes is the ultimate determinant of menopausal age under physiologic conditions. Depletion of the oocyte reserve starts in utero and continues thereafter [39, 40]. At the time of puberty, there are on average 200,000 primary oocytes remaining in the ovary [41].

Follicles provide support for the oocyte, and folliculogenesis occurs concomitantly with oocyte development . Initially, primordial follicles (immature oocytes surrounded by flat granulosa cells) develop and reach their maximal numbers around the same time as the peak in primary oocytes, around 20 weeks gestational age. These follicles will either continue to develop or spontaneously regress or undergo apoptosis, with only a fraction remaining by puberty [39]. Primordial follicles, also known as pre-antral follicles , are not responsive to gonadotropins and therefore rely on other factors for their development. The factors that initiate follicular development prior to attainment of gonadotropin sensitivity have not been fully determined; however, kit ligand, LIF, EGF, KGF, BMP-4, AMH, and bFGF have been shown to contribute to this process [42,43,44,45,46,47]. AMH is expressed in granulosa cells of small growing follicles and inhibits transition of primordial follicles to primary follicles. It also reduces follicle sensitivity to FSH , thereby inhibiting FSH-induced pre-antral follicle growth. In animal models, it also inhibits kit ligand and bFGF, which are known stimulatory factors for primordial follicle recruitment [48, 49]. Therefore, AMH seems to play a pivotal role in preventing follicle exhaustion and recruitment at a younger age by suppressing primordial follicles [49, 50].

As early follicular development is independent of gonadotropin stimulation, this stage of follicular development can occur before puberty, as well as during reproductive ages. However, they spontaneously regress or undergo apoptosis [51]. Only after the development of antrum, the follicle becomes responsive to the gonadotropins [44]. With puberty, maturation of the hypothalamic pituitary axis and pulsatile release of FSH and LH, antral follicles continue their development until either ovulation or atresia [52, 53]. With puberty, maturation of the hypothalamic pituitary axis and pulsatile release of FSH and LH allow antral follicle development to progress until either ovulation or atresia [53].

4.6 Cumulus Cells and Oocyte Interactions During Ovulation

Follicles contain both an oocyte and a number of cells surrounding it, including an inner layer of cumulus and outer layer of granulosa cells. The oocyte actively regulates adjacent cumulus cell (CC)/granulosa cell (GC) metabolism and creates the optimal environment for its own development. The oocyte–CC interaction is achieved by direct contact via gap junctions and by a paracrine effect of oocyte-secreted factors (OSF). Because cumulus cells lie closer to the oocyte than granulosa cells, the oocyte regulates the adjacent CC cells more than the distant granulosa cells. Two distinct factors that have been determined as OSF are growth differentiation factor 9 (GDF9) and BMP-15 [54].

The effect of OSF on granulosa and cumulus cells can be summarized as follows:

  1. 1.

    OSF increases DNA synthesis in both CC and GC and increases cell proliferation

  2. 2.

    Inhibition of CC luteinization

  3. 3.

    Inhibition of CC apoptosis

  4. 4.

    Regulation of CC metabolism

  5. 5.

    Promotion of CC mucification and expansion

In conclusion, the oocyte tightly controls the adjacent microenvironment for its optimal development. Under the effect of OSF, CC/GC are transformed into supportive cells for oocyte development. CC/GC have different physiological properties than mural granulosa cells, which are not affected by the OSF. Mural granulosa cells express FSH receptors and later in follicular development they are involved in steroid hormone secretion, follicular expansion, and finally, ovulation.

4.7 Late Follicular Development and Oocyte Pickup

In the follicular phase, under the influence of hypothalamic GnRH pulse frequency, anterior pituitary gonadotrophs release FSH . FSH binds to its receptors on the primary follicular granulosa cells and induces proliferation. Under continuous FSH stimulus, pre-antral follicles escape from follicular atresia and continue to develop. A dominant follicle is then selected and continues to grow and develop under continued FSH and eventually LH stimulus, while other follicles begin to undergo atresia. After the LH surge in midcycle, the oocyte of the dominant follicle completes its first meiotic division (it had been arrested in meiosis prophase I since initial development in gestation) and shortly thereafter is expelled from the ovary. At this stage, the oocyte is surrounded by a thick glycoprotein layer, the zone pellucida, and overlying granulosa cells, which altogether form the cumulus oophorus complex. The oocyte and granulosa cells are functionally connected through gap junctions, which are thought to play an important role in local regulation of the oocyte. Shortly after ovulation, the cumulus oophorus complex is taken up by the infundibular part of the fallopian tubes. The infundibula contain fimbriae that are finger-like projections that constantly sweep the ovarian surface. The fimbriae guide the ovulated COC into the fallopian tube. Myometrial contractions together with tubal epithelial ciliary beatings are thought to contribute to this process. Within minutes, the cumulus oophorus oocyte complex can be found in the ampullary region of the tube.

During oocyte transport, the spermatozoa are moving up the fallopian tubes to meet the cumulus oophorus oocyte complex. Unlike spermatozoa which maintain fertilizing capability for days, the oocyte loses its capability to become fertilized after 12 hours in the female reproductive tract. The differential timing of oocyte and sperm viability demonstrates the clinical importance of proper timing intercourse to assure sperm availability at ovulation.

4.8 Fertilization

4.8.1 Sperm Penetration through the Cumulus Oophorus

To fertilize the oocyte, the capacitated sperm has to pass through the cumulus oophorus, a specialized layer of cuboidal granulosa cells that surround the oocyte (◘ Fig. 4.1). These cells are formed by follicular cells, which are adherent to the oocyte prior to ovulation and originate from the squamous granulosa cells present at the primordial stage of follicular development. These cumulus cells are attached to each other with an extracellular matrix that is mainly composed of hyaluronic acid, heparin sulfate, and chondroitin sulfate [55]. Although cumulus-free oocytes surrounded only by a zona pellucida are able to induce an acrosomal reaction, cumulus cells seem to foster the reaction before the sperm reach the zona pellucida [56]. Sperm hyperactivated motility also helps penetration through this initial barrier.

