INTRODUCTION
Reproduction is the creation of a new individual from previously existing individuals. The union of sperm and egg is the essential step in the process of reproduction.
In the historical milestones, the factors involved in reproduction remained a mystery till the earlier part of seventeenth century. Leeuwenhoek of Holland for the first time in 1677, with his home-made microscope described fairly accurately the anatomy of the sperm. Thereafter in the 18th century, there was insignificant progress in the knowledge of biology. In 1822, Karl Dernt von Baer "Father of Modern Embryology" documented observation on eggs and their developmental stages. It was Oscar Hertwig in Germany who first demonstrated in 1875 by his observation on sea urchins that the union of sperm and egg was the essential step for reproduction. However, in humans, the remote site of these events and the secluded place of origin of the participants (gametes) made fertilization a difficult subject for study. Most of the knowledge about human reproduction was achieved through experiments in animal species. Scope of study directly in humans has only been possible following the advent of assisted reproduction. The process of gametogenesis, their maturation, sperm-egg interaction, fertilization and implantation are a few of the many major benefits, which have been derived through clinical application of the assisted reproductive technologies. Some of the information about the basics of human reproduction gathered so far through these technologies backed up by studies on animal models will be discussed in this chapter.
Fundamental requirements for sexual reproduction can be classified under three broad groups:
- Development of morphologically and functionally competent gametes (oocytes and spermatozoa).
- Existence of anatomically and physio-logically normal male and female reprod-uctive organs.
- Delicately balanced intricate interactive events leading to release of gametes (male and female), their transport, fusion and fertili-zation, cleavage and implantation.
DEVELOPMENT OF MORPHOLOGICALLY AND FUNCTIONALLY COMPETENT GAMETES
The Gametes
The female gamete (oocyte) resides within the graafian follicle in the ovary and the male gamete (spermatocyte) within the seminiferous tubules lined by Sertoli cells in the testis. Ovaries and testes are known as female and male gonads respectively.
Embryogenesis of Gonads and Gametes
Initially fetal gonad remains in an undifferentiated form (neither female nor male). In the third week of embryonic development, the intraembryonic mesoderm covered by coelomic epithelium differentiates into three distinct segments: cervical, thoracic and caudal parts. The urogenital system of the embryo develops in the caudal part of the mesoderm covered by coelomic epithelium. Three paired ridges develop in this part of mesoderm due to coelomic condensation (Fig. 1.1). The most medial one is the gonadal or genital ridge. Lateral to gonadal ridge is the mesonephric ridge and the most lateral is the paramesonephric ridge. Pronephros at the cranial end, mesonephros in the midsegment and metanephros at the caudal end form the mesonephric ridge. Pronephros disappears in the course of embryonic development; mesonephros gives rise to wolffian system and metanephros is the precursor of the cortical part of kidney. Paramesonephric duct is the embryologic progenitor of mullerian system.
The gonad originating from gonadal ridge is composed of primitive germ cells, coelomic surface epithelial cells and an inner core of medullary mesenchymal tissue. Apart from germ cells, the origin of somatic cells in the gonad (granulosa-theca cells in the ovary and Sertoli-Leydig cells in the testis) is controversial. One view suggests that gonad is formed by invasion of germinal epithelium which gives rise to gonadal tissue. The other view suggests that gonadal tissues develop from mesonephros.1 The germ cells develop within the primitive ectoderm but the specific cell of origin has not been identified. Germ cells have been detected 3rd week after fertilization in the primitive endoderm at the caudal end and in the dorsal wall of the adjacent yolk sac. They have also been found in the splanchnic mesoderm of the hindgut.2 Though originating in these areas, the germ cells can only survive in the gonadal ridges. Therefore, they have to migrate by the process of displacement because of growth of embryo and also by active amoeboid movement, along the dorsal mesentery to the genital ridges. The germ cells migrate from the yolk sac through the hindgut to their gonadal sites between 4th and 6th week of gestation. The factors involved in the process of migration are perhaps mediated through chemotactic activity and adhesive peptides.
Fig. 1.1: Undifferentiated embryonic ridges which will ultimately differentiate into gonads (gonadal ridge); male and female reproductive organs (wolffian or mesonephric and mullerian or paramesonephric ridges)
The germ cells are the precursors of ova or sperm. These germ cells multiply by the process of mitosis during migration. Before the gonads have differentiated either as testes or as ovaries (approximately 6th gestational week) the number of germ cells has reached a total of 10,000.36
The differentiation of the gonad either to a testis or to an ovary will be completed between 6th and 9th gestational weeks. This will depend on the sex chromosome complement of the developing embryo. If the chromosome complement is XY, testis will form and if it is XX, the gonad will be an ovary. The fetal testis produces two types of hormones while ovary produces only one. Hormones produced by testis are testosterone and antimullerian hormone (AMH) while ovaries produce only estrogen. The male phenotype is dependant on the products of the fetal testis while the female phenotype is primarily the result of the absence of testis and consequently the testicular product.4 Traditionally, it has been suggested that development of testis is an ‘active’ process, whereas development of ovary is a ‘passive’ procedure. This means that presence of ‘Y’ chromosome (SRY gene on Y-chromosome) will lead to testicular development while the absence of ‘Y’ chromosome will lead to development of ovary. However, any process of differentiation will require gene expression and therefore, ovarian development too may be also under the influence of yet some unidentified gene or genetic expression.
In the male fetus, Sertoli cells appear at 7 weeks gestation (either from coelomic epithelial or from mesenchymal cells) and by aggregation, they form the seminiferous tubules. The primordial germ cells are embedded within the seminiferous tubules. The Sertoli cells produce ABP (androgen-binding protein), necessary for transport of androgens produced by Leydig cells outside the lumen to within the lumen of seminiferous tubules necessary for spermatogenesis. Sertoli cells also produce inhibin.
Leydig cells are the steroidogenic cells of the testis. They develop from the mesenchymal cells at about 8 weeks of gestation.
The differentiation of the wolffian system begins with testosterone production. All androgen-sensitive organs do not require prior conversion of testosterone to dihydrotesto-sterone (DHT). In the process of masculine differentiation, development of epididymis, vas deferens and seminal vesicles is dependent on testosterone while, development of penis, urethra and prostate is under the influence of DHT. In the female, the regression of wolffian system is due to lack of locally produced androgens and antimullerian hormone (AMH).
MATURATION AND FERTILIZING COMPETENCE OF THE GAMETES
Oogenesis
Immediately following ovarian differentiation as female gonad at 6 to 8 weeks of gestation, the germ cells start rapid mitotic multiplication reaching to 6-7 million oogonia by 16-20 weeks.5 From this period, the germ cell content of the ovary will continue to decrease by a process known as physiological atresia (apoptosis), till the age of menopause around the age of 50 years or earlier when the stock of oocytes will be finally exhausted.
By mitosis, the germ cells will give rise to oogonia. The oogonia are transformed to oocytes as they enter the first meiotic division (at around 11-12 weeks) and arrest in prophase. The arrest of meiosis at the end of prophase continues throughout pregnancy till puberty or thereafter until the oocyte comes out of the follicle by the process known as ovulation.
