- Basics of Human Reproduction: As we Understand it Now
- Role of History Taking and Clinical Examination in an Infertile Couple
- Preconceptional Evaluation of a Couple
- Assessing Ovulatory Function and Ovarian Reserve
- Tubal Evaluation: An Eternal Dilemma
- Mechanical Issues in Fertility: How to Evaluate?
- How to Assess the Receptivity of the Human Endometrium
- Assessment of the Male
- Role of Semen Analysis in Understanding Fertility Potential
- Role of Endoscopy in Infertility
- Ultrasound and Color Doppler in Fertility
INTRODUCTION
Reproduction as we understand happens when a sperm fertilizes the ovum to form a zygote which would further develop into an embryo, fetus, and deliver as a child in future. But the process of development and maturation of the gametes and fertilization is very complex. Understanding of biology of gametes and human reproduction has led to evolution of technologies and techniques to help the infertile couples, over the years. The techniques used, try to mimic the natural way of conception. Thus, it is very important to understand the anatomy of the reproductive tract and physiology of the conception to evaluate the cause of infertility and to provide proper necessary treatment.
The union of sperm and egg is one of the essentials of reproduction; however, the remote site of occurrence of this event and microscopic dynamic nature, made it difficult to study for many years. Greater understanding of sperm and egg development and union provides major benefits of the clinical application of the assisted reproductive technologies. This chapter includes understanding of gametogenesis, sperm and egg transport, fertilization, implantation with associated genetics and immunology.
HISTORY
In 1677, Anton van Leeuwenhoek of Delft, Holland, by Galileo's microscope described the “little animals of the sperm.” After 198 years, Wilhelm August Oscar Hertwig, in Germany, demonstrated the union of sperm and egg, fertilization, in the sea urchin. In 1951, Min Chueh Chang and Colin Russell Austin made discovery of sperms spending some time in the female reproductive tract before acquiring ability to fertilize the egg was made in rat and rabbit respectively.1,2
MALE REPRODUCTIVE SYSTEM
Male reproductive system consists of scrotum and testes within it, epididymis, vas deferens, ejaculatory duct, urethra, penis and accessory glandular structure (SVs: seminal vesicles, bulbourethral glands, and prostate).
The testes contain two anatomical units:
- A network of seminiferous tubules comprised of:
- Sertoli cells: They contain follicle-stimulating hormone (FSH) receptors, and synthesize inhibin B, anti-Müllerian hormone (AMH) and androgen-binding protein.
- Germ cells: This develops into sperm by spermatogenesis.
- An interstitium containing Leydig cells that produce androgens (testosterone mainly) and peritubular myoid cells. Luteinizing hormone (LH) receptors are found on Leydig cells (Flowchart 1).
Tight junctions between the Sertoli cells separate the tissue into two functional compartments (basal and adluminal), which are distinct structure and have limited permeability for diffusion of macromolecules between them. The basal compartment consists of the outer layers of the seminiferous tubules containing the spermatogonia. The adluminal compartment contains the inner portion of the seminiferous tubules, including primary spermatocytes and more advanced stages of spermatogenesis.
Spermatogenesis (Flowchart 2)
The male gamete is called as spermatozoa or sperm. Spermatogenesis/spermiogenesis is a complex, multistep process involving the proliferation and differentiation of spermatogonia into mature sperm, taking place in testes.4
After the primary germ cells (PGCs) reaches the testes in the intrauterine life, they divide into Type A1 spermatogonia, which has ovoid nucleus with chromatin and nuclear membrane. They are found near the basement membrane and are sperm stem cells. They are capable to divide into Type A2 spermatogonia or self-renewal into Type A1 spermatogonia. Type A2 spermatogonia forms Type A3 spermatogonia and successively Type A4 spermatogonia. All these have capacity of self-renewal. If Type A4 spermatogonia do not undergo self-renewal, it can undergo either cell death by apoptosis or differentiates into intermediate spermatogonium stage with the onset of puberty. Those who enter the process of spermatogenesis divide mitotically into Type B spermatogonia. From this, they divide mitotically to form Primary spermatocyte, which each undergo first meiotic division to form a pair of Secondary spermatocytes, which complete the second division of meiosis. The haploid cells thus formed are called spermatids. With the division from Type A1 spermatogonium to spermatid, the cells move farther from the basement membrane toward the lumen. The spermatids located at the border of the lumen, loose their cytoplasmic connection and become sperm.3 The seminiferous tubules empty into the network of tubules called as rete testes, through which sperm move to the epididymis.
Optimal spermatogenesis requires temperatures lower than body temperature, hence mammalian testis are located outside the body cavity.
Microscopic structure of sperm consists of:
- Head: Containing nucleus and acrosome
- Mid-piece: Containing mitochondria
- Tail: Containing axial filament and end piece.
Sperm Maturation and Transport
Sperm formation takes approximately 65 days from the spermatocyte stage,3 and the transport of sperm through the epididymis to the ejaculatory ducts requires about 14 days. Maturation of sperm begins during their passage through the epididymis, as evidenced by enhancement of motility, but the final maturation (or capacitation) of sperm take place in the female genital tract after ejaculation. In epididymis, the sperm acquire surface glycoprotein (beta-defensin 126) that protects the sperm while they travel through the cervical mucus. The caudal epididymis stores sperm and make them available for ejaculation, thus providing repetitive fertile ejaculations. Adequate testosterone levels in circulation and maintenance of normal scrotal temperature is required for optimal sperm-function preservation and storage.4 During ejaculation epididymal content is mixed with secretion from the prostate gland and seminal vesicle (SV).5
Otherwise the epididymis is believed to play only the storage role, as the sperm obtained by testicular biopsy is directly injected into an oocyte in intracytoplasmic sperm injection (ICSI), and is very successful in achieving fertilization and pregnancy.6
Semen forms a gel almost immediately following ejaculation but then liquefies in 20–30 minutes by prostatic enzymes. The alkaline pH of semen provides temporary protection for the sperm from the acidic environment of the vagina, but most sperm left in the vagina are immobilized within 2 hours. The most motile and fortunate sperms gain entrance into cervical mucus and then enter into the uterus. This entry is rapid, and sperm have been found in mucus 5within 90 seconds of ejaculation.7 Contractions of the female reproductive tract occur during coitus and male forces (the flagellar activity of the sperm) are responsible for successful transport. The sperm swim and migrate through pores in the cervical mucus that are smaller than the sperm head; therefore, the sperm must actively push their way through the mucus. Those sperm incapable in motility are held at the level of cervical mucus.