Fig. 4.1
figure 1

Fertilization process. 1 Sperm penetration of cumulus cells, 2 attachment to zona, 3 exocytosis of acrosomal contents, 4 penetration to the zona pellucida, 5 entry into perivitelline space, 6 binding and fusion with the egg plasma membrane, 7 cortical reaction, and 8 block to polyspermy. Reproduced with permission from Esfandiari N. In: Hurd WW, Falcone T, eds. Clinical reproductive medicine and surgery. St. Louis, MO: Mosby/Elsevier; 2007

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4.9 Structure of Zona Pellucida and Sperm Penetration

Once spermatozoa pass through the cumulus oophorus, they bind zona pellucida, which is the thick extracellular coat of the egg. (◘ Fig. 4.2) [57]. The sperm to zona pellucida binding is a species-specific process. This concept is the basis of the “hamster zona binding test.” Human sperm cannot bind to hamster eggs with an intact zona pellucida, which led to the thought that the zona pellucida contains species-specific receptors. Human sperm can only bind to hamster eggs after this glycoprotein layer is removed. Although there are some species–species exceptions, the zona pellucida is an important barrier between interspecies fertilization. In the clinical setting, the sperm penetration assay, the sperm–zona pellucida binding, the acrosome reaction, and the hyaluronan binding can be utilized in workup of subfertile men [58].

Fig. 4.2
figure 2

Light microscopy of mouse sperm binding the zona pellucida of an unfertilized egg. (Source: Wassarman PM, Jovine L, Litscher ES. A profile of fertilization in mammals. Nat Cell Biol. 2001 Feb;3(2):E59–64. Used with permission from Nature Publishing Group)

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With the aid of electron microscopy and advanced molecular techniques, our understanding of the zonal structure has increased. There are three major glycoproteins that compose the zona pellucida and have distinct roles in this structure: ZP1, ZP2, and ZP3 [59]. The ZP2 and ZP3 proteins form a filamentous structure that is then cross-linked with ZP1 proteins [60, 61].

In a classical model, ZP3 binds sperm and initiates the acrosomal reaction (see ◘ Fig. 4.1). Mutagenesis of O-glycosylation sites of ZP3 has been shown to decrease sperm receptor activity, suggesting that ZP3 serves as the sperm receptor in zona pellucida [62]. As sperm binds to ZP3, the outer acrosomal membrane fuses with the sperm plasma membrane that subsequently causes membrane blebs and results in the releasing of acrosomal enzymes that lyse the zona pellucida. This reaction exposes the inner acrosomal membrane that can bind to ZP2 (◘ Fig. 4.3) [63]. Eventually, sperm penetrate the zona pellucida and enter into the perivitelline space. Various other models have shown that this acrosomal reaction could occur when sperm encounter cumulus cells [64]. However, as mentioned above, some oocytes do not have cumulus cells and can still be fertilized.

Fig. 4.3
figure 3

Sperm and ZP3 binding. Acrosome intact spermatozoon shown with red crescent on its head, whereas acrosome reacted spermatozoon does not. A- ZP3 binds spermatozoon and induces acrosome reaction, thereby releasing of acrosomal enzymes that lyse the zona pellucida. Acrosome-reacted spermatozoon binds ZP2 via their exposed inner acrosomal membrane and penetrate the zona pellucida, ultimately fusing with the oocyte. B- Immediately after fertilization cortical granules release proteases to the perivitelline space that clip ZP2 and converts it to cleaved ZP2 (ZP2c) that can no longer bind acrosome-reacted sperm. Cleaved ZP2 dissociates from ZP3, resulting in subtle modification of ZP3 to convert it ZP3f that lacks sperm receptor and acrosome inducing capability. (Source: Clark GF, Reproduction. 2011 Sep.; 142(3):377–81. Used with permission from Bioscientifica Ltd.)

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4.10 Cortical Reaction to Block Polyspermy

As the spermatozoa enter the perivitelline space, they initiate the cortical reaction (see ◘ Fig. 4.1). Release of proteolytic enzymes from egg cortical granules causes cleavage of ZP2, with subsequent dissociation of ZP2 from ZP3 [65]. Thus, after the cortical reaction, sperm can no longer bind to ZP3 (◘ Fig. 4.3) [63]. Additionally, cleaved ZP2 cannot bind a spermatozoon that had previously undergone an acrosomal reaction. In conclusion, neither a sperm with an intact acrosome nor a sperm that has undergone an acrosomal reaction would be able to bind to the zona pellucida after the cortical reaction [63]. This is the principal mechanism preventing polyspermy.

Although through murine models we have learned an impressive amount about the process of fertilization, there are still many questions that need to be answered and further research on the exact mechanism of sperm binding is needed.