Phases of Oocyte Maturation
Oocyte maturation includes both nuclear and cytoplasmic maturation. Basically the procedure involves reduction division of a diploid primitive germ cell into a haploid mature oocyte.7
Reduction division is specific for germ cells, which involves extrusion of half of the genetic material (DNA component) from chromosome. This means that after reduction division, a diploid germ cell will become a haploid gamete. This does not happen with other somatic cells, where a diploid mother cell will always divide to form a diploid daughter cell. Reduction division of germ cell leading to production of mature oocyte is known as meiosis.
Premeiotic Chromosomal and Genetic Components of Germ Cell
Before entering into the phase of meiosis, each germ cell just like any other somatic cell contains 46 chromosomes arranged in 23 pairs (n) and 46 strands of DNA material (c) in each chromosomal bar (n and c denote chromosome number and DNA component respectively). Hence in humans, chromosome number and DNA components are represented as follows:
- 2n = 2 n = 2 23 = 46 chromosomes
- 2c = 2 c = 2 23 = 46 DNA strands; i.e. each chromosomal bar contains one DNA strand.
Meiosis
In the 3rd gestational month, the germ cells of the female fetus enter into the phase of meiosis.
Meiosis occurs in two phases; Meiosis-I and Meiosis-II.
Meiosis-I
During this phase, nuclear and cytoplasmic maturation proceed simultaneously. There are four phases of maturation (Fig. 1.2) through which a diploid germ cell matures into a haploid fertilizable oocyte. These phases are (a) G1 – GAP-1 (b) S – synthesis of DNA (c) G2 – GAP-2 (d) M – metaphase.
G1 Phase: During this phase, proteins are incorporated within the cytoplasm. These proteins will organize into DNA strands within each chromosomal bar (2n, 2c; Fig. 1.3). Chromosomal bar with DNA strands are covered by nuclear membrane and exist at the center of the cell.
S Phase: One DNA strand of each chromosome will replicate into two. But the replicated DNA strands still remain in one chromosome. Thus at S phase, total chromosome and DNA strands are 2n, 4c (Figs 1.3 and 1.4).
G2 Phase: Uptil now, nuclear maturation was continuing and nucleus is now ready for first phase of reduction division.
M Phase: Replication of DNA strands within each chromosomal bar indicates onset of Meiosis-I, i.e. onset of nuclear maturation.
But further nuclear maturation remains arrested because of lack of maturation-promoting factor (MPF) proteins in the cytoplasm. Cytoplasmic maturation-promoting factor is the key force for complete nuclear maturation and reduction division (Meiosis-I arrest).
Now germ cell nucleus still contains 2n chromosomes and 4c DNA material (DNA strands).
Maturing Oogonia Forming Primordial Follicles
The mitotic (multiplying) germ cells enter into the phase of meiosis after they have migrated from their place of origin (splanchnic mesoderm of the hind gut and the dorsal wall of the yolk sac) to the gonadal ridges, where these cells will permanently reside and grow. After this stage, each maturing oogonium (arrested at Meiosis-I) is surrounded by one or two layers of somatic or stromal cell (future granulosa cells). The whole mass is now covered by a membrane called basal lamina and this will form the primordial follicle (Fig. 1.5). Those germ cells which do not acquire granulosa cell covering to form primordial follicles undergo degeneration.
Further Maturation of OocyteCompletion of Meiosis-I
The female fetus is born with the ovaries full of primordial follicles. But the maturation of oocyte remains incomplete till the child reaches the age of puberty. At puberty, under the influence of LH surge, cytoplasmic metaphase-promoting factor (MPF) appears which leads to germinal vesicle break down (GVBD). GVBD means disappearance of nuclear membrane (Fig. 1.6A). Chromosomes are now exposed in the cytoplasm. In the cytoplasm, a spindle-shaped equational plate is formed. Chromosomes arrange themselves at the equatorial plate of the spindle (Fig. 1.6B). Cytoplasm at the periphery of the oocyte becomes thick and will be known as zona pellucida. The area immediately beneath zona pellucida is known as perivitelline space. Outer covering of perivitelline space is zona pellucida and inner covering is thin condensation of cytoplasm.
Fig. 1.6A: Germinal vesicle nuclear membrane break down (GVBD). Chromosomes are exposed in the cytoplasm
At this stage, cytoplasmic division starts. One portion will get scanty cytoplasm to form the first polar body and the bigger one will form the nucleus of actual oocyte. By this time, the chromosome in each cytoplasmic segment has acquired a faint nuclear membrane. Extrusion of first polar body into the perivitelline space indicates completion of first meiotic division (Figs 1.7A and B). Oocyte now enters into metaphase II stage with complete nuclear maturation and is ready to be fertilized. This is the beginning of Meiosis II, which will be completed after fertilization with extrusion of second polar body.
2nd Meiotic Division
At metaphase-II, nuclear maturation is again arrested (arrest-II). Arrest is released after fertilization when sperm head enters into ooplasm. Centromere of each chromosome will divide and one set of chromatid will go to one pole and the other set will move to the opposite pole (1n, 1c). One set of chromatid will enter into perivitelline space (2nd polar body) and the other set will form the female pronucleus (Figs 1.8A to C).
Follicle Formation
The pattern and control of follicle formation and growth varies in different phases of a woman's life.
Early Intrauterine Period
Around 20 weeks of fetal life, follicle formation begins. Procedure of primordial follicle formation has already been described. Primordial follicles consist of an oocyte arrested at prophase of meiosis-I enveloped by a single layer of spindle-shaped pregranulosa cells surrounded by a basement membrane. Eventually, all oocytes are covered in this manner. This process of primordial follicular development continues until all oocytes in the diplotene stage can be found in follicles shortly after birth. By change of cell type, pregranulosa cells assume the shape of typical granulosa cells and a primary follicle is formed. By further differentiation of granulosa cells, a preantral follicle is formed.
At this stage, surrounding mesenchymal cells (future stroma of ovary) are compressed to form thin thecal layer. Appearance of Call-Exner body (coalescence of granulosa cells to form an antrum) will lead to formation of antral follicles. Antral follicles are found by the end of pregnancy but not in large numbers. During last stage of pregnancy, theca cells are found surrounding the follicles.6
The process of follicle formation, variable degree of ripening and atresia starts from intrauterine fetal life. In the first half of pregnancy, follicle formation and growth are autonomous and not gonadotropin dependant. Estrogen production is insignificant at this stage. Unlike the male, gonadal steroids are not essential for development of female internal genital organs. The development of the mullerian duct into fallopian tubes, the uterus and the upper third of vagina is totally independent of the ovary and is due to the absence of testis.
Late Intrauterine Period
In the second half of pregnancy, some of the follicles become gonadotropin dependant.7 The ovary develops receptor for gonadotropin in second half of pregnancy. But development of primordial follicles and the process of meiosis are not gonadotropin dependant.8 After birth, the ovary contains only 1-2 million follicles. Most of these follicles are in the stage of primordial follicles. A few are in the antral stage.