FEMALE REPRODUCTIVE SYSTEM
- It comprises of two ovaries (female gonads) on each side of the body, two fallopian tubes, uterus, cervix and the vagina. Vagina is the female copulating organ and the sperms are deposited in the vagina after ejaculation.
- The function of the ovaries is to cyclically produce ovum for fertilization and of steroidogenesis by secreting cyclical estrogen and progesterone.
Oogenesis (Flowchart 3)
- Embryologically ovaries appear as early as 5th intrauterine week, and the PGCs start dividing by mitosis as early as 6–8th intrauterine week, reaching to 6–7 million oogonias by 16–20th intrauterine week. This represents the maximum ovarian capacity for the germ cells, mitosis does not occur after that.
- With the commencement of meiosis I by 7–8th intrauterine week the oogonia get arrested at the diplotene stage of prophase and they are now called as primary oocytes. The oogonia not undergoing meiosis are degenerated by apoptosis. So at the time of birth ovaries are left with only 2 million, and then 300,000 primary oocytes till puberty, out of which approximately only 400 will ovulate in reproductive years.
- Onset of puberty is marked by activation of HPO axis, and a cohort of oocytes is recruited by nonhormonal followed by hormonal selection for development and maturation by ovarian cycle every month. The oocyte of the dominant follicle undergoes resumption and completion of meiosis I with the onset of LH surge and the first polar body (containing 23X chromosomes and small amount of oocyte cytoplasm, in the perivitelline space) is released; now it is called as secondary oocyte. Just before the ovulation, it gets arrested at metaphase stage of second meiotic division, which is completed after sperm penetration.
- Thus, the ovum available for fertilization has 23X, and the rest 23 (X or Y) chromosomes come from sperm after fertilization.6The second polar body containing 23X chromosomes from the oocyte with cytoplasm is released in the perivitelline space. Nowadays polar body biopsy from the metaphase II ovum, for patients undergoing IVF is possible to detect chromosomal abnormality of the ovum in case of high maternal age.8
- There is continuous loss of the oogonias and the oocytes by apoptosis from the intrauterine life to 45–50 years after birth. When the ovarian reserve gets exhausted it leads to menopause. The amount of the ovarian reserve can be measured by a marker AMH that is secreted by the preantral and small antral follicles in the ovarian cycle.9 It suggests the number of the primordial follicles recruited from the primary pool of ovary. It is believed to be constant marker throughout the cycle.10
Physiology of Menstruation
The studies and ideas, over the past few decades have made a scientific progress in understanding the nature of the normal menstrual cycle and role of higher center and gonadal hormones in it. The normal cycle can be divided into:
- Follicular phase
- Ovulation
- Luteal phase.
Follicular Phase
This phase occurs over a period of 10–14 days, featuring action of hormones and local autocrine-paracrine peptides, leading to growth of the cohort of primordial follicles containing the follicle destined to ovulate through various stages of development.
The primordial follicle:
- The primordial follicle is resting stage of follicle containing oocyte, arrested in the diplotene stage of prophase of meiosis I, surrounded by single layer of spindled-shaped granulosa cells.
- The recruitment is nonhormonal and the selection of a cohort probably depends on residual pool of primordial follicles.
- Onset of follicular maturation is marked by increase in the oocyte size, change in the granulosa cells from spindle to cuboidal, formation of small gap junctions in between granulosa cells and oocyte which acts as pathway for exchange of nutrition, metabolite and signal interchange.
- Oocyte depends on the glycolysis end product, pyruvate for energy source from granulosa cells.11
- Activin and BMP are promoters and inhibin and AMH are inhibitors of primordial follicular development and growth.12
The preantral follicle:
- With the increasing follicular growth, the oocyte enlarges and is surrounded by a membrane called as zona pellucida, and the granulosa cells undergo multilayer proliferation and the theca cells form the surrounding stroma.
- Once the granulosa cells become 3–6 layered, they are separated from the stromal cells by basement lamina. The stromal cells close to the basement lamina are called as theca interna cells, and those in the outer region are called as theca externa cells.
- This growth of follicle is gonadotropin dependent.
- Follicle-stimulating hormone receptor number rise quickly till 1,500 receptors per granulosa cells in the preantral stage.13
- Role of androgens, in early follicular development is very complex. In low androgen concentration, by FSH stimulation, androgens are converted to estrogen by the process of aromatization, but higher concentration stimulates the 5α-reductase activity that reduces androgen to more potent 5 α -androgens. These further inhibits conversion to estrogen and also inhibits FSH- induced LH receptors formation14–16 (Flowchart 4).
The antral follicle:
- Under synergistic influence of estradiol and FSH, there is increase in follicular fluid in the intracellular spaces of the granulosa cells, eventually coalescing to form a cavity, called as antrum, and the follicle is now called as antral follicle.
- The granulosa cells surrounding the oocyte are now called as cumulus oophorus.
- The antrum is rich in hormones mainly estrogen, growth factors and cytokines, and provides environment and nutrition for development and maturation of oocyte.
- The follicle with greatest rate of granulosa cells proliferation; produce highest estradiol concentration acquires dominance amongst them and is believed to yield a healthy oocyte.
- In contrary, those follicles with least estrogenic concentrations and higher androgenic levels, inhibits granulosa cell proliferation and undergo atresia and degeneration of oocyte.
- In case of premature LH rise, leads to excess androgen production that favors degeneration.
Two-cell, two-gonadotropin system (Flowchart 5):
- In preantral and antral follicles, the FSH receptors are located only on granulosa cells, and LH receptors on the theca interstitial cells located in the theca interna cells.
- The LH acts on the theca cells and facilitates conversion of low-density lipoprotein (LDL) cholesterol to androgens and these androgens are transported to granulosa cells and acts as a substrate to get converted into estrogens under the influence of FSH. This conversion depends upon follicular sensitivity to FSH, brought about by the action of FSH and estrogen.
- Thus, in the final stages of follicular maturation, LH is important for increasing amount of androgens, which are the substrate for estrogen production and promoting growth of the dominant follicle, with the regression of the smaller ones.
Selection of the dominant follicle:
- With the increasing concentration of estrogen, it exerts a negative feedback on the HPO axis and withdraws the release of FSH. This leads to regression of the all follicles by apoptosis, except the dominant follicle.
- The dominant follicle is selected by 5–7th day of the cycle and escapes the suppression, because of previous higher sensitivity and longer exposure to FSH. It has its own estrogen production and continues to grow.