4.11 Sperm-Oocyte Membrane Fusion

After a spermatozoon penetrates the zona pellucida and enters the perivitelline space, the oocyte membrane and the spermatozoon membrane unite (see ◘ Fig. 4.1). At this stage, the spermatozoon has already undergone an acrosomal reaction, which exposed the inner acrosomal membrane and modified the membrane composition of both equatorial and post-acrosomal regions of the spermatozoon. The fertilizing spermatozoon binds to the microvillar region of the oocyte membrane with its equatorial segment [66]. Sperm tail movement decreases or stops within a few seconds of sperm-oocyte fusion [67]. Subsequently, the posterior region of the sperm head and the tail are incorporated into the egg. Unfortunately, the details of molecular interactions in sperm-egg fusion are not fully known. Initially, ADAM family members that are found on the sperm membrane, specifically fertilin and cyritestin, gained much attention. However, gene knockout studies questioned their fundamental roles in sperm-egg fusion. Currently, cyritestin, fertilin α(alpha), fertilin β(beta), CRISP1, izumo proteins, α(alpha)6β(beta)1 integrin, GPI-anchored proteins, CD151, CD9, and CD81 on the plasma membrane are thought to be involved in sperm-oocyte membrane fusion and are the subjects of ongoing research [68, 69] (◘ Fig. 4.4).

Fig. 4.4
figure 4

Model for molecular interactions during sperm-egg binding. GPI-anchored proteins, integrins, CD151, CD9, CD81 on the oocyte membrane and ADAM proteins, Pdi3a chaperone refolding Izumo on the sperm membrane are involved in sperm-oocyte membrane fusion. (Source: Nixon B, Aitken RJ, McLauglin EA. Cell Mol Life Sci. 2007 Jul;64(14):1805–23. Used with permission from Springer)

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4.12 Oocyte Activation

Mammalian oocytes become arrested at the metaphase of the second meiotic division. After sperm-oocyte fusion, the oocyte continues meiotic division, releases cortical granules, progresses cell cycle, forms its pronucleus, and recruits maternal mRNA that are all essential for gamete formation [70]. These morphologic and biochemical changes that occur in the oocyte are collectively called “oocyte activation.” Another important hallmark of oocyte activation is calcium oscillations. It has been shown that injecting calcium into mice oocytes is enough to trigger embryo development up to the blastocyst stage [71]. In a mammalian oocyte, the calcium oscillations are a direct result of inositol triphosphate-mediated calcium release. Sperm-derived phospholipase-zeta (PLC-ζ(zeta)) is also responsible for oocyte activation [72]. Another protein that has been shown to activate oocytes is post-acrosomal sheath WW domain-binding protein. Its exact signaling mechanism is not clearly known, but it presumably acts through calcium signaling [73]. Regardless of the signaling pathway, oocyte activation is essential for pronucleus formation and subsequent embryo formation.

Oocyte activation clearly has clinical importance. A deficiency in oocyte activation was regarded as the principal cause of fertilization failure or low fertilization rate after ICSI. Recently it has been suggested that PLC-ζ(zeta) could be used as an alternative oocyte-activating agent, including male factor infertility, similar to other artificial oocyte activators [74, 75].

4.13 Male Pronucleus Formation and Genomic Union

The final step of fertilization is the union of sperm and egg pronuclei, producing a diploid cell, the zygote. Dynactin, nucleoporins, vimentin, dynein, and microtubules are involved in bringing the two pronuclei together. It was proposed that a nuclear pore complex is inserted into the nuclear envelopes of the newly forming pronuclei. Dynactin and vimentin filaments are then incorporated into this nuclear pore complex. Formation of the complex probably starts after egg activation. The sperm aster then extends the microtubule “plus ends” away from the male pronucleus, some of which reach the female pronuclear envelope. With the aid of the dynactin-dynein motor complex, the two pronuclei are apposed [76]. Subsequently, the two nuclear envelopes disappear and the DNA undergoes replication. Homologous chromosomes are paired and aligned on the newly formed mitotic spindle. Eventually, the zygote is ready to undergo its first mitotic division.

4.14 Early Embryonic Development

In mammals, the zygote undergoes mitotic division (known as cleavage) as it travels through the fallopian tube, and eventually develops into a blastocyst once in the uterus (◘ Fig. 4.5). The symmetrical cell divisions and cleavage create a ball of totipotent cells (blastomeres) that are still enclosed in the zone pellucida. When the zygote is approximately 16 cells, blastomeres form a closely packed group of cells with a smooth outer surface. This early developmental event is called compaction. The smooth surface is created by the formation of adherens and tight junctions between the blastomeres. At this time, two types of polarity originate in the zygote. The first type of polarity is cellular polarity. Cellular polarity occurs as the formation of microvilli on the external surface of the outer blastomeres separate from the basolateral surface [77].The second type of polarity is developmental polarity. Developmental polarity is represented by the ability of the blastomeres in the internal compartment, the inner cell mass, to remain pluripotent, whereas the outer blastomeres begin to form trophoblast cells as they continue to divide [78]. This begins formation of the blastocyst and typically occurs around day 5 of fertilization. As cleavage continues, outer blastomeres express tight junction proteins, including ZO-1 and uvomorulin, gap junction proteins such as Connexin-43, and differentially position Na-K ATPase pumps selectively along the apical-basolateral axis. The outer blastomeres have a highly restricted developmental fate, eventually becoming the cells of trophoectoderm . The polarized expression of Na-K-ATPase in trophoectoderm creates a trans-trophoectoderm sodium gradient, which drives the osmotic accumulation of water into the nascent blastocoelic cavity. Growth factors like TGF-α(alpha) and EGF increase expression of Na-K-ATPase, which subsequently stimulate further expansion of the blastocoelic cavity (blastocoel). Meanwhile, the inner blastomeres continue to divide, and with the expansion of the blastocoel, they create a cluster of cells that impend into blastocoel . This totipotent cell cluster is commonly called the inner cell mass. The inner cell mass will eventually give rise to the embryo and extraembryonic tissues . The outer layer of blastomeres, which have developed into trophoblasts/trophoectoderm, eventually give rise to the placenta. It is at this point that the developing embryo is called a “blastocyst.”