Neonatal and Childhood Period
In the neonatal and childhood period, the gonadotropin level is low, hypothalamic activity is suppressed and there is absence of pituitary response to GnRH. The ovarian follicles, however, are not quiescent. Though majority are in the primordial follicular stage, a few antral follicles, respond to gonadotropins by forming follicular cysts. However, they are not pathological; they form and regress. The follicles begin to grow and because of absence of gonadotropin support cannot reach the stage of full maturity and ultimately become atretic. The continued follicular growth followed by atresia leads to increase of the stromal compartment of the ovary. This leads to prepubertal enlargement of ovary both in size and in volume.
At Puberty and during Reproductive Years
With the advent of puberty because of continued atresia the germ cell population has been reduced to a total of 3,00,000 to 5,00,000 units. Out of these only 400 to 500 will be selected for ovulation. For every follicle that ovulates, approximately 1,000 will grow for variable length of time to achieve incomplete grades of maturity and will eventually become atretic.
It is now apparent that the time required for growth of primary follicle to an ovulatory follicle is approximately 85 days9 (Fig. 1.9). Major part of the growth occurs in environment deficient of gonadotropin. The number of follicles that will mature will depend on amount of gonadotropin available to the gonad and the sensitivity of the 11follicles to the gonadotropins. During reproductive years, the recycling process of follicle recruitment and growth, ovulation and corpus luteum formation are dependant on the complex but well-defined regulatory sequence of hypothalamic pituitary, gonadal interactions.
Recycling of menstruation and ovulation
From menarche to menopause, recycling of menstruation and ovulation will depend on delicately balanced interactions of 5 endocrine glands (unless interrupted by pregnancy and lactation). Of these, three are directly and two indirectly involved. The directly involved endocrine glands are CNS-hypothalamus, pituitary and ovary. The supporting endocrine glands, thyroid and adrenal cortex indirectly support events in menstruation and ovulation. It is essential that these glands should remain intact meaning thereby that they should not be damaged or their function should not be retarded by tumor, disease, chromosomal, genetic, autoimmune or receptor dysfunction. Even if they remain intact, the biological expression of their function will depend on regulatory ‘feedback’ mechanism of their products namely, endocrine, autocrine and paracrines.
Endocrine control of menstruation and ovulation
Elaborate description of endocrine control of ovulation is beyond the scope of this chapter. The important events leading to ovulation are summarized below:
Hypothalamus releases gonadotropin-releasing hormone (GnRH) under the influence of neurotransmitter, norepinephrine. GnRH stimulates the pituitary through receptors to synthesize and release two gonadotropins namely follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH in different periods of menstrual cycle stimulate two cells in the follicle, granulosa and theca to produce estrogen and progesterone respectively. The mechanism of follicular maturation and ovulation is primarily controlled by this "two cell-two gonadotropin" system, working through feedback mechanism. Ovulatory cycle is slightly different from menstrual cycle. Menstrual cycle starts from first day of one menstrual cycle and ends on first day of next menstrual cycle. But ovulatory cycle starts with decline of E2 and, therefore, starts in midluteal phase of one menstrual cycle and ends with rise of E2 in late follicular phase of the subsequent menstrual cycle.
Feedback Mechanism in Different Phases of Ovulatory Menstrual Cycle
From ovulation point of view, menstrual cycle is divided into four distinct phases; (a) Late luteal phase of previous menstrual cycle, (b) Menstrual and early follicular phase, (c) Mid follicular phase, (d) Late follicular phase and periovulatory events.
The events which occur in these different phases of ovulatory menstrual cycle are:
- Late luteal phase of previous menstrual cycle: Estrogen and progesterone start declining. Anterior pituitary is released from negative feedback effect of estrogen and progesterone. FSH primarily and LH to a some extent start rising. Under the influence of FSH, fresh batch of follicles are recruited which will compete for maturity in the next menstrual cycle.
- Menstrual and early follicular phase: Rise of FSH continues. LH rise is relatively slower than FSH. There are four functions of FSH: 12(i) Multiplication of granulosa cells—thereby increasing the size of the follicles, (ii) Production of enzyme aromatase which will convert androgen to estrogen, (iii) Increasing FSH and LH receptors in the follicles (more receptors for both FSH and LH in follicle destined to become dominant compared to those which will become atretic), (iv) Production of inhibin-B.Rise of FSH in early follicular phase will lead to increase in size of follicles and increased concentration of estrogen in the intrafollicular environment. Small amount of LH in early follicular phase is responsible for production of androgen primarily from the theca cells, which will be converted to estrogen by the enzyme aromatase. Excess androgenization of intrafollicular environment will lead to arrested follicular growth and improper oocyte maturation.
- Midfollicular phase: During the process of follicular growth, one of the follicles gets maximum FSH exposure. As a consequence, this follicle grows at a much faster rate than its fellow follicles. This follicle is known as dominant follicle (DF). The criterion for selection of this dominant follicle is not very clear. The dominant follicle is destined to rupture and ovulate. Selection of dominant follicle is completed by d5 or d6. As the levels of follicular estrogen and inhibin-B increase, there is negative feedback effect on the pituitary. This results in decline of FSH and gradual rise of LH. The growth of dominant follicle continues in spite of declining FSH. Obviously, the control of development of dominant follicle is taken over by rising level of LH.Endocrine events and folliculogenesis in ovulatory menstrual cycle have been diagrammatically represented in Figures 1.10 to 1.12).
- Late follicular phase and periovulatory events: FSH in the early and midfollicular phase has already produced receptors for LH in the dominant follicle. Under the influence of rising LH, the dominant follicle continues to grow. The remaining follicles become atretic. There is luteinization of granulosa cells with release of small amount of progesterone (0.8-1.8 ng/ml). This will lead to oocyte maturation by completing the first meiotic division. In spite of decline of FSH level in the late follicular phase estradiol continues to rise under the influence of LH and reaches a peak level when dominant follicle becomes functionally mature. Each mature follicle produces about 100 pg of estradiol. Estradiol while at the plateau level exerts positive feedback effect on hypotha-lamus. This peak level is maintained for about 48 hours (plateau). This peak level of estradiol is achieved when mean follicular diameter (MFD) measures between 17 mm and 23 mm. The peak level of estradiol during the period of plateau, through positive feedback on pulsatile GnRH, will stimulate the pituitary to release a bolus amount of LH (about 75 to 90 ng/ml)10 which is known as ovulatory ‘LH surge”. The follicle ruptures with release of mature oocyte. This is known as ovulation. Besides the dynamic event of ovulatory LH surge, intrafollicular prostaglandin and an enzyme collagenase, generated by plasminogen activator, plasmin are also involved in the mechanism of follicular rupture. The liberated mature oocyte is picked up by fimbria of the fallopian tube and proceeds towards ampulla, which is now ready to be fertilized if a mature sperm is available.
The entire mechanism of ovulatory menstrual cycle is schematically represented in Table 1.1.