- Also, the vascularity of theca cells of the dominant follicle increases as compared to others, thus delivering more gonadotropins to it than other.
- With the decreasing concentration of FSH, and mid-follicular estrogen rise, exerts a positive feedback on HPO axis and influences LH release. LH levels rises steadily over the late follicular phase, stimulating more androgen production in the theca cells.
The preovulatory follicle:
- The granulosa cells of the preovulatory follicle enlarge and acquire lipid inclusions and the theca cells becomes vacuolated and vascular.
- The oocyte now proceeds with meiosis I.
- Estrogen rises slowly at first, and then rapidly, marking its peak 24–36 hours prior to the ovulation. Peak estradiol level stimulates the onset of LH surge.
- In preovulatory follicle, LH inhibits further growth of the follicle and promotes luteinization of the granulosa cells that results in progesterone production19 and stimulates progesterone receptor expression on the granulosa cells, unlike traditionally believed it to be expressed mediated by estrogen.208
- The rising midcycle 17α hydroxy progesterone causes positive feedback on pituitary and leads to midcycle FSH surge and peak.
- Under the influence of rising LH, there is midcycle increase in local and peripheral androgens which increase follicular atresia in the ovary and increases libido systemically.
Ovulation
- The process in which mature oocyte along with its cumulus cells (COC) are expelled out of the ovary into the peritoneal cavity is called as ovulation.
- Ovulation occurs:
- 10–12 hours after LH peak
- 34–36 hours of the onset of LH surge
- 24–36 hours after peak estradiol levels
- Causes of ovulation:
- FSH surge causes conversion of plasminogen to more potent plasmin that causes activation of collagenase, which causes disruption of the follicular wall. This results in movement of the follicle, destined to ovulate to come to the surface of the ovary. Proteolytic digestion of the follicular apex is called as stigma.
- Synthesis of prostaglandins and other eicosanoids: They are produced under the influence of LH. LH along with prostaglandin E (PGE2) leads to cumulus expansion and resumption of meiosis. PGE2 causes contraction of the smooth muscle cells and expels the COC.
- Stretching factor: Passive stretching causes flattening of the surface by necrobiosis and rise in the intrafollicular pressure more than 10–15 mm Hg causes ovulation.
- Thus, the oocyte (arrested in metaphase stage of second meiotic division with a polar body) along with its cumulus cells and some follicular fluid ovulates, but the meiosis is completed only after fertilization by the sperm (marked by formation of the second polar body).
- The time that elapses from a primary follicle to ovulation is approximately 85 days.23
Oocyte transport:
- Oocyte transport begins from the ovulation and ends when the morula stage of the embryo reaches the uterine cavity.
- It reaches the ampulla of the fallopian tube within 15–20 minutes of ovulation.
- The tubal transport is toward the uterus and is dependent on the flow of the secretory fluids by the ciliary movement and the tubal smooth muscle contraction.
- Interaction and the sperm penetration take place in the ampullary region of the fallopian tube.
Luteal Phase
Luteal phase starts after ovulation and ends with the onset of menstruation. It corresponds to the secretory phase of the endometrial cycle and lasts for 14 days. It is divided into early and late luteal phase.
- Before ovulation, the granulosa cells starts to increase in size and assume a vacuolated appearance with deposition of yellow pigment, lutein, thus called as luteinization, and the follicle is called as corpus luteum after ovulation.
- Corpus luteum is made up of luteal cells, endothelial cells (constitutes 35% of total cell mass), leucocytes (mainly neutrophils), and fibroblasts.
- Angiogenesis is the hallmark of the luteal phase, after the cessation of the LH surge; the capillaries penetrate reaching till the central cavity and filling it with blood. It is mediated by vascular endothelial growth factor (VEGF) and angiopoietins produced by the luteinized granulosa cells.24–26 The corpus luteum has one of the highest blood flow per unit mass in the body.
- Luteinizing hormone mediates steroidogenesis in the luteal phase from LDL cholesterol. LH regulates LDL-receptor binding, internalization and postreceptor processing, in the luteinized granulosa cells during the early luteal phase in response to midcycle LH surge. This leads to production of progesterone and estrogen both (Flowchart 6).
- Progesterone rises to peak after 8 days of ovulation. Under the action of LH, inhibin B of the follicular phase changes to inhibin A that contributes to suppression of FSH in luteal phase and thus prevention of new follicular growth.
- There is a short wave of follicular recruitment, mostly due to midcycle FSH surge, but there is no follicular growth because of luteal phase FSH suppression.
- The corpus luteum rapidly declines after 9–11 days of ovulation due to luteolytic action of estrogen, prostaglandin F (PGF2α) produced in the luteal phase, nitric oxide, endothelin and other factors.
- Late luteal phase is marked by rapidly declining levels of estradiol and progesterone as an effect of luteolysis.
Luteal follicular transit: This is time extending from the onset of late luteal phase, marking rapidly declining levels of progesterone and estradiol to the selection of the dominant follicle for the next cycle. It is marked by menstruation.
- The demise of the corpus luteum results in nadir of progesterone, estrogen and inhibin A. Thus, this removes the suppression of the release of FSH secretion from the pituitary.
- Also the decrease in the estradiol and progesterone removes the negative feedback mechanism on the hypothalamus and pituitary, favoring increase in the frequency of gonadotropin-releasing hormone (GnRH) and gonadotropin release.
- This leads to release of FSH and recruitment of new follicular cohort of primordial follicles and again the dominant follicle is selected for next cycle.
FERTILIZATION
Mammalian fertilization comprises of sperm migration through the female reproductive tract and sperm-egg interaction in the ampullary portion of the fallopian tube.
It is completed in five stages:
- Sperm capacitation
- Zona binding
- Acrosomal reaction
- Zona reaction (Cortical reaction)
- Completion of meiosis and zygote formation.
Sperm Capacitation
- The sperms ejaculated in the vagina, do not have ability to fertilize unless they undergo a process called capacitation.
- Capacitation is the process of cellular changes occurring in the female reproductive tract after ejaculation by which sperm become competent to fertilize an ovum, by releasing surface cholesterol and other glycoproteins acquired from epididymis.
- The sperms then enter the fallopian tube and wait for the ovum at the isthmic region.
- After ovulation, when the ovum is in vicinity it attracts the sperms by chemotactic factors and the capacitated sperms acquire hypermobility to help them gain velocity to enter ZP.
Zona Binding
- Sperm penetrate the matrix of the cumulus oophorus, which is rich in proteins and carbohydrates such as hyaluronan, an unsulfated glycosaminoglycans, this is called as sperm-cumulus interaction.