Fig. 4.5
figure 5

Schematic drawing showing the major events from ovulation to the implantation of blastocyst during the first week of human life. Reproduced with permission from Esfandiari N. In: Hurd WW, Falcone T, eds. Clinical reproductive medicine and surgery. St. Louis, MO: Mosby/Elsevier; 2007

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Although difficult to completely exclude, this initial exponential division and formation of the blastocyst seems to be relatively independent of maternal contribution.

Around day 6 after ovulation, the embryo/blastocyst reaches the uterine cavity and is initially still covered with zona pellucida. For proper implantation, it must shed the zona pellucida. Trophoblast-derived trypsine-like enzymes, strypsin and plasmin, are thought to lyse the zona pellucida, allowing the embryo to hatch from the zona pellucida and begin to attach to the uterine endometrium [79, 80].

4.15 Trophoblastic Development and Invasion

The blastocyst is lined with a layer of trophoectoderm , which, as stated above, will give rise to the placenta. Although the inner cell mass is destined to produce embryonic and extraembryonic tissues, it stimulates trophoectodermal growth. In vitro, the removal of the ICM causes maturation of the trophoblastic cells, inducing them to turn into trophoblastic giant cells that are unable to invade the endometrium. For proper endometrial attachment, the blastocyst should remain attached to the trophoectoderm cells that are adjacent to the inner cell mass. Attachment of trophoblastic cells remote from the ICM has been associated with abnormal placental shape and eccentric insertion of the umbilical cord [81].

Prior to blastocyst attachment, for a successful pregnancy in the window of implantation, the uterine epithelium has to retract its cilia and express pinopodes. If all necessary molecular events occur, the blastocyst is firmly attached to the uterine epithelium around 6–7 days postconception.

The trophoblastic cells in contact with the inner cell mass start to proliferate and invade the uterine epithelium. As they invade, they fuse with each other and form multinucleated giant trophoblastic cells, known as syncytiotrophoblasts. An inner layer of mononucleated trophoblastic cells also develops called cytotrophoblasts. With fusion, the multinucleated syncytiotrophoblastic cells cannot proliferate, so the cytotrophoblastic cells function as a reservoir. Throughout the pregnancy, cytotrophoblastic cells divide and replenish the mature syncytiotrophoblastic cells. In addition to replenishing syncytiotrophoblasts, cytotrophoblasts give rise to various other cell types of the placenta, which are discussed below [82]

The fusion kinetics of cytotrophoblasts changes during pregnancy. In early pregnancy, two mononuclear cytotrophoblasts fuse to become a syncytiotrophoblast. However, later in the pregnancy, cytotrophoblasts fuse with already formed syncytiotrophoblasts [82]. Here we will discuss the process of early embryo development; the process of implantation will be discussed in more detail below.

Around 14 days postconception, cytotrophoblastic cells invade beyond the syncytiotrophoblastic cell layer and come into contact with maternal decidual cells. They form a column of cells with a proliferating core, and as the cells proliferate, more mature cells are passively pushed towards the maternal decidua [83]. More immature cells have α(alpha)6β(beta)4 integrin on their surface, which help bind basal membrane components like collagen IV and laminin. However, as they move further in the column and become closer to maternal decidual cells, they change their expression of surface integrins (integrin α1/β1, α5/β1, or α-v/β3/5), which helps them attach to the maternal extracellular matrix [82, 84].

In addition to adhesion molecules, trophoblasts secrete variety of enzymes that regulate invasion. MMP-2 and MMP-9 degrade collagen IV, which is the main collagen component of the basement membrane, and are therefore regarded as key enzymes in the implantation process, enabling the invasion of the trophoblast cells through the decidua and into the maternal vasculature [85, 86]. Tissue inhibitor of matrix metalloproteinases (TIMP), particularly TIMP-1, TIMP-2, and TIMP-3, were also detected in the trophoblastic cells and decidual tissues. TIMPs are normally inhibitory metalloproteinases and their regulation through trophoblastic and decidual cytokines control MMP activity [87,88,89]. Other lytic enzymes involved in extracellular matrix degradation are urokinase and tissue-type plasminogen activator (uPA and tPA, respectively). Both uPA and tPA are produced by trophoblasts, and their activity is controlled by plasminogen activator inhibitors (PAI) [88]. Another trophoblast protein, adrenomedullin, decreases PAI levels and subsequently increases plasminogen activators. Additionally, adrenomedullin increases trophoblastic proliferation [90].

Trophoblasts also secrete proangiogenic factors, which stimulate new vessel formation during invasion. Neovascularization is essential for the growth and maintenance of the developing embryo. VEGF, PDGF, and PAF are the main angiogenic factors that have been shown to be secreted by trophoblasts. TGF-β(beta) and TNF-α(alpha), which are present in decidua, further stimulate trophoblastic secretion of these angiogenic factors [91].

Complex molecular interactions take place between the decidua and trophoblasts to regulate trophoblastic invasion. In addition to those factors mentioned above, cytokines like EGF, HB-EGF, IGFBP-1, LIF, IL-1 and hormones like hCG and progesterone have also been shown to regulate trophoblast invasion [88].

A number of other important types of trophoblast cells are involved in implantation—namely, extravillous, endovascular, and endoglandular. Small extravillous trophoblasts invade maternal decidua up to the inner one-third of uterine myometrium and reach the maternal spiral arteries. EVTs replace the spiral arteries’ tunica media, which contains mainly the smooth muscle, and transform the spiral arteries into low resistance vessels that are no longer reactive to maternal vasomotor substances. This transformation aims to allow adequate maternal exchange with the developing fetus, particularly in the second trimester when maternal blood flow increases to the uterus to support the developing fetus. Apart from replacing the smooth muscle, endovascular trophoblasts, a subset of EVTs, replace the intimal layer of the spiral arteries [92]. Disturbances in this remodeling can result in IUGR and preeclampsia. Lastly, endoglandular trophoblasts invade the uterine glands, orient them towards the intervillous space, and replace the uterine epithelial cells (◘   Fig. 4.6) [81].