Main Events in normal ovulatory cycle
Spermatogenesis
Following migration from hindgut to genital ridge during embryogenesis, total number of spermatogonia in each gonad will be approximately 3,00,000.
They undergo mitotic division and at puberty, number of spermatogonial stem cells has increased to about 600 millions.
The maturation of spermatozoa (described subsequently) starts at puberty and takes about 70 days to complete.11 After maturation, sperm requires further 12-21 days for transport to ejaculatory duct. During transit through seminal pathway, sperm acquires further maturity and capacity for sustained motility.12 Spermatogenesis may be considered under two broad headings:
- Molecular events
- Anatomical and endocrine consideration
Molecular Events in Spermatogenesis
There are three phases:
- Spermatogonial differentiation from prolife-rating diploid spermatogonial stem cells
- Meiosis: During this phase chromosome pairing and genetic recombination occurs, leading to formation of haploid spermatocyte and spermatid.
- Spermiogenesis: During this phase, series of changes take place which involve develop-ment of compact nuclear DNA, formation of acrosomal cap, midpiece and tail and ultimately resulting in the development of an adult spermatozoa.
Spermatogonial Differentiation
Germ cells migrating into male gonad (testis) will behave as spermatogonial stem cells. Two types of spermatogonial stem cells have been recognized. Type A and Type B. Spermatogonial Type A stem cells proliferate producing three types of spermatogonia:
- Proliferating and replicating spermatogonia which can repopulate testes after damage.13 They remain as reserve cells and only differentiate into adult spermatozoa on demand (Type A spermatogonial cells)
- Differentiating spermatogonial stem cells – precursors of future spermatozoa (Type B spermatogonial cells)
- Large number of cells which undergo cell death by apoptosis14
Once Type A spermatogonial stem cells differentiate into intermediate Type B and thereafter to adult spermatozoa, they become incapable of re-entering the pathway that produced Type A spermatogonial stem cells. A large number of spermatogonial stem cells undergo apoptosis. This indicates existence of a natural sophisticated monitoring mechanism by which many defective stem cells are removed.
Phase of Meiosis
During this phase, diploid proliferating and differentiating spermatogonial stem cells are converted into haploid gametes. This is a critical and unique event of genetic recombination.
Following events occur in sequence in the phase of meiosis (Fig. 1.13):
- Differentiating Type B spermatogonial stem cells will have diploid chromosomes with single DNA strand in each chromosomal bar (2n, 2c). At the beginning of Meiosis-I, the DNA strand in each chromosomal bar will replicate but the chromosome complement is still diploid. A primary spermatocyte is formed (2n, 4c). Chromosome number is represented by letter n and DNA strand by letter c.
- Chromosome pairing and crossing over of genes occur during this phase
- Crossing over is crucial in gametogenesis; this may lead to genetic defect and structural anomaly of sperm
- After chromosomal pairing and crossing over, the first meiotic division is completed. At this stage, each separated chromosome still contains two DNA strands
- Two secondary spermatocytes are formed; number of chromosome is reduced to haploid complement but the DNA content is still diploid (1n, 2c)
- The second meiotic division starts, giving origin to four spermatids with haploid number of chromosomes and haploid DNA (1n, 1c)
- For comprehensive meiotic division to occur, meiotic cells contain many novel proteins and enzymes
- These are essential for chromosome and DNA alignment, DNA breakage, recombination and DNA repair
- Occasionally, DNA repair mechanism in the phase of meiosis may be defective; e.g. there may be anomalies in chromosomal segregation, pairing and crossing over of genetic material, leading to germ cell arrest at the spermatocyte level, either primary or secondary
- Anomalies in pairing and chromosome segregation may lead to either structural or genetic abnormalities of spermatozoa
- Phase of meiosis continues till round spermatid is formed
Phases of Spermiogenesis (Maturation of Different Segment of Spermatozoa)
During this phase, the male gamete matures from round spermatid form into a highly elongated and polarized (sperm head in front, flagellum at rear) spermatozoa.
These changes occur at a time when repair capabilities of spermatozoa are deficient compared to those observed during the phase of meiosis.
Specific changes of different segments and their impact on anatomical and molecular integrity of spermatozoa are as follows:
- Head nuclear protein consisting of histone is replaced by protamine, producing a tightly compacted nucleus
- Chromatin condensation during spermio-genesis results in DNA occupying about 90% of the total volume of sperm nuleus. This is something special for sperm cell. Because in other somatic cells, nucleus occupies only 5% of the cell volume
- Displacement of histone and replacement by protamines may result in haploid genome damage
- If there is damage to these delicate changes, repair mechanism is defective (unlike in the phase of meiosis)
- In addition to nuclear DNA structuring, axoneme, tail with its outer dense fiber develop during this phase
- Mitochondria, the storehouse of spermato-zoal energy, also develops during the phase of spermiogenesis
Possible Adverse Impact of these Changes
- Massive changes during spermiogenesis may put tremendous load on haploid spermatozoa, leading to germ cell developmental blockage. This is one of the potential causes of male infertility
- Defects in synthesis or development of midpiece, tail, mitochondria may result in structurally abnormal spermatozoa with poor motility
- Mutation in protein, essential for compaction of sperm head nuclei, may result in spermatozoa with abnormal head
- Minor genetic defect may not alter spermatozoal morphology but may lead to production of genetically defective spermatozoa
- This will be a great concern for spermatid injection in ICSI
Anatomical Consideration and Endocrine Control of Spermatogenesis
Spermatogenesis occurs inside seminiferous tubules within the testes. Seminiferous tubules exist within interstitial cell compartment of testes in which Leydig cells, blood vessels and lymphatics are the more prominent components. Details of anatomical consideration of sperma-togenic development have been discussed in subsequent part of this chapter.
Endocrine Control of Spermatogenesis
Sertoli cells are directly under FSH control. FSH stimulates Sertoli cells for synthesis of androgen binding globulin (ABG) and inhibin. Androgen binding globulin (ABG) helps in transport of androgen from outside to inside the lumen of seminiferous tubules. Androgen is synthesized by Leydig cells which lie outside the seminiferous tubules. The other protein hormone inhibin, produced by Sertoli cells, controls pituitary FSH release. Lyedig cells have receptors for both LH and human chorionic gonadotropin (hCG). These gonadotropins stimulate testosterone synthesis. Therefore, testosterone synthesis by Lyedig cells is under LH control (Fig. 1.14).
ANATOMY OF HUMAN SPERMATOZOA (FIGS 1.15A AND B)
The different parts of spermatozoa are: (a) Heads, (b) Neck (also known as connecting piece) and (c) Tail.
Head
The sperm head contains nucleus. Nucleus occupies major area of sperm head with scanty space left for cytoplasm. There may be vacuoles in the nucleus. Existence of multiple vacuoles indicate abnormal sperm head. Anterior aspect of sperm head is covered by the acrosomal cap. Acrosomal cap has two layers—outer acrosomal layer and inner acrosomal layer. Acrosomal cap contains various types of enzymes of which two are significant namely, acrosin and hyaluronidase. Covering the outer acrosomal layer is the plasma membrane, which is the outermost layer of the sperm head.