Acrosomal Reaction
- The acrosome is a Golgi-derived exocytotic organelle that covers the tip of the sperm head.
- Acrosomal reaction is the acrosomal exocytosis that releases the acrosomal enzymes necessary for zona penetration and fertilization.
- It happens only in the capacitated sperm bound to ZP. This process is mediated by influx of calcium ions and efflux of hydrogen ions.
- After penetration of the ZP, sperm immediately meet and fuse with the oocyte plasma membrane.
Zona Reaction (Cortical Reaction)
The hardening of ZP after one sperm penetration to avoid entry of another sperm is called zona reaction. The hardening of the extracellular layer is by cross-linking of structural proteins, and inactivation of ligands for sperm receptors.30
Completion of Meiosis and Zygote Formation
- Fusion of the sperm and oocyte causes metabolic activation of the oocyte.
- The meiosis of the oocyte is completed about 3 hours of sperm insemination, marked by the release of second polar body containing haploid (23X) number of chromosomes.
UTERUS, ENDOMETRIUM, AND IMPLANTATION
The uterus is an important female reproductive organ, which holds feto-placental unit during pregnancy. Endometrium is the dynamic inner mucosal lining of the uterus, which responds to the hormonal changes of ovarian cycle and sheds cyclically as well as provides favorable physiological environment for embryo implantation, thus it is considered essential for reproduction.
Layers of Endometrium
- Functionalis layer (upper two-thirds): Consists of epithelial cells and glands. Its main purpose is to provide receptive environment for embryo (blastocyst) implantation.
- Basalis layer (lower one-third): Consists of endometrial stem cells and basal cells, to regenerate functional endometrium following menstrual loss.31
Endometrial Changes during an Ovulatory Menstrual Cycle
Cyclical sequence of changes is essential to prepare receptive endometrium approximately every month for implantation, failing which the endometrium breaks down and sheds in the form of menses.
These changes occur in three main phases:
- The menstrual phase
- The proliferative phase
- The secretory phase.
Menstrual Phase
- It is a transitional phase marked by exfoliation of endometrium till onset of endometrial proliferative phase of the next cycle.
- In histology, epithelial reparative changes along with remnants of menstrual shedding are seen.
- The epithelial repair is fast; by day 4 of the cycle, more than two-thirds of the cavity is covered with new epithelium.32 By day 5–6, the entire cavity is re-epithelialized and stromal growth begins.
- Ultrasound findings: The endometrium is relatively indistinct during menses with menstrual blood collection seen sometimes and then becomes a thin hyperechoic line after cessation of menses.
Proliferative Phase
- It coincides with ovarian follicular phase and increased estrogen secretion by growing follicles. This estrogen is responsible for reconstruction, growth, and proliferation of the endometrium. Estrogen also induces progesterone receptors on endometrium.
- In histology, all of the tissue components (glands, stromal cells, and endothelial cells) demonstrate proliferation, which peaks on days 8–10 of the cycle, reflecting rising estradiol levels in the circulation and maximal estrogen receptor concentration in the endometrium.33,34 Depending on the advancement of these histological changes, this phase is divided into early and late proliferative phase.
- Ultrasound findings: Endometrium becomes thicker, grows from approximately 1 mm to 7–10 mm and develops in “triple line” pattern.
Secretory Phase
- It is associated with the luteal phase of ovarian cycle after ovulation and it is a constant phase of 14 days; the endometrium now demonstrates a combined effect of estrogen and progesterone activity.
- Endometrial height is fixed now at its preovulatory extent (approximately 7–10 mm) despite continuous availability of estrogen.
- Progesterone secreted by corpus luteal cells is responsible for cessation of proliferative changes and it induces secretory changes in endometrial glands.
- The first histologic sign that ovulation has occurred is the appearance of subnuclear intracytoplasmic glycogen vacuoles in the glandular epithelium on cycle day 17–18.
- The glands acquire tortuosity and creates “cork-screw pattern” while spiral vessels become more coiled.
- The peak secretory changes are seen approximately 7 days after LH surge and it generally coincide with blastocyst implantation.
- Ultrasound findings: Endometrium loses its “triple line” image and becomes more uniformly hyperechoic.
Fate of Endometrium after Secretory Phase (Flowchart 7)
- In the absence of fertilization and implantation, corpus luteum regresses which causes hormonal support withdrawal.
- Estrogen and mainly progesterone withdrawal is responsible for various events which leads to endometrial breakdown and shedding.
Flowchart 7: Fate of endometrium after secretory phase.Source: Modified from Fritz MA, Leon Speroff L. Clinical Gynecologic Endocrinology and Infertility, 8th edition. Lippincott William and Wilkins; 2010. p. 134.
- Matrix metalloproteinase is activated under influence of increasing VEGF concentration (in response to progesterone withdrawal), which causes vasoconstriction and hypoxia in the endometrial layers to be shed.
- Also tumor necrosis factor (TNFα)-inhibits endometrial proliferation and induces apoptosis.
- Vasoconstriction and myometrial contractions associated with the menstrual events are believed to be significantly mediated by prostaglandins (PGF2α and PGE2).
- The basalis endometrium remains as it is during menses, and repair takes place from this layer.
Endometrial Preparation for Implantation
From the 7th to 13th day postovulation, with thickness of 7–10 mm, the endometrium is differentiated into three distinct zones, which after implantation forms the decidua of the pregnancy:
- Stratum basalis (less than one-fourth of the tissue) is the unchanged basalis fed by its straight vessels and surrounded by indifferent spindle-shaped stroma.
- Stratum spongiosum (mid-portion, approximately 50% of the total) is lace-like, composed of loose edematous stroma with tightly coiled spiral vessels and glands.
- Stratum compactum (superficial one-fourth) containing all the components but tightly compacted.
- The change from proliferative to secretory endometrium and preparing it for the arriving blastocyst is a very well synchronized process.
- This process involves temporary expression of many biochemical and molecular events in the endometrium for a time being called implantation window.
- The endometrial receptivity array consists of a customized array containing 238 differentially expressed genes in endometrial samples that are within the window of implantation regardless of their histologic appearance, is a recent diagnostic tool to predict receptive behavior of endometrium to blastocyst.37
Implantation
Implantation is defined as the process by which an embryo attaches to the endometrial lining and penetrates first the epithelium and then the circulatory system of the mother to form the placenta.