Fig. 4.6
figure 6

Trophoblastic invasion of maternal decidua. Small interstitial extravillous trophoblasts invade maternal decidua up to the inner one-third of uterine myometrium and replace the tunica media maternal spiral arteries to create low resistance blood flow. Endovascular trophoblasts replace the intimal layer of the blood vessels while endoglandular trophoblasts invade the uterine glands. (Source: Huppertz B, Berghold VM, Kawaguchi R, Gauster M. Am J Reprod Immunol. 2012 May;67(5):349–57. Used with permission from John Wiley and Sons)

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Despite all these early trophoblastic changes, free transfer of maternal blood is only established towards the end of the first trimester. The large number of endovascular trophoblasts plugs the distal segments of the spiral arteries during initial invasion. Rather than maternal blood, the intervillous space ultimately contains glandular secretion products and maternal plasma filtrate, which are responsible for intrauterine nutrition, up until approximately 10 weeks gestation [81]. The reasoning behind the initial spiral artery plugging is believed that it helps keep a low oxygen environment and thus decrease free-radical formation during early embryogenesis.

After 10 weeks, the trophoblastic plugs dissolve and maternal blood contributes to intervillous fluid, which provides the appropriate amount of nutrients and oxygen for the developing fetus. These carefully regulated interactions between the invading trophoblasts and the maternal decidua eventually create a functional placenta, which is the main organ of nutrition, respiration, metabolite excretion, and hormone production in the developing fetus.

4.16 Implantation

Implantation can be divided into three stages: apposition, adhesion, and invasion. Apposition is the initial adhesion of the blastocyst to the endometrial surface. Apposition is unstable and with uterine flushing the blastocyst can be detached from the endometrial surface. Apposition is followed by the adhesion stage, when a stronger connection is established between the embryo and endometrium. Finally, in the invasion stage, trophoblastic cells invade the endometrium.

4.17 Endometrial Receptivity

Successful implantation requires a properly developed blastocyst , a receptive endometrium, and series of molecular interactions. In humans, 75% of the failed pregnancies are considered to be secondary to implantation failure, therefore it is essential to understand the basic molecular interactions involved in the process [93]. Under the influence of estradiol, the endometrium proliferates and reaches a critical thickness to support implantation. After ovulation, in response to progesterone, the endometrium differentiates and becomes receptive to the newly hatched blastocyst.

Implantation occurs around 6 days after ovulation, ranging between 6 and 12 days [94]. The ideal time for implantation is thought to be around day 7 to day 9 after the LH surge and is called the “window of implantation.” This period is characterized by structural and secretory changes in endometrial cells, providing the most favorable conditions for successful blastocyst implantation. The endometrium increases in thickness, becomes more vascularized, and glands become tortuous and increase their secretions rich in cholesterol, fat-soluble vitamins, lipid, and protein. These secretions will serve as an energy source for the embryo, which has no connection to uterine vessels at this point in development. In addition, there is a decrease in uterine fluid content to allow greater contact between the embryo and endometrium. These drastic changes in the uterine environment are mostly the result of progesterone stimulating the differentiation of endometrial cells into decidual cells. Decidual cells contain more intracellular lipids and glycogen deposits than endometrial cells, which cause them to get a polygonal shape as opposed to more rounded endometrial cells.

4.18 Pinopodes

One characteristic feature of receptive endometrium is the presence of pinopodes on the apical surface of endometrial cells. Pinopodes are bleb-like protrusions into the uterine lumen and are found in large numbers between days 19 and 21 in an idealized 28-day menstrual cycle. Although they are expressed throughout the mid- and late secretory phase, they show different morphological features. This suggests that their morphology, rather than their presence, is important for successful implantation [95]. Blastocyst attachment has been shown to occur preferentially on the top of pinopodes, which suggests that receptors necessary for attachment are located on the pinopode surface [96].

Pinopode development has been associated with progesterone [95], HOXA-10, LIF [97], and aVβ3 integrin [98]. HOXA-10, a homeobox gene, is necessary for blastocyst implantation, endometrial stromal cell proliferation, and epithelial cell morphogenesis [95]. Blocking HOXA-10 expression greatly decreases the number of pinopodes.

4.19 Selectins

Selectins are glycoproteins that have a glycosylated extracellular domain, single spanning transmembrane domain, and a cytoplasmic tail. There are three distinct selectins: P selectin, L selectin, and E selectin. Selectins are commonly known for their role in initial leukocyte attachment and subsequent rolling on the endothelial surface. In addition to leukocytes, however, selectins are thought to be responsible for the initial blastocyst -endometrium attachment.

Strong L selectin expression has been shown on the blastocyst surface, whereas on the maternal site, its ligands, namely MECA-79 and HECA-452, are up-regulated during the window of implantation [99]. Although L selectin is found on both luminal and glandular epithelium, expression of L selectin is higher on the luminal epithelium [100]. Initial trophoblast attachment to endometrium is thought to occur with trophoblastic L selectin and endometrial oligosaccharide interactions [101].

