Pregnancy in human female, Thesis of Zoology

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Reproduction

Pregnancy

Umesh Rai

Sunil Kumar

Department of Zoology, University of Delhi, Delhi 110 007

INTRODUCTION

Pregnancy is the state of carrying and nurturing the developing embryo(s) in the uterus of the mother. In therians (marsupials and eutherians), ovum is devoid of adequate amount of yolk proteins and, therefore, the embryo stays inside the uterus of the mother for deriving nutrients during the development. Pregnancy begins with the fertilization of ovum and ends with the parturition or birth of the offsprings. The period through which a female carries a developing fetus in her body is known as gestation period, and it varies from species to species.

Human pregnancy

In humans, pregnancy lasts for approximately 38 weeks from the date of fertilization or 40 weeks from the date of last menses. The duration of human pregnancy is divided into three equal segments called trimesters. The first trimester extends upto the twelfth week of preganancy and encompases fetal organogenesis. This is the period when all organs, nerve and brain cells develop. The menses ceases to recur during pregnancy. Therefore, the first and most obvious sign of pregnancy is the absence of menses for 10 or more days after the usual length of the menstrual cycle. However, this might not hold true for those women in whom the cycle is irregular. This may be accompanied by tiredness, nausea, frequent urination, constipation, tenderness of breast, and the weight gain by the maternal body. Miscarriages, also called spontaneous abortion, occur most frequently during the first trimester. The second trimester lasts from thirteenth to twenty seventh weeks of gestation and embraces the development of fetal bones, fingernails, toenails and hair. The differentiation of external genitalia occurs in the second trimester. Although nausea and chances of miscarriage decreases in second trimester, a mild swelling in feet, ankles, hands and face occurs due to an increase in blood volume and fluid retention. The breast enlarges in response to female sex steroids, prolactin and placental lactogen. The third trimester from 28 to 40 weeks is a period of rapid fetal growth. During this period, the protective sheath of fat covers the nerve fibers and allows brain impulses to travel faster. Thus, the learning ability of the fetal brain enhances. Air sacs are developed in fetal lungs during the third trimester. In the last four weeks of pregnancy, the baby puts on a lot of weight and develops a thick layer of fat called ‘baby fat’. The increased size, weight, and activity of baby causes heartburn, shortness of breath, heart palpitations, and leg cramps in mother. The fetal movement can be felt by the mother in the third trimester though movement of fetus is initiated around 18-22 weeks of pregnancy.

EVOLUTION OF VIVIPARITY

Viviparity in mammals is believed to have originated from oviparous reptilian ancestors. It has evolved many times in squamates (lizards and snakes), so that around 20% of existing species exhibit viviparity (Andrews, 1997). In lacertilians, oviparity to different degrees of viviparity such as simple placenta with little nutrient uptake from mother (lecithotrophic viviparity), intermediate placental complexities to the highly developed placenta (obligate placentotrophy) are reported. This transition from oviparity to viviparity is seen associated with the reduction in egg size and shell, increased vascularization of uterus for nutrient transfer and respiratory gas exchange, prolonged retention of egg in oviduct, and development of extraembryonic membranes for the protection of developing embryo (Thompson et al., 2004). The reptiles, birds and mammals share the common extraembryonic membranes namely, chorion, amnion, allantois and yolk sac, and hence, are kept in the same group, amniota. However, extraembryonic membranes of viviparous reptiles, unlike mammals, develop into two structurally and spatially separated placentome, one at the embryonic pole (choriallantoic placenta) which is most likely the site of gaseous exchange,

female pronuclei occurs to form the zygote. The entire process is known as syngamy. The fertilization in all eutherian mammals takes place in the ampullary region of the oviduct.

CLEAVAGE AND BLASTULATION

Zygote formation is followed by a series of quick cell divisions called cleavage. It involves rapid cell division without the cell growth. Thus, total cellular volume of embryo remains similar to the zygote. Unlike mitosis, cleaving cell shows a modified cell cycle with completely omitted G1 and G2 phases. Therefore, the cells cycle rapidly between S and M phases. After third cleavage (8-cell stage), blastomeres begin to flatten. As a result of this, cleaving embryo changes into a smooth ball of cells with indistinguishable cell boundaries. This developmental phenomenon is known as compaction (Watson et al., 2004). It is stabilized by expression of tight and gap junctions that enable the smooth exchange of ions and small regulatory molecules between the blastomeres. Further cleavage of compacted blastomeres forms a ball of solid cell mass (16-32 blastomeres) known as morula. It consists of a small group of internal cells known as inner cell mass (ICM) (Barlow et al., 1972). This gives rise to the embryo and its associated yolk sac, allantois and amnion. The cells of the outer ring are known as trophoblast. This group of cells forms the tissue of the chorion, the embryonic portion of the placenta. Thus, trophoblasts provide protective covering and helps in supplying nutrients to the embryo from the mother. Initially, the morula does not have an internal cavity. Later, a cavity is formed in which the fluid secreted by the trophoblast is accumulated. This cavity is called blastocoel and the process is known as cavitation. Now, the resulting structure is called the blastocyst. While traveling through the oviduct en route to the uterus, trophoblast secretes a trypsin like proteases that lyse the zona pellucida which prevents the adherence of blastocyst to oviduct. However, if such adherence takes place, it is called as ectopic or tubal pregnancy.