- Implantation begins with the loss of the ZP (hatching) about 2–3 days after, morula enters the uterine cavity.38 Thus, implantation occurs 5–7 days after fertilization (at the 32–256 cells-blastocyst stage).
- Successful implantation requires invasion of healthy and genetically normal blastocyst and secretory receptive endometrium simultaneously. But there are various hormonal and immunological factors affecting implantation.
Implantation takes place in three stages:
- Apposition
- Adhesion
- Invasion (and placentation).
Apposition and adhesion:
- Apposition begins at blastocyst stage as development of inner cell mass and trophectoderm are essential for initiating implantation.
- A prerequisite step for apposition to endometrial lining is hatching of blastocyst from ZP which occurs in vitro by contraction and expansion of blastocyst while in vivo by the presence of lytic enzymes in uterine fluid dissolve ZP.
- The polar trophoblast cells besides the inner cell mass are primarily involved in adhesion with endometrial epithelial cells.
- Various cytokines (CSF-1, LIF, and IL-1) and adhesion molecules (including integrins, selectins, and trophinin) play their role in the process of adhesion by increasing capillary permeability near implanting blastocyst and providing extracellular matrix support.
Invasion and placentation:
- The purpose of invasion (migration) is to remodel uterine vasculature by replacing maternal endothelium and converting high resistance, low flow vasculature in to high flow, low resistance vascular system in order to provide high blood flow interchange system between mother and fetus.
- Deep endometrial stromal invasion by human embryo is called interstitial implantation.
- The early embryo secretes a variety of enzymes (e.g. collagenase and plasminogen activators), that are important for digesting the intercellular matrix that holds the epithelial cells together.
- By invading endometrial stromal cells and basement membrane, blastocyst then erodes the endothelium of maternal capillaries to make sinusoidal sacs lined with endovascular trophoblast, thus providing connection with maternal blood.
- Further invasion is limited by various inflammatory cytokines secreted from the infiltrated lymphocytes in the endometrial cells and immunological factors like endometrial NK cells.
The first hormonal evidence of implantation (the appearance of hCG) occurs on 8, 9, or 10 days after ovulation; the earliest was 6 days and the latest 12 days.39
NEUROENDOCRINOLOGY
The HPO axis (Flowchart 8) plays a very important role by interplay of hormones. The direction signals sent by hypothalamus, and the operating signals given by anterior pituitary causes production of steroid hormones and ovulation. Depending upon, the amount of the hormone production, the lower centers send the positive or negative feedback signals in order to produce or reduce the signal for its hormone production respectively. This axis can be disturbed by various factors including increase in prolactin and TSH levels.
There are three types of feedback mechanisms:
- Long feedback mechanism: From the ovary to pituitary and hypothalamus depending upon steroid hormone production.
- Short feedback mechanism: From pituitary to hypothalamus depending upon gonadotropin secretion.
- Ultrashort feedback mechanism: From pituitary or hypothalamus for their autoregulation.
Neurohormone/Gonadotropin-releasing Hormone
- It is a decapeptyl hormone, released from the arcuate nucleus of the hypothalamus into the portal circulation of anterior pituitary, secretion of which stimulates the release of gonadotropins (FSH and LH) from the anterior pituitary.
- It is released in pulsatile manner (also called as LH pulse), and changes its amplitude and frequency depending upon the cycle day and requirement of the gonadotropin (Table 1).
- Lower GnRH pulse frequencies and less amplitude favor FSH secretion, higher GnRH pulse frequency with higher amplitude favors LH secretion. Functions of GnRH on anterior pituitary:
- It causes synthesis and storage of gonadotropins.
- Activation: Movement of the gonadotropins from the storage site to the pool for direct secretion; a self-priming action.
- Direct secretion of gonadotropins.
- Catecholaminergic system has direct effect on release of GnRH. Norepinephrine stimulates the release while dopaminergic pathway inhibits the release of GnRH.
- GnRH antagonist injections are now available by multiple amino acid substitution. They suppress gonadotropin secretion immediately by blocking the receptor site of GnRH on pituitary and thus making it unavailable for it. Nowadays, this is widely used as short protocol for COH in cases of patients undergoing IVF, in precocious puberty, endometriosis and prostate cancer. They degrade rapidly if given oral.
Gonadotropin (FSH and LH)
- They are water-soluble glycoproteins of higher molecular weight.
- They are secreted by the beta cells of the anterior pituitary.
- They have two subunits α and β. The amino acid composition of α subunit of FSH, LH, and hCG is the same; they differ in their β-subunits.
- We have already understood the functions of FSH and LH in ovarian cycle.
- Injectable gonadotropins are widely used for COH in treatment of infertility.
Prolactin
- It is a polypeptide.
- Its use in reproductive physiology is not clearly understood.
- It is secreted by the alpha cells of anterior pituitary.
- Its secretion remains under chronic inhibition by the dopaminergic pathway.
- But if the prolactin levels increases in the blood, it stimulates hypothalamic-dopaminergic pathways, thus suppressing prolactin secretion but also GnRH secretion.
- That results into hypogonadotrophic hypogonadism.
- Mild hyperprolactinemia (20–50 ng/mL) causes poor preovulatory follicular growth and thus short luteal phase.43,44 Moderate hyperprolactinemia (50–100 ng/mL) causes anovulation, oligomenorrhea, or amenorrhea. Severe hyperprolactinemia (> 100 ng/mL) causes hypogonadism and estrogen deficiency and related consequences.45,46
Thyroid Hormone
- Thyroid stimulating hormone (TSH) is released from the beta cells of the anterior pituitary.
- Higher TSH causes disturbed folliculogenesis and defective spermatogenesis and embryogenesis. Hypothyroidism can affect fertility due to anovulatory cycles, luteal phase defects, hyperprolactinemia, and sex hormone imbalance.47
We have already studied about estrogen and progesterone in ovarian cycle in this chapter.
GENETICS IN REPRODUCTION
Genetic abnormality is often one of the causes of infertility, failure of infertility treatment, miscarriage, congenital abnormalities in child and some of the comorbidity. Genetics contributes to reproduction by influencing varieties of physiological process including gametogenesis, quality of gametes and hormonal homeostasis.
Reproduction genetic is a branch which deals with the relationship between genotype and reproduction. It includes studying the transmission of the genetic material and its epigenetic modification from one generation to next and also the effects of abnormalities in the genetic material on reproduction.