4.20 Integrins

Integrins are transmembrane glycoproteins composed of noncovalently linked α(alpha)- and β(beta)-subunits. Each subunit has an extracellular, intracellular, and transmembrane domain. The intracellular domains are linked to the cytoplasmic cytoskeleton and intracellular signaling pathways [96]. They are paired to compose integrin heterodimers; 24 functionally distinct integrins have been identified [102]. Among various other functions, they are mainly involved in cell-to-cell and cell-to-extracellular matrix interactions. Among the many different types of integrins that are expressed constitutively in the endometrium, α1β1, α4β1, αvβ3 are co-expressed between days 20 and 24 in the menstrual cycle. β(beta)3 integrin deserves special attention among other subunits, because its expression starts at cycle day 19 and increases thereafter [103]. Moreover, it is mainly expressed on the endometrial luminal surface, which suggests that αvβ3 integrin, and its endometrial ligand osteopontin, might serve as a receptor for embryonic attachment [103, 104]. Various studies showed that the αvβ3 integrin is regulated in both a hormonal and paracrine manner. For example, estrogens down-regulate integrin expression, but increasing levels of progesterone in the luteal phase counteract the estrogen effect. Rather than a direct effect, progesterone increases epidermal growth factor and heparin binding growth factor in the uterine stroma, which results in increased αvβ3 levels [103]. The embryo is also actively involved in the β3 subunit regulation, probably with the embryonic IL-1 system [96].

Additionally, HOXA-10 increases the expression of the β3 subunit in endometrial cells [105]. This subunit is the rate-limiting step in αvβ3 integrin production. Considering the important role of αvβ3 integrin in the implantation process, it’s not surprising that it is used as a clinical marker of endometrial receptivity [96, 106].

4.21 Mucins

Mucins are heavily glycosylated proteins. Carbohydrates constitute 50–90% of their molecular weight. To date, 18 mammalian mucin genes have been identified [107]. Mainly mucin-1 (MUC1) and to lesser extent mucin-6 (MUC6) are expressed in the human endometrium. They are found on the luminal surface of the epithelial cells in the reproductive tract. Their proposed physiological role in the reproductive tract is to trap bacteria and viruses and expel them. They are resistant to digestive enzymes. Their extracellular portion can be cleaved, and those cleaved molecules can join via sulfide bonds to create a mucin gel. Altogether, mucins produce a formidable barrier in microbial defense. Estrogens increase mucin production. Progesterone has no independent effect on mucin production; yet by counteracting the effects of estrogens, the net effect of progesterone is to decrease mucin levels. Cytokines, particularly TNF-α, have also been shown to be involved in mucin regulation.

Although they provide an important barrier in microbial defense, mucins also constitute a barrier against blastocyst implantation. Mucins extend their projections well beyond endometrial surface receptors, thereby hindering blastocyst access to them [108]. At the site of implantation, mucins’ extracellular domain needs to be cleaved. The sheddase family of enzymes, particularly TACE/ADAM17 and MT1-MMP, has been suggested to play a role in this cleavage process [109]. The blastocyst, through the action of secreted cytokines, up-regulates sheddases that cleave mucins in endometrium [108].

Interestingly, during the implantation period, mucin production is increased [110]. It seems to be a paradoxical phenomenon; however, two possible explanations have been suggested. First, after sexual intercourse, the ejaculate may introduce microbial pathogens into the endometrium and the increased mucin levels may act as an additional protective barrier . Second, as the blastocyst is actively involved in sheddase induction, it has to be competent to do so. Mucins may be a protective mechanism against the attachment of unhealthy embryos that would otherwise have resulted in pregnancy failure [110]. Consistent with this view, women with recurrent pregnancy failure have decreased mucin levels compared to a fertile control group [111].

To summarize, mucins prevent embryo attachment and need to be cleaved at the site of embryonic attachment. This process involves a series of interactions that requires a healthy embryo as well as a functional endometrium.

4.22 Cytokines

Cytokines are soluble proteins that have a variety of functions in inflammation, the menstrual cycle, ovulation, and implantation. A disturbance in the normal expression or action of several cytokines results in implantation failure and abnormal placental development in humans. Of known importance are members of the gp130 family, such as LIF, IL-1, IL-11, and IL-15 system [112].

4.23 Leukemia Inhibitory Factor

A member of the gp130 cytokines, LIF acts through its surface receptor complex, LIF receptor (LIFR), and the gp130 receptor chain. Binding of LIF to LIFR results in heterodimerization with gp130 and subsequent activation of downstream signaling pathways that include the JAK/STAT, MAP kinase, and PI3 kinase pathways [96]. Other members of the gp130 cytokine family, including oncostatin M, ciliary neurotrophic factor, cardiotrophin-1, IL-6, and IL-11, can also bind to the LIFR [112].

LIF was the first cytokine shown to be critical for implantation in mice [113]. Wild-type mice embryos failed to implant in the endometrium of homozygous LIF mutant female mice, and the implantation failure was reversed after LIF supplementation .

LIF mRNA is expressed between menstrual cycle days 18 and 28 in fertile women , and it is expressed by both glandular and luminal epithelium [114]. Among many LIF regulators, progesterone is probably responsible for endometrial LIF induction. When treated with a selective progesterone receptor modulator, mifepristone, decreased levels of LIF are observed in endometrium [115]. In addition to progesterone, IL-1α, TNF, PDGF, TGF-β1, and HB-EGF stimulate LIF expression in cultured endometrial stromal cells. The embryo secretes hCG, IGF-1, and IGF-2 that also increases LIF levels [116].

LIF protein expression is maximal in uterine flushings in the midlate secretory phase of the menstrual cycle at the time of expected implantation. Considering the ease of performing uterine flushings, LIF has been suggested as a marker of uterine receptivity [117, 118]. In women with recurrent implantation failure, LIF levels are lower than in controls, emphasizing the importance of LIF in successful implantation [118, 119]. rhLIF has also been suggested to improve endometrial receptivity in recurrent implantation failure patients; however, the efficacy has not been demonstrated in clinical trials [120].