Neck
Neck contains proximal and remnants of the distal centriole. Sperm centriole has an important function during fertilization. Following entry of sperm head into the oocyte, sperm centriole triggers up formation of female pronucleus inside the ooplasm.
Fig. 1.15B: Biochemical anatomy of sperm head and neck
PM —Plasma membrane
OAM —Outer acrosomal membrane
IAM —Inner acrosomal membrane
AS —Acrosomal sac containing enzymes; acrosin and hyaluronidase
C —Centriole
This area has been termed as the ‘black box’ (preserving all the information for fertilization) of the spermatozoa.
Sperm Tail
This is formed at spermatid stage. Sperm tail consists of three different segments: (a) Mid-piece (b) Main or Principal piece (c) Endpiece
Midpiece
Midpiece consists of 11 central axis fibrils surrounded by outer ring of 9 coarse fibrils. Mitochondria surround outer fibrils. Mitochondrium is involved in oxidative mechanism and also offers energy necessary for sperm motility.
Principal Piece and Endpiece
Coarse nine fibrils of outer ring diminish in thickness and terminate in endpiece.
Presence of dyenin bands (a type of protein) in the tail is the source of energy and offers propellant force for the spermatozoa. Absence of dyenin or deformity of tail are the factors responsible for sperm immotility.
Unique Characteristics for Human Spermatozoa
- Smallest cell in the body (length of sperm head is 4-5 µm)
- These cells (adult sperm cells) do not grow or divide
- Most polarized cell (head in front, flagellum at rear) in the body
- Fulfill their function outside the body in different individual (female genital tract)
- Sperm head has a large nucleus but lacks large cytoplasm
- Unique among mammals for presence of plenty of abnormal forms of spermatozoa found in the ejaculate
- Sperm chromosome structure is very complex
- Position of chromosomes in sperm nucleus is different from other somatic cells
- Two strands of DNA which make each chromosome are attached to a sperm-specific structure which is called "nuclear annulus" (NA)
- The NA-DNA sequences are located at the base of the nucleus
- Centromeres are located centrally and telomeres peripherally
- Folding of chromosomal p and q arms are flexible
- This specific chromosomal arrangement may be responsible for increased frequency of abnormal sperm shape and increased frequency of aneuploidies
It has been observed that sex chromosome and ‘g’ group (Chromosomes 21 and 22) are more susceptible romosomes to nondisjunction during spermatogenesis. Morphologically abnormal sperms (large head, round head, etc.) have either numerical or structural abnormalities of chromosome.
Existence of Anatomically and Physiologically Normal Male and Female Reproductive Organs
Embryogenesis
The wolffian (mesonephric) and mullerian (paramesonephric) ducts develop during the ambisexual period of embryonic development (up to 8 weeks). Thereafter one duct will persist and will give rise to sex-specific internal and external genital organs and the other will disappear by the third fetal month except for rudimentary vestiges.
The crucial factor, which will determine as which duct will persist or regress, is the presence or absence of antimullerian hormone (AMH) secreted by the testis. If antimullerian hormone is present, paramesonephric duct will disappear and wolffian duct will start developing. On the other hand, in the absence of antimullerian hormone, the paramesonephric duct will develop and wolffian duct will disappear.
Internal genital organs have the intrinsic tendency to feminize. In the absence of a Y-chromosome, a functional testis and antimullerian hormone, the mullerian system will develop into fallopian tubes, uterus and upper vagina. Therefore, development of female reproductive organ is passive and perhaps does not require an active stimulus.
On the other hand, differentiation of wolffian system requires active stimulation through testosterone production by testis.
Developmental Anatomy of Female Reproductive Organs
In the absence of antimullerian hormone, the two paramesonephric ducts come into contact in the midline to form a Y-shaped structure, which will form the uterus, tubes and upper part of vagina.15 The fallopian tubes, uterus and the upper portion of the vagina are created by fusion of the mullerian ducts by 10th week of gestation. By 22nd week of gestation, canalization of the uterine cavity, cervix and vagina is completed. Smooth muscle cells and uterine stroma will originate from the mesenchymal tissue underneath the epithelium. By 20th week, uterine mucosa has differentiated into the endometrium. Endometrium, one of the most complex tissues of the human body, is essential for reproduction. Endometrium is cyclically changing in response to estrogen and progesterone of the ovulatory menstrual cycle and to a complex interaction among its own autocrine and paracrine factors.
Functional Anatomy of Male Reproductive Organs (Fig. 1.16)
Male reproductive organs consist of: (a) Testis, (b) Epididymis, (c) Vas deferens, (d) Ampulla of vas, (e) Seminal vesicles, (f) Prostate, (g) Cowper's and urethral glands, (h) Penis.
Testis
Human testis is ovoid in shape and is located within the scrotal sac. The length and weight are approximately 4.5 cm and 34-45 g respectively. The reproductive components within the testis are: (a) Leydig cells, (b) Seminiferous tubules, (c) Sertoli cells.
Leydig cells are found in clusters and form about 5-15% of the total volume of testis. They lie outside the lumen of seminiferous tubules and are the source of androgen production. The number of Leydig cells in both testis in a 20-year-old male is 700 million and diminishes by one-half by the age of 60.
Seminiferous Tubules
Seminiferous tubules are the sites of spermato-genesis. They are long, loop-like convoluted ducts with both ends terminating in the rete testis.
The number of seminiferous tubules is 600-1200 with an estimated total length of 250 m.16 Spermatozoa and fluid originating in the tubules are transported to the rete testis and then to the epididymes. Rete testis acts as a valve that controls the flow.17
Sertoli Cells
Sertoli cells line the inner aspect of the basement membrane of seminiferous tubules. The germinal cells are arranged and scattered in between the Sertoli cells. The undifferentiated spermatogonia are located near the basement membrane, and the more advanced forms are arranged successively at higher levels of Sertoli cells near the tubular lumen. Sertoli cells are more than nursing cells to the adjacent germinal cell. The adjacent Sertoli cells are joined to each other by inter-Sertoli ‘tight’ junctions. Sertoli-cell ‘tight’ junctions subdivide the seminiferous tubules longitudinally into basal and adluminal compartments. These tight junctions of the adjacent Sertoli cells form an impermeable "blood-testis" barrier. Germ cells develop up to the stage of primary spermatocytes within the basal compartment which has free access to the extratubular environment. Secondary spermatocytes continue their development in the adluminal compartment. Because of the blood-testis barrier, any factor influencing the latter stages of spermatogenesis must be mediated through the Sertoli cells.
Epididymis
This is an elongated structure which extends from cranial to caudal pole of testis. It begins from efferent ducts and continues up to vas deferens. The epididymis consists of three parts—caput, corpus and cauda. Coiled efferent ducts emerge from the rete testis and constitute most of the caput epididymis. The length of epdidymes and associated ducts has been measured to be 5-6 m.18 Epididymis synthesizes certain compounds that are secreted into the lumen of the canal. These include protein, carnitine, lipids, glycerophosphoryl choline (GPC), carbohydrates, steroids and other 20small molecules. Carnitine is an epididymal marker and helps in preserving sperm viability and stimulating motility after ejaculation.