Genetic Tests of Infertile Couple
Availability of genetic tests from basic to advance to diagnose any genetic abnormalities in infertile couples/patients helps selecting further treatment plans like ICSI with preimplantation genetic diagnosis (PGD) or donor gametes/zygote. Basic genetic evaluation includes:
- Karyotyping
- In infertile female
- In infertile male
- In recurrent implantation failure
- In recurrent miscarriage
- Cystic fibrosis gene mutation
- Fragile-X mental retardation gene mutation
- Y-chromosome microdeletion test.
Karyotyping
Karyotyping in infertile female:
- The prevalence of chromosomal abnormalities in women with regular ovulation, as reflected by a regular menstrual cycle, is reported to be 0.58%.48
- Women carrying extra X-chromosomes are at risk of reduced ovarian reserve and premature ovarian insufficiency (POI).
- Assessment of ovarian reserve is essential whenever numerical chromosomal translocations are involved.
- Women lacking an X-chromosome (Turner syndrome, 45XO) are known to be infertile with streak ovaries.
Karyotyping in infertile male:
- Chromosomal abnormalities account for approximately 5% of causes of infertility in males, and the prevalence increases to 15% in the population of azoospermic males.49
- Aneuploidy is the most common abnormality seen in infertile male, specifically of sex chromosome.
- Klinefelter's syndrome (nonmosaic, 47, XXY; and mosaic, 47, XXY/46, XY), is the most common chromosomal abnormality caused by aneuploidy with 5% prevalence in severe oligospermic men and 10% in azoospermic men.50 The syndrome causes arrest of spermatogenesis at primary spermatocyte level. The affected men are sterile but sperm can be seen in the ejaculate of some men with nonmosaic pattern.
- Chromosomal translocation is another source of aneuploidy. Robertsonian translocations are more common in oligozoospermic and azoospermic men, with rates of 1.6% and 0.09%, respectively.51,52 Carriers of Robertsonian translocations may exhibit a normal phenotype but could be infertile because of a lack of gamete production.
Karyotyping in recurrent implantation failure:
In couples presenting with RIF the prevalence of chromosomal abnormalities on karyotyping is 2.11%, more specifically 2.73% in women with RIF compared to 1.89% in male partners of women suffering from RIF.53
Karyotyping in recurrent miscarriage:
The prevalence of structural balanced chromosomal translocation in either partner of a couple experiencing RM increases according to the number of miscarriages; 2.2% after one miscarriage; 4.8% after two miscarriages; and 5.2% after three miscarriages.54 However, the frequency of numerical chromosomal abnormalities in miscarriages with advancing age is also high.
Y-chromosome Microdeletion Test
- Y-chromosome microdeletions, in the AZF region, have been observed in between 3% and 15% of men with severe oligozoospermia and non-obstructive azoospermia.55
- Proper genetic counseling should be done, as this can be transmitted to the male offspring.
Cystic Fibrosis Gene Mutation
- Cystic fibrosis is the most common autosomal recessive condition in northern Europeans with a carrier rate of 1 in 25 Caucasians.
- In contrast to patients with AZF deletions, these patients typically have normal spermatogenesis with normal testicular size and consistency, normal FSH, absence of the vas deferens, and/or an indurated epididymis.
- However, there has been no study to date showing that these genetic conditions are mutually exclusive. Therefore, the possibility exists that a single patient can genetically demonstrate both conditions.
Fragile X Mental Retardation 1 Gene Mutation
Fragile X syndrome has a prevalence of 1 in 4,000 males and 1 in 6,000 females and is the most common cause of mental retardation in men.59 This syndrome is caused by mutation in fragile X mental retardation 1 (FMR-1) gene, which codes for fragile X mental retardation protein (FMRP), it might be associated with poor ovarian response during ovarian stimulation.
Couples with high maternal age or with RM and RIF can be offered IVF/ICSI with PGS. This involves taking a biopsy of a single blastomere from day 3 embryo or trophectoderm biopsy from a blastocyst and checking for any aneuploidy.60,61 In this case there is a choice of transferring only euploid embryo. In case of couples with single gene disorders, IVF/ICSI along with PGD can be offered, which tests the single cell biopsied from the embryo for that specific genetic abnormality. The embryo, which is free from the disorder, can be transferred.62
IMMUNOLOGY IN REPRODUCTION
- Autoimmunity influence reproduction by:
- Affecting the reproductive life and fertility of both sexes, and
- Affecting implantation of the embryo by either failure of implantation or rejection of implantation resulting in abortion.
- Autoimmune disorders involve an immune response directed against a specific part of host or self, like systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome (APLA) syndrome.
- Mechanisms involved in autoimmune disorders:
- Direct action of antibodies (antiphospholipid, antithyroid, antinuclear antibodies) without any clinical disorder.
- By affecting process of spermatogenesis, oogenesis, implantation and pregnancy loss.
- By causing vasculitis, impairing the blood circulation.
The major immunological disorders affecting fertility are:
- Antisperm antibodies (ASAs)
- APLA
- Human leukocyte antigen-killer cell immunoglobulin- like receptor (HLA-KIR)
Antisperm antibody: The presence of ASAs in serum and in the secretions of the reproductive system is seen in some infertile patients. Its role, however, in reproduction remains debatable as many authors have revealed ASA in a relatively high percentage of both infertile as well as fertile couples. Thus, ASA test is not an essential workup in the evaluation of infertile patients.
The role of ASAs is in various mechanisms have been proposed affecting male fertility: sperm agglutination and impaired cervical mucus penetration, complement-mediated sperm injury through the female genital tract, and interference with gametes interaction.63
Various tests for qualitative detection (like agglutination tests, MAR, complement-mediated sperm immobilization or cytotoxicity assays, anti-human antibody-coated immunobeads, IFA and immunogold assay) and for quantitative detection (like ELISA, radiolabeled antiglobulin assays, radioisotopes and enzymes are quantitative probes and FCM) and tests for identification of specific bond (like immunoblotting and affinity chromatography) have been evolved but have so far not been used in the routine laboratory investigation of the subfertile couple.
APLA syndrome or APS: APS is a systemic autoimmune inflammatory thrombotic disorder, characterized by the presence of antiphospholipid (aPL) antibodies in the serum of the patient and associated with an array of obstetric complication with or without vascular thromboembolic events.
Antiphospholipid antibodies are directed against various phospholipid-binding proteins. The most important of these proteins is β-2 glycoprotein but some less frequent targets include prothrombin, thrombin, tPA, annexin-2, etc.
The exact mechanism through which these antibodies mediate the prothrombotic state is not clear but proposed mechanisms of action include:
- Activation of cellular elements (platelets, endothelial cells, and monocytes)
- Inhibition of the fibrinolytic system
- Activation of the coagulation cascade, and
- Activation of the complement system.