4.24 Interleukins

IL-1 is one of the key regulatory mediators of the inflammatory response. IL-1α, IL-1β, and the IL-1 receptor antagonist are members of the IL-1 cytokine family. Stromal cells, glandular cells, and macrophages are the reservoir of IL-1 in the endometrium. In vitro, treating endometrial cells with IL-1 increases integrin β3 expression in the epithelial cells [121]. IL-1α knockout mice are, however, fertile, suggesting redundancy in the effects these interleukins have in implantation. IL-1 receptor antagonist expression is decreased during the implantation window. It is possible that down-regulation of IL-1 antagonist works synergistically with IL-1 to affect implantation [122]. Exogenous IL-1 receptor antagonist treatment during implantation can block blastocyst implantation [121]. Overall, the IL-1 system is clearly involved in implantation; however, its exact role in implantation remains unclear.

IL-6 is involved in many immune interactions, and it has been also suggested to play a role in implantation. Endometrial IL-6 mRNA expression increases during the mid- to late secretory phase and decreases in the late secretory phase. Strong immunoreactivity has been observed in uterine glandular and luminal epithelium during the window of implantation [123]. While controversial, IL-6-deficient mice appear to have reduced fertility and decreased implantation rate [124]. The IL-6 receptor is found on the surface of the blastocyst , and IL-6 is probably involved in paracrine/autocrine interactions in the window of implantation . Decreased levels of mid-secretory IL-6 mRNA are found in patients with recurrent spontaneous abortions, also supporting this hypothesis [125].

Another cytokine that has gained attention is IL-11. IL-11 has anti-inflammatory activities, and it is expressed in endometrial glandular and luminal epithelium. Estrogen, progesterone, and local factors increase IL-11 levels. IL-11 advances progesterone-induced decidualization of human endometrial stromal cells. IL-11 and its receptor IL-11R were immunolocalized to decidualized stromal cells in the mid-late secretory phase epithelium. They were also shown on the trophoblastic cells, suggesting a role in normal placentation [112]. Additionally, inadequate IL-11 signaling was found to result in dysregulation in trophoblastic invasion [126].

4.25 Prostaglandins

Prostaglandins (PGs) are lipid mediators of inflammation, and they have a variety of functions in inflammation, menstrual cycle regulation, ovulation, embryo attachment, trophoblastic invasion, and labor. Prostaglandins, leukotrienes, and thromboxanes are members of the eicosanoid family. They are produced from membrane lipids by phospholipase A2 (PLA2) and cyclooxygenase (COX) enzymes. To date, three isoforms of COX enzymes have been discovered: COX-1, COX-2, and COX-3. COX-1 is constitutive and expressed under normal physiological functions, whereas COX-2 is involved mainly in inflammatory responses. COX-3 is expressed in the human brain and is responsible for fever and response to pain.

Murine studies have shown the importance of prostaglandins (PGs) in implantation. Lack of either PLA2 or COX2 in mice leads to defective PG synthesis; PLA2 knockout mice show pregnancy failure [127]. COX expression is maximal in the menstrual and proliferative phases . Among many regulators, IL-1 deserves further consideration. IL-1 increases COX enzymes and PG production, resulting in increased endometrial integrin levels that are essential for blastocyst implantation [96].

Although prostaglandins’ role in the menstrual cycle and pathophysiology in endometriosis is well known, their role in human blastocyst attachment and subsequent invasion needs to be explored further.

4.26 HOX Genes

Homeobox (HOX) genes are highly conserved genes that are involved in embryonic development as well as endometrial growth, differentiation, and receptivity [128]. Both estrogen and progesterone increase HOXA10 and HOXA11 expression. Additionally, HOXA10 and HOXA11 expression reach the highest levels during the window of implantation [129]. Wild-type mice embryos cannot implant to the uteri of HOXA10 or HOXA11 knockout mice. These findings suggest that HOXA10 and HOXA11 play an essential role in endometrial receptivity [130]. In parallel with these findings, pinopodes, β3 integrin, and insulin-like growth factor binding protein were shown to be regulated by HOX genes [131]. As discussed above, these genes are among the few proven to be essential for endometrial receptivity. In humans, there are no documented HOXA10 or HOXA11 mutations. However, in various gynecologic disorders such as endometriosis, PCOS, hydrosalpinx, and uterine fibroids, endometrial HOXA10 and HOXA11 mRNA levels are reduced [132,133,134]. These findings demonstrate that HOX genes contribute to the defective endometrial receptivity that is observed in those disorders.

4.27 Immune Response to Trophoblast Invasion: Trophoblast–Leukocyte Interactions

As mentioned above, to achieve a successful pregnancy, the blastocyst must be able to attach to the endometrial decidua without complication. The blastocyst has to invade the endometrium and maternal blood vessels in order to ensure adequate blood supply for nutrients and gas exchange. However, because a blastocyst receives half of its genome from the father and the other half from the mother, it is treated as a semiallogenic by the maternal immune system. Therefore, alterations in the reactivity of the maternal immune system must occur at the maternal–fetal interface.

As the blastocyst attaches to the uterine epithelium, the trophoectoderm differentiates into two layers, as previously mentioned, an outer syncytiotrophoblast and an inner cytotrophoblast layer. Two weeks after implantation, the cytotrophoblast layer protrudes through the syncytiotrophoblasts and forms cytotrophoblastic buds. The buds then differentiate into both villious trophoblasts and extravillous trophoblasts. Villous trophoblasts cover the chorionic villi which form the main interface for gas and nutrient exchange between the fetus and mother, and as discussed above, extravillous trophoblasts invade and remodel the spiral arteries.