Vas Deferens
It is a tube, 35-45 cm long with a diameter of 0.85 ± 0.7 mm. It extends from the tail of the epididymes, runs along the medial side of the spermatic cord, through the inguinal canal and ends in a glandular enlargement on the medial side of the seminal vesicle. This terminal glandular enlarged portion is known as ampulla of vas. Ampulla of vas finally fuses with the neck of the seminal vesicle to form the ejaculatory duct. Ejaculatory duct passes through the prostate and opens into the floor of prostatic urethra at the level of verumontanum. Ampulla of vas helps in continuous maturation process of spermatozoa which has started in the epididymis.
Prostate Gland
Prostate gland is the largest accessory male sex gland. In young and middle-aged adults, the gland is 3-4 cm in diameter and approximately 20 gm in weight. The two ejaculatory ducts pierce the prostate obliquely and pass into the interior of the gland. Within the prostate, they converge, decrease in diameter and terminate in the floor of the prostatic urethra, in the region known as verumontanum.
Prostate fluid accounts for 15-30% of the total ejaculate volume. Prostatic fluid contains a number of constituents of which acid phosphatase, plasminogen activator, seminin, zinc, magnesium and calcium are significant. Plasminogen activator and seminin cause lysis of seminal clot. Acid phosphatase can be used as prostatic marker.
Seminal Vesicles
These are paired, highly convoluted pyriform glands. Each vesicle is 5-6 cm long and 1-2 cm wide. They lie lateral to ampulla of vas deferens, posterior to urinary bladder and superior to prostate. About 70% ejaculate originates in seminal vesicles.
Of the various secretion of seminal vesicles, fructose and prostaglandins are important. Though constituents of seminal plasma are not absolutely essential for fertilization, the secretion may optimize conditions for sperm motility, survival and transport in both the seminal pathway and the female reproductive tract.
Cowper's (Bulbourethral) and Urethral Glands
They are paired bodies 3-5 mm in diameter which are homologous to Bartholin's glands in the female. They secrete droplets of mucin which help in urethral lubrication. Scattered accessory glands can be found throughout the male reproductive tract.
Penis
Penis is composed of three cylindrical masses of erectile cavernous tissue, blood vessels, lymph and nerves. The erectile tissue within the penis is a labyrinth of irregular blood sinus and spaces. Male sexual function consists of: (a) Erection: This is both reflexogenic and psychogenic, (b) Accessory glandular secretion and seminal emission and (c) Ejaculation.
EVENTS LEADING TO RELEASE OF GAMETES, THEIR TRANSPORT, FUSION AND FERTILIzATION, CLEAVAGE AND IMPLANTATION
Egg Release and Transport
Prior to ovulation, the oocyte completes its first meiotic division under the influence of midcycle ‘surge’ of LH. Thereafter, it enters into second meiotic division and is arrested at second metaphase. The dominant follicle gradually moves up to the surface of the ovary. After follicular rupture, the ovum is picked up by fimbrial end of the fallopian tube by sweeping movement. Entry into the tube is facilitated by 21muscular movements that bring the fimbriae into contact with the surface of the ovary. Variations in the method of ovum pick up surely exist because women may achieve pregnancy even with one ovary and a single tube located on contralateral side. Furthermore, pregnancies have been recorded following direct intraperi-toneal insemination (DIPI).19
Egg and subsequently, zygote and embryo transport involves the time that elapses from ovulation up to the time of entry of compacted morula into the uterus. The egg is fertilized in the ampulla of the fallopian tube.
The epithelium of fallopian tube consists of two types of cells—ciliated and nonciliated. They undergo cyclic changes of the menstrual cycle.20 The nonciliated cells secrete cytoplasmic components during passage of the egg or embryo providing important metabolic factors essential for transport and implantation. Ciliary movement and tubal muscular contractions are both involved for transport of egg from ampulla towards the uterus.
Following ovulation, the egg is inside the ampulla and subsequently when fertilized, in the proximal segment of the fallopian tube for 2-3 days. The transport time from ampulla to uterus for the fertilized oocyte is approximately 3 days. In human, 80% of this time, period is spent in the ampulla (Fig. 1.17)
In most species, the fallopian tube appears to be essential for full development of embryos. Because uterine fluid during the first 48 hours following ovulation remains toxic to the egg. In humans also, if the endometrium is in the reduced or advanced stage of development compared with the developmental stage of the fertilized egg, implantation may fail. This may not be always true. Because, pregnancies have been recorded following Estes operation where the eggs are ovulated directly inside the uterine cavity. Moreover, when fertilized donor eggs are cleavage and implantation transferred to women, who are receiving hormone supplementation, a larger implantation window is created in the endometrium when the blastocyst will implant. Hence, a perfect synchrony between the incoming embryo and developing endometrium is not absolutely essential.
Sperm Release and Transport
The sperms reach caudal epididymis approximately 72 days after initiation of spermato-genesis. The caudal epididymis is the storehouse of the sperms, which should be available for ejaculation. Semen coagulates immediately following ejaculation. But this is liquefied 20 minutes to 30 minutes following ejaculation by an enzyme derived from prostate gland. Most of the sperms become immotile in the acid pH of vaginal secretion. The alkaline pH of semen offers some transient protection for the spermatozoa to survive but majority of sperms are immobilized within 2 hours. The more active sperms by their own motility enter into the cervical canal and then into the uterine cavity. It is generally believed that cervical mucous has a filtering action. Sperm antibodies on the sperm head interacts with cervical mucous and inhibits sperm motility and entry into uterine cavity. Similarly, less active 22sperms are unable to swim up into the uterine cavity. The number of active sperms remaining within the cervical mucous remains constant for 24 hours and after 48 hours only few sperms are left behind in the cervical canal.21 In many animals isthmus of the fallopian tube is believed to be the storehouse of sperms but in humans, cervical mucous rather than fallopian tube seems to be the reservoir of sperms.22 Approximately 80 to 100 million sperms are deposited in the vagina and out of these, only a few are able to achieve proximity of the egg in the ampulla.22 Majority of sperms are lost in the vagina either by enzymatic digestion or by phagocytosis.
In the uterus and in the fallopian tube, the sperms acquire two very important functions viz. capacitation and hyperactivated motility.
Capacitation and Hyperactivation
While in the female genital tract, (cervix, uterus or in the fallopian tube) the sperms undergo some physiobiochemical modifications which is called "Capacitation".23 Capacitation involves removal of seminal plasma factors coating the surface of the sperm, and modification of some biochemical properties of the sperm head membrane. Capacitation will help in acrosome reaction and acquisition of hypermotility. These factors are the basic requirements for sperm penetration through cumulus cells and zona pellucida; the outer vestments surrounding the oocyte.