Anticoagulation and antiplatelet medications have been the mainstay of the treatment of this disease.
Human leucocyte antigens-killer immunoglobulin-like receptor: Blastocyst adheres and implants into the decidualized endometrial cell lining. HLA-KIR is an antigen antibody interaction, which if favorable results into successful correct implantation and placentation and if incorrect results into implantation failure or incorrect placentation leading to abortion, or pregnancy complications like preeclampsia, prematurity, etc.
HLA system is the locus of genes, which encodes for the proteins on the surface of the cells associated with the regulation of the immune system. EVT cells of the blastocyst express a large repertoire of class I HLA-C and nonclassical HLA-G and HLA-E antigens, whereas the class I antigens HLA-A and HLA-B and class II antigens are absent.65,66
|
16HLA-C genotypes are the main one and could be HLA-C1 or HLA-C2.67 Of the two, C2 is a stronger ligand than C1. HLA genes are inherited one from mother and one from father in each cell. So, EVT cell would express HLA C1C1, HLA C1C2, or HLA C2C2. HLA C1C1 is the most compatible, where as HLA C2C2 is the least.
Uterine NK cells express receptor KIR for ligand HLA of the EVT cells. The maternal KIR genotype could be haplotype AA (no activating KIRs), AB, or BB (1–10 activating KIRs).67
Interaction and compatibility between KIR and its HLA (ligand) facilitates migration followed by invasion of EVTs into maternal vasculature. Thus, maternal immune system is not going to reject the semiallogenic fetus. The recognition of HLA-C expressing trophoblasts by maternal KIR positive NK cells during pregnancy is important for successful placentation.68–71
Noncompatibility of expression of genotypes of HLA and KIR results in failure of trophoblastic invasion which might be correlated with an increased risk of RM, preeclampsia and fetal growth retardation.70,71
CONCLUSION
It is essential to understand reproductive biology, physiology and reproductive endocrinology to help in making differential diagnosis from various causes of infertility in evaluation of both the partners. Reproductive Genetics and immunology play a very important role in case of understanding reason for failure of infertility treatment, and thus helps to provide a treatment suitable to the conditions.
REFERENCES
- Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature. 1951;168(4277):697–8.
- Austin CR. Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B. 1951;4(4):581–96.
- Dym M. Spermatogonial stem cells of the testis. Proc Natl Acad Sci USA. 1994;91:11287–9.
- Foldesy RG, Bedford JM. Biology of the scrotum. I. Temperature and androgens as determinants of the sperm storage capacity of the rat cauda epididymis. Biol Reprod. 1982;26:673–82.
- Cornwall GA. New insights into epididymal biology and function. Hum Reprod Update. 2009;15:213–27.
- Devroey P, Liu J, Nagy Z, et al. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril. 1994;62:639–41.
- Sobrero AJ, MacLeod J. The immediate postcoital test. Fertil Steril. 1962;13:184–9.
- Montag M, Köster M, Strowitzk T, et al. Polar body biopsy. Fertil Steril. 2013;100:603–7.
- Visser JA, de Jong FH, Laven JS, et al. Anti-Müllerian hormone—a new marker for ovarian function. Reproduction. 2006;131(1):1–9.
- van Disseldorp J, Lambalk CB, Kwee J, et al. Comparison of inter- and intra-cycle variability of anti-Müllerian hormone and antral follicle counts. Hum Reprod. 2010;25(1):221–7.
- Su YQ, Sugiura K, Eppig JJ. Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism. Seminars Reprod Med. 2009;27:32–42.
- Trombly DJ, Woodruff TK, Mayo KE. Roles for transforming growth factor beta superfamily proteins in early folliculogenesis. Semin Reprod Med. 2009;27:14–23.
- Erickson GF. An analysis of follicle development and ovum maturation. Semin Reprod Endocrinol. 1986;4:233.
- McNatty KP, Makris A, Reinhold VN, et al. Metabolism of androstenedione by human ovarian tissues in vitro with particular reference to reductase and aromatase activity. Steroids. 1979;34:429–43.
- Hillier SG, Van Den Boogard AM, Reichert LE, et al. Intraovarian sex steroid hormone interactions and the regulation of follicular maturation: aromatization of androgens by human granulosa cells in vitro. J Clin Endocrinol Metab. 1980;50:640–7.
- Jia X-C, Kessel B, Welsh TH, et al. Androgen inhibition of follicle-stimulating hormone—stimulated luteinizing hormone receptor formation in cultured rat granulosa cells. Endocrinology. 1985;117:13–22.
- Jia X-C, Hsueh AJ. Homologous regulation of hormone receptors: luteinizing hormone increases its own receptors in cultured rat granulosa cells. Endocrinology. 1984;115:2433–9.
- Kessel B, Liu YX, Jia X-C, et al. Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells. Biol Reprod. 1985;32:1038–50.
- Yong EL, Baird DT, Yates R, et al. Hormonal regulation of the growth and steroidogenic function of human granulosa cells. J Clin Endocrinol Metab. 1992;74:842–9.
- Chandrasekher AY, Brenner RM, Molskness TA, et al. Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. II. Progesterone receptor expression in luteinizing granulosa cells. J Clin Endocrinol Metab. 1991;73:584.
- Zelinski-Wooten MB, Hutchison JS, Chandrasekher YA, et al. Administration of human luteinizing hormone (hLH) to Macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles. J Clin Endocrinol Metab. 1992;75:502–7.
- Hoff JD, Quigley ME, Yen SS. Hormonal dynamics at midcycle: a reevaluation. J Clin Endocrinol Metab. 1983;57:792–6.
- Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17:121–55.
- Christenson LK, Stouffer RL. Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial factor production by macaque granulosa cells from pre and periovulatory follicles. J Clin Endocrinol Metab. 1997;82:2135–42.
- Dickson SE, Fraser HM. Inhibition of early luteal angiogenesis by gonadotropin-releasing hormone antagonist treatment in the primate. J Clin Endocrinol Metab. 2000;85:2339–44.
- Bedford JM. Puzzles of mammalian fertilization—and beyond. Int J Dev Biol. 2008;52:415–26.
- Zaneveld LJ, Polakoski KL, Williams WL. Properties of a proteolytic enzyme from rabbit sperm acrosomes. Biol Reprod. 1972;6:30–9.
- Jones R. Identification and functions of mammalian spermegg recognition molecules during fertilization. J Reprod Fertil. 1990;42(Suppl):89–105.