Maternal and fetal cells are in direct contact during this invasion process, and the immune response deserves a detailed explanation. Maternal leukocytes reside in the uterine endometrium, and it has been estimated that approximately 40% of the decidua is leukocytes. Fortunately, trophoblasts have a distinct MHC expression profile. They do not express the most common HLA antigens such as HLA-A and HLA-B, and even with potent stimulators like IFN-α, they do not express MHC class II antigens recognized by certain leukocytes. The dominant MHC types expressed on trophoblasts are HLA-C, HLA-G, and HLA-E.

Villous syncytiotrophoblasts line blood-filled lacunae and are in direct contact with maternal blood. They do not express MHC-I antigens and are therefore protected from T-cell-mediated responses. Interstitial trophoblasts invade the decidua and express HLA-C, HLA-G, and HLA-E. Endovascular trophoblasts that line the maternal spiral arteries express HLA-C, HLA-G, and HLA-E. The expression of HLA-G and HLA-E on this cell population confers protection from maternal immune rejection.

Leukocytes are normally found in endometrium and actually 40% of the decidua consists of leukocytes. In endometrial infection, there are a multiple types of leukocytes that are found in the endometrial lining, and they all act through different mechanisms. B-cells respond to antigenic stimulation and produce antibody secreting plasma cells.

Additionally, macrophages can be found and represent approximately 20% of the endometrial leukocytes; they can recognize and respond to HLA-G antigens [135].

T cells represent 10% of the leukocytes found in the endometrium. They require an MHC-II antigen presentation for immune response and, as trophoblasts do not express MHC-II antigens, they cannot directly stimulate T-cell responses. This is how villous syncytiotrophoblasts, which line the maternal blood-filled lacunae, are protected from the maternal T cells. However, maternal endometrial dendritic cells and macrophages can process paternally derived antigens by migrating to lymph nodes where they can initiate an immune response.

Interestingly, antibodies against paternal HLA antigens can be found during pregnancy; however, they are likely formed during birth due to fetal cells crossing the placenta. Fortunately, these antibodies are mainly against HLA-A and HLA-B. As these HLA types are not expressed by the trophoblasts, the presence of these antibodies is not correlated with pregnancy success [92].

Natural killer T (NKT) cells are a subset of T cells that have an immunomodulatory role in infection through cytokine production. Invading trophoblasts are protected from blood NK cells through multiple different mechanisms. Villous syncytiotrophoblasts are likely protected by the absence of NK-activating ligands on the syncytiotrophoblastic surface. Similarly, endovascular and interstitial trophoblasts, which line maternal spiral arteries and decidua, respectively, express HLA-C, HLA-G, and HLA-E. The expression of HLA-G and HLA-E on these cell populations confers protection from blood NK cells.

Uterine NK cells (uNK) are among the most studied endometrial leukocyte type and are known to be involved in endometrial renewal, differentiation, and breakdown in menstrual cycle. Although their exact role in implantation is unknown, their dysregulation has been shown to be associated with recurrent pregnancy loss, preeclampsia, and implantation failure. uNK cells are CD56brightCD16dim and functionally distinct compared to their circulating counterparts. They are spatially and temporally correlated with the implantation site of the embryo and modulate the cytokine, chemokine microenvironment, thereby contributing to physiological changes within the uterine stroma during pregnancy [136]. Their origin is still unknown, yet they are thought to arise from in utero proliferation and differentiation of CD34+ stem cells. They are found in deeper layers of decidua and are not shed during menstruation. Another alternative to their origin is recruitment from CD56+ cells in the blood into the endometrium. Regardless of their origin, their quantity correlates with maternal progesterone levels. Additionally, they are found to accumulate in large numbers at the site of implantation. Their close proximity to trophoblasts suggests that they may be involved in regulating trophoblastic invasion [92]. In addition, lower uNK counts in the endometrium have been correlated with decreased IVF-ET success [137]. In summary, although dysregulation of uNK cells and their cytokine production profile was shown to be related with recurrent pregnancy loss, preeclampsia, and implantation failure, their exact role in implantation is not known [138].

4.28 Clinical Relevance

Infertility is classically defined as the failure of a couple to conceive after 12 months of frequent intercourse in women under 35 years, and after 6 months in women over the age of 35 [139]. Infertility can be due to male factors, female factors, or both. The availability of ICSI/IVF has resulted in pregnancy rates in couples with male factor infertility that are comparable to those without male factor infertility [140].

Key steps for successful fertilization are the oocyte quality and appropriate oocyte maturation. As our understanding of oocyte biology has increased, we are able to mimic endogenous oocyte developmental steps in vitro. One such success in reproductive medicine is improving in vitro maturation (IVM) , in which immature oocytes are collected and then maturated for in vitro fertilization. This technique provides an invaluable opportunity for many infertile patients. Additionally, IVM and IVF provide an opportunity for fertility preservation, including use in patients undergoing gonadotoxic chemotherapy for various cancers [141]. As will be discussed further in the book, morphokinetics of the developing embryo have been used to select viable embryos that are more likely to implant the endometrium and result in successful pregnancy [142].

Defective endometrial receptivity is a significant cause of ART failure [143]. Therefore, it is essential to correctly assess the endometrial receptivity state for successful implantation. Among many others, pinopodes and αvβ3 integrin have been suggested as candidate biomarkers that reflect the window of implantation [144]. Endometrial scratching and G-CSF have been used to improve endometrial receptivity and implantation, however strong evidence favoring these methods is lacking [145,146,147]. Endometrial receptivity arrays based on the transcriptomic data have been developed to predict the window implantation in efforts to increase implantation success [148, 149]. However, there is currently insufficient evidence from adequately powered prospective clinical trials to validate these markers. Patients with endometriosis, fibroids, PCOS, and hydrosalpinx frequently present with infertility due at least in part to defective endometrial receptivity. Recognizing and treating the underlying etiology can at least partly improve endometrial receptivity in these patients.