Preparatory changes in Gametes before Fertilization
Further Sperm Maturation (Acrosome Reaction)
The fusion of plasma membrane with outer acrosomal layer followed by breakdown of the membranes to allow escape of acrosome cap contents is known as ‘acrosome reaction’.24 Sperm capacitation is a prerequisite change for acrosome reaction. While passing through cumulus, the sperms do not release acrosin.25 The acrosome cap contains enzymes, the important ones are hyaluronidase and acrosin. Hyaluronidase digests the cumulus cells (Figs 1.18 and 1.19) and acrosin helps penetration of zona pellucida. Sperm hypermotility induced by capacitation is also an essential step for rapid sperm entry through zona pellucida.
Further oocyte maturation
Apart from meiotic divisions of the oocytes, there is an influx of extracellular calcium in response to estradiol which improves the chances of fertilization. This is followed by secondary rise in calcium ions from intracellular stores. This is characterized by wave-like oscillations within the ooplasm.26 This transient increase in intracellular calcium, which is estrogen dependant, improves quality of oocyte and increases the chances of fertilization.
23These events are not related to oocyte meiosis. However, improved fertilization following ‘estradiol induced calcium increase’ indicates the important role of intrafollicular estradiol for overall oocyte maturation.
FERTILIZATION
Fertilizable life span of oocyte ranges between 12 hours to 24 hours. Similarly, fertilizable life period of the spermatozoa ranges between 48 and 72 hours. Majority of pregnancies occur when coitus takes place within 3 days prior to ovulation.27 The process of fertilization consists of a series of events occurring in both sperms and eggs. Contact of a single sperm with egg is due to chemotactic activity exerted by the egg on the sperm.
Events in Sperm-Egg Interaction
There are three types of glycoproteins in zona pellucida. These are known as ZP1, ZP2 and ZP3 of which ZP3 is the most abundant.28 ZP3 is the primary ligand for the sperm and ZP2 is responsible for zona reaction following sperm penetration to prevent polyspermy. Penetration through the zona is rapid and mediated by acrosin, a trypsin-like proteinase.29
Spermatozoa enters perivitelline space at an angle. Then there is binding between inner acrosomal membrane of the sperm head and oolemma (the outer membrane of ooplasm). This induces cortical and zona reactions which prevent entry of another spermatozoa into the oocyte, thereby blocking polyspermy.
Pronucleus Formation—Syngamy—Embryonic Cleavage
Approximately, 3 hours after entry of sperm head in the oocyte, the second meiotic division is completed and the second polar body is released with a haploid complement of oocyte chromosomes. The remaining haploid number of chromosomes in the oocyte will form the female pronucleus. The nucleus of the sperm head undergoes decondensation and the male pronucleus is formed. The male and female pronuclei migrate towards each other. When they come in close proximity, the limiting membranes break down. There is exchange of chromosome material between the male and female pro-nucleus. The process is known as syngamy. A spindle is formed on which chromosomes become aligned. The stage for first mitotic cell division has now been organized and with first cell division a zygote is formed. Embryonic genomic activity starts between 4 and 8 cell stages of cleavage, 2-3 days after fertilization.30 Normal embryonic genomic activity will now control further cell division into morula and blastocyst.
Preimplantation preparatory changes
Prior to implantation both developing endo-metrium and incoming embryo undergo some preparatory changes.
ENDOMETRIAL PREPARATION FOR IMPLANTATION (ENDOMETRIAL RECEPTIVITY)
Endometrial preparation for implantation (endometrial receptivity) is a complex procedure which involves interaction of several molecules generated by following changes within the endometrium.
- Endocrine regulation (E2 and progesterone receptors)
- Physiological changes (pinopod formation)
- Biochemical changes (integrin, selectin, cadherins)
- Immunomodulatory alteration (formation of protective cytokines interleukin–3, 5, 6, 10, 13 and suppression of natural killer (NK cells)
- Endometrial genetic expression (Hoxa-10 genes, troponin, transformation growth factor alpha, PDG-a, etc.).
Summary of Molecular Events Leading to Initiation of Optimal Endometrial Receptivity
- Endocrine regulation is the primary event in the process of commencement of endometrial receptivity31
- Estradiol in the follicular phase induces development of estrogen receptors (commonest is ER alpha). Endometrial receptivity depends on estrogen receptor (ER) development, which is genetically coded for each individual. ER development varies from woman to woman and in the same woman from cycle to cycle
- The classic estrogen receptor (ER-alpha) increases in response to estrogen during the proliferative phase, and is diminished in response to progesterone during the secretory phase
- Estrogen stimulates a large number of genes including C-FOS and C-JUN – cell cycle protein in addition to estrogen receptor itself. ER alpha, however, disappears at the time of implantation. This indicates that continued ER alpha expression may be detrimental to the development of endometrial receptivity. Persistence of ER alpha in the preovulatory period indicates lack of endometrial receptivity (as in endometriosis)
- Progesterone response in augmenting receptivity is possible only on estrogen primed endometriun. Progesterone suppresses ER alpha activity. Progesterone stimulates endometrial responsiveness through estrogen primed gene expression, one of the best known genes is endometrial Hoxa-10 gene expression which rises at the time of ovulation.32
- Progesterone is also involved in immunomodulatory changes through PIBF (progeste-rone induced blocking factor). This involves accelerated response of protective cytokines (IL-3, 4, 5, 6, 10, 13) and suppression of natural killer (NK) cells
- In addition, progesterone also induces several implantation related biochemical molecules—also known as adhesion molecules. Most important one is beta-integrin. Basement membrane and matrix substrate include collagen, fibronectin, laminin, enactin and tenascin which help to guide the trophoblast through basement membrane and stromal matrix for anchorage on maternal decidua
- Physiological changes induced by pro-gesterone lead to formation of ‘pinopodes’ on the surface epithelium. Pinopodes form as a result of cystic changes in the surface epithelial microvilli. Pinopodes serve to absorb uterine fluid from the uterine cavity forcing the blastocyst to be in contact with the endometrial epithelium
- In summary, E2 induces and progesterone activates potential markers of endometrial receptivity. But both E2 and progesterone receptors must disappear to allow these markers to generate optimum uterine receptivity
Embryonic preparation for implantation and preimplantation signals
Signals
Embryo while still in the fallopian tube signals to the mother as it prepares for implantation. In response to this signal, mother produces early pregnancy factor (EPF).35 EPF has immuno-suppressive property and is associated with cell proliferation and growth.
After reaching the uterine cavity in the compacted morula or blastocyst stage, the embryo produces βhCG which is essential for embryo hatching and embryonic implantation. hCG liberated by the embryo (even before implantation) will signal the corpus luteum to 25secrete higher level of estradiol and progesterone which can be detected in the maternal serum.36 Optimum function of corpus luteum is essential for endometrial bed preparation, implantation and maintenance of pregnancy during the first 9-10 weeks of gestation.