- Sathananthan AH, Trounson AO. Ultrastructure of cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro. Gamete Res. 1982;6:225.
- Chan RW, Schwab KE, Gargett CE. Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod. 2004;70:1738–50.
- Ludwig H, Spornitz UM. Microarchitecture of the human endometrium by scanning electron microscopy: menstrual desquamation and remodeling, In: Bulletti C, Gurpide E (Eds). The Primate Endometrium. New York: The New York Academy of Sciences, New York; 1991. p. 28.
- Bergeron C, Ferenczy A, Shyamala G. Distribution of estrogen receptors in various cell types of normal, hyperplastic, and neoplastic human endometrial tissues. Lab Invest. 1988;58:338–45.
- Tabibzadeh SS. Proliferative activity of lymphoid cells in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab. 1990;70:437–43.
- Fata J, Ho ATV, Leco KJ, et al. Cellular turnover and extra-cellular matrix remodelling in female reproductive tissues: functions of metal-loproteinases and their inhibitors. Cell Mol Life Sci. 2000;57:77–95.
- Zhang J, Salamonsen LA. In vivo evidence for active matrix metalloproteinases in human endometrium supports their role in tissue breakdown at menstruation. J Clin Endocrinol Metab. 2002;87:2346–51.
- Díaz-Gimeno P, Horcajadas JA, Martínez-Conejero JA, et al. A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril. 2011;95:50–60.
- Navot D, Scott RT, Droesch K, et al. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril. 1991;55:114–8.
- Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. New Engl J Med. 1999;340:1796–9.
- Kaiser UB, Conn PM, Chin WW. Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev. 1997;18:46–70.
- Norwitz ER, Xu S, Jeong KH, et al. Activin A augments GnRH-mediated transcriptional activation of the mouse GnRH receptor gene. Endocrinology. 2002;143:985–97.
- Bilezikjian LM, Corrigan AZ, Blount AL, et al. Pituitary follistatin and inhibin subunit messenger ribonucleic acid levels are differentially regulated by local and hormonal factors. Endocrinology. 1996;137:4277–84.
- Seppala M, Ranta T, Hirvonen E. Hyperprolactinaemia and luteal insufficiency. Lancet. 1976;1:229–30.
- Corenblum B, Pairaudeau N, Shewchuk AB. Prolactin hypersecretion and short luteal phase defects. Obstet Gynecol. 1976;47:486–8.
- Biller BM, Baum HB, Rosenthal DI, et al. Progressive trabecular osteopenia in women with hyperprolactinemic amenorrhea, J Clin Endocrinol Metab. 1992;75:692–7.
- Schlechte J, Walkner L, Kathol M. A longitudinal analysis of premenopausal bone loss in healthy women and women with hyperprolactinemia. J Clin Endocrinol Metab. 1992;75:698–703.
- Verma I, Sood R, Juneja S, et al. Prevalence of hypothyroidism in infertile women and evaluation of response of treatment for hypothyroidism on infertility. Int J Appl Basic Med Res. 2012;2(1):17–9.
- Papanikolaou EG, Vernaeve V, Kolibianakis E, et al. Is chromosome analysis mandatory in the initial investigation of normoovulatory women seeking infertility treatment? Hum Reprod. 2005;20:2899–903.
- Ferlin A, Raicu F, Gatta V, et al. Male infertility: role of genetic background. Reprod Biomed Online. 2007;14:734–45.
- Foresta C, Garolla A, Bartoloni L, et al. Genetic abnormalities among severely oligospermic men who are candidates for intracytoplasmic sperm injection. J Clin Endocrinol Metab. 2005;90:152–6.
- Johnson MD. Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening. Fertil Steril. 1998;70:397–411.
- Meschede D, Lemcke B, Exeler JR, et al. Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection—prevalence, types, sex distribution and reproductive relevance. Hum Reprod. 1998;13:576–82.
- De Sutter P, Stadhouders R, Dutré M, et al. Prevalence of chromosomal abnormalities and timing of karyotype analysis in patients with recurrent implantation failure (RIF) following assisted reproduction. Facts Views Vis Ob Gyn. 2012;4:59–65.
- Hook EB, Healy NP, Willey AM. How much difference does chromosome banding make? Adjustments in prevalence and mutation rates of human structural cytogenetic abnormalities. Ann Hum Genet. 1989;53:237–42.
- Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosome of infertile men. Hum Reprod. 1997;12:1635–41.
- Anguiano A, Oates RD, Amos JA, et al. Congenital bilateral absence of the vas deferens (A primarily genital form of cystic fibrosis). JAMA. 1992;267:1794–7.
- Kaplan E, Shwachman H, Perlmutter AD, et al. Reproductive failure in males with cystic fibrosis. N Engl J Med. 1968;279:65–9.
- Mak V, Zielenski J, Tsui LC, et al. Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia. JAMA. 1999;281:2217–24.
- Crawford DC, Acuña JM, Sherman SL. FMR1 and the fragile X syndrome: human genome epidemiology review. Genet Med. 2001;3(5):359–71.
- Schoolcraft WB, Fragouli E, Stevens J, et al. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril. 2010;94(5):1700–6.
- Magli MC, Gianaroli L, Ferraretti AP. Chromosomal abnormalities in embryos. Mol Cell Endocrinol. 2001;183:S29–34.
- Francavilla F, Santucci R, Barbonetti A, et al. Naturally-occurring anti-sperm antibodies in men: interference with fertility and clinical implications. An update. Front Biosci. 2007;12:2890–911.
- Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006;4:295–306.
- King A, Allan DS, Bowen M, et al. HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Eur J Immunol. 2000;30:1623–31.
- Apps R, Murphy SP, Fernando R, et al. Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies. Immunology. 2009;127:26–39.
- Uhrberg M, Valiante NM, Shum BP, et al. Human diversity in killer cell inhibitory receptor genes. Immunity. 1997;7:753–63.
- Hiby SE, Walker JJ, O'shaughnessy KM, et al. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med. 2004;200:957–65.
- Hiby SE, Regan L, Lo W, et al. Association of maternal killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage. Hum Repro. 2008;23:972–6.
- Hiby SE, Ashrafian-Bonab M, Farrell L, et al. Distribution of killer cell immunoglobulin-like receptors (KIR) and their HLA-C ligands in two Iranian populations’. Immunogenetics. 2010;62:65–73.
- Hiby SE, Apps R, Sharkey AM, et al. Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2. J Clin Invest. 2010b;120:4102–10.