Embryo Hatching
A prerequisite change of the embryo for implantation is "hatching". After its entry into the uterine cavity in the morula (16 to 64 cells) or blastocyst stage (30 to 200 cells), it remains in the uterine cavity for two days still encapsulated by zona pellucida. Zona must be lyzed before the embryo can attach to the maternal decidua. Lysis of zona and escape of embryo is known as zona hatching. Zona hatching is accomplished by components of uterine fluid as well as by blastocyst movement. By this time, blastocyst has differentiated into inner cell mass (embryo) at one pole and trophectoderm (placenta) at the other pole with a cystic cavity (blastocele) in between. Zona pellucida becomes thin and ultimately disrupts through which the inner cell mass wriggles out to differentiate into three primitive layers of future fetus, namely ectoderm, mesoderm and endoderm. Initially, primitive embryonic plate is bilaminar consisting of ectoderm and endoderm. After certain period of growth, a third layer, mesoderm originating from ectoderm insinuates between ectoderm and endoderm so that embryonic plate now becomes trilaminar. All tissues and organs of the growing fetus will develop from these three layers. Trophoectoderm and inner cell mass are essential for implantation. Even if the endometrial preparation is adequate for implantation, this may not occur if the embryo is not at the proper stage of development. This dys-synchrony is often observed in in vitro fertilization procedure when there is a risk of failure of zona hatching after the embryo has been transferred into the uterine cavity. In in vitro procedure, the zona may become hard because of its exposure to culture medium. However, under favorable circumstances, the process of implantation starts with embryo-endometrial contact.
Implantation
Implantation occurs in four stages: apposition, adhesion, penetration and invasion (Fig. 1.20). This will occur when there is synchrony between optimal endometrial receptivity and appropriate blastocyst development. The appropriate interaction between the preimplantation embryos and the maternal endometrium is at least partly controlled by cytokines, growth factors and growth factor receptors. Both the endometrium and the preimplantation embryo exhibit several of these cytokine/growth factor receptor pairs during implantation.37,38
While cytokines help in attachment and adhesion, the growth factors are involved in stomal invasion, vascular penetration and finally nidation. The cytokines and growth factors have wide range of family members. Some of them help in implantation others antagonize implantation. Accordingly they are broadly known as ‘adhesive’ and ‘antiadhesive’ molecules.26
Cytokines consist of different members of interleukin family (IL) while growth factor consists of VEGF (vascular endothelial growth factor), tumor necrosis factor (TNF), integrin, laminin, fibronectin, selectin, cadherin, entactin, tenascin, etc.
Both these cytokine and growth factor may react through ‘embryo-endometrial’ dialogue by ‘helpful’ or ‘harmful’ response. According to these responses, either the pregnancy is maintained or this is rejected.
Embryo-Endometrial Contact
Apposition and Adhesion
As the blastocyst comes into close contact with the endometrium, the microvilli on the surface of the trophectoderm will interdigitate with those on the luminal surface of the decidual epithelial cells. At this stage, the cell membranes are in close contact and junctional complexes have been formed. Thereafter the early embryo cannot be easily dislodged.
Penetration and Invasion (Anchorage and Placentation)
Trophoblast is invasive in nature. The early embryo secretes a variety of enzymes (e.g. collagenase and plasminogen activators) and these are important for digesting the intracellular matrix that holds the decidual cells together. This highly proliferative phase of trophoblast in early embryogenesis is regulated by many growth factors and cytokines produced in both fetal and maternal tissues. This is essential for effective anchorage and at the same time limiting the extent of trophoblastic invasion of maternal decidual tissue. Invasion requires the expression of integrins stimulated by insulin-like growth factor II and inhibited by transforming growth factor-2.39
The trophoblast differentiates into two layers—cytotrophoblast (the cellular layer) and the syncytiotrophoblast (the acellular layer). Cytotrophoblast invades the uterine spiral arterioles and allows the maternal blood to enter into the decidual lacunae created by trophoblastic invasion. These lacunae are built up by cytotrophoblastic cells which remain in contact and are constantly bathed by maternal blood for fetal nutrition and exchange of gaseous materials. Penetration of the maternal decidua will depend on factors which are capable of suppressing the maternal immune response to paternal antigens. The endometrial tissue is responsible for immune suppression by synthesizing proteins in response to the blastocyst even before implantation.40 Usually, the genetically abnormal embryos are rejected by the decidua. It may be possible that the abnormal embryo cannot produce a signal in early pregnancy that can be recognized by the mother.
CONCLUSION
The fundamentals of normal reproduction involves the fulfillment of three basic requirements. These are availability of mature male and female gametes, anatomically and functionally competent reproductive organs of both the partners and finally, the ability of the gametes to achieve biological competence for fertilization and implantation in the female genital tract. The gametes, originating as germ cells in the hindgut and yolk sac, migrate to the genital ridge (future gonad) to become either oocyte or spermatocyte. Genital ridge differentiates into ovary or testis in response to chromosomal complement of the embryo. For spermatocyte, the maturational changes are nearly completed within the testis as the diploid spermatogonia are reduced to haploid gametes before leaving the testis. In case of oocytes, the first meiotic division which starts in the intrauterine life remains arrested at prophase and is completed before ovulation. The 27maturational changes of gametes and their periodic release from testis and ovary are influenced by endocrine, autocrine and paracrine factors. DNA in the nucleus of the sperm head, centriole in the neck of the sperm and release of mature haploid oocyte from the dominant follicle are the vital segments of gametes which are actively involved for successful fertilization.
Anatomically normal and physiologically active male and female reproductive organs are essential for further maturation and for accelerating fertilizing potential of the gametes. For this, sperm requires additional energy because it has to travel a long distance to penetrate the outer vestments of the oocyte namely the cumulus matrix and zona pellucida. The additional energy is acquired by the sperm in different phases. As it travels through the seminal pathway in the male genital tract, sperm acquires motility and other biochemical back up from secretion of epididymis, vas deferens, seminal vesicles and prostate. In the female genital tract climax fertilizing potential of the sperm is completed by two very significant changes namely capacitation and acrosomal reaction. Oocyte after ovulation, travels relatively a shorter distance before it reaches the sperm for sperm-ovum interaction. During this short journey, there is intracellular influx of calcium which accelerates its fertilizing ability.
The biological competence of gametes induces sperm-ovum interaction, fertilization, formation of pronucleus and cleavage into a two-celled zygote. Zygote, cleaving into an embryo, sends signals to the maternal tissues, which now prepare to receive the incoming embryo for implantation.
Implantation is a complex procedure, which occurs in four stages namely, attachment, adhesion, penetration and anchorage. Effective implantation requires optimal synchrony between developing embryo and receptive endometrium. Besides the influence of steroid hormones viz. estrogen and progesterone, the process of implantation is additionally influenced by various biochemical, immunomodulatory molecules and physiological changes occurring within the endometrium during the period of implantation window. These events are basically controlled by molecular expression of the genetic coding of the endometrial cell induced by estrogen in the proliferative phase and activated by progesterone in the luteal phase. The genetic coding expression varies from woman to woman and in the same woman from cycle to cycle.
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