Fig. 1. Stages of early embryonic development in mammalian model (departments.weber.edu/.../blastocyst.html)

During pre-implantation, different genes are implicated in regulation of various developmental processes such as compaction, trophectoderm differentiation and blastocoel formation (Watson et al., 2004). The predominant expression of IQGAP (IQ GTPase- activating protein), rac1 (ras-related C3 botulin toxin substrate 1) and cdc42 (cell division cycle 42 homolog) in cytoplasm of outer blastomeres of early 8-cell embryo facilitate the cell-cell adhesion and thus, regulate the compaction. In addition, p38 MAPK (p 38 mitogen- activating protein kinase) may be involved in compaction since it upregulates the formation of actin filaments. The trophectoderm differentiation is reported to be associated with down regulation of Oct-4 (Octamer-4) transcription factor. In case of blastocoel formation, Na/K- ATPase generates the trans-trophectoderm ionic gradients that promote the accumulation of water across the epithelium. This, along with the formation of tight junction which block the paracellular movement of water between adjacent trophectoderm cells, results in the formation of a fluid- filled cavity. In recent years, the role of aquaporins or water channels is demonstrated as physiological mediators of fluid movement across the trophectoderm.

IMPLANTATION

At the blastocyst stage, the trophectoderm acquires the competence to attach to the receptive uterine endometrium. The duration required for implantation differs among species (hours in rodents to days in humans) primarily due to variation in degree of endometrial invasion by

ovareictomy prior to luteal E2 secretion (on the morning of day 4) results in inhibition of implantation. The embryos at blastocyst stage become dormant. This condition is termed as delayed implantation (DI) and can be maintained for certain period by daily P4 treatment. A single injection of E2 can terminate the state of blastocyst dormancy and make the uterus receptive for implantation. Thus, the time period in receptive phase during which the uterus is most conducive for blastocyst attachment is referred to as “window of implantation” (Dey and Lim, 2006). Subsequent to receptive phase, the uterus become refractory, as it fails to respond to the presence of blastocysts.

The primate model baboon has been extensively studied to understand the hormonal control of blastocyst implantation in humans. In baboon, the changes in uterine receptivity during implantation can be divided into three distinct phases (Kim et al., 2004). The first phase extends from days 8 to 10 postovulation. During this phase, the blastocyst implantation usually occurs. E2 and P4 are primarily involved in regulating the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. The second phase of uterine receptivity is induced by blastocyst-derived ‘signals’, importantly, chorionic gonadotropin (CG) that superimposes its effect on the E2/P4-primed receptive endometrium. The third phase of uterine receptivity is initiated after attachment of blastocyst. On day 23 of the menstrual cycle in human, stromal edema is observed irrespective of whether implantation occurs or not. This is followed 3 to 4 days later by pre-decidual reaction. In case of implantation, the reaction is intensified and becomes the decidua of pregnancy. In baboon, the pre-decidual reaction does not occur during menstrual cycle though the stromal fibroblasts undergo extensive modification following implantation to form the decidua (Kim et al., 2004). Decidualization is the major change that occurs in the primate endometrium after conception. E2 and P4 fail to induce decidualization in the absence of conceptus. In decidualization, the fibroblast-like stromal cells express specific decidual proteins, importantly, insulin-like growth factor binding protein-1 (IGFBP-1). Apart from sex steroids, 3’:5’cyclic adenosine monophosphate (cAMP) is also needed to complete the decidualization in baboon.

TYPES OF IMPLANTATION BASED ON:

(A) Implantation mechanism

The interaction between trophoblasts and uterine epithelial cells is the common event of implantation in all the species. However, the degree of fusion of their apices and invasion by trophoblasts in uterine wall is species specific. In pig, though the apices of trophoblasts and LE cells closely interdigitate with each other, their fusion does not occur. In this species, trophoblast never penetrates through the LE cells. Therefore, the conceptus remains superficial in uterine lumen for the growth and development (Carson et al., 2000). The placenta also remains superficial in ruminants, but there is a fusion of trophoblasts with LE cells (Carson et al., 2000). In case of superficial implantation, blastocyst occupies the central position in the uterine lumen, and hence, implantation is known as centric implantation.

In rabbit, an intricate phenomenon is involved in implantation. The trophoblasts after fusion with each other form the syncytiotrophoblast which further fuses with LE cells. After that, syncytiotrophoblast invade through LE cell to reach to the basal lamina and stroma (Carson et al., 2000).

A distinct type of implantation is observed in rodents wherein uterine epithelium adjacent to the decidual area undergoes apoptosis. The trophoblast cells phagocytose the sloughed tissues. During this shedding of uterine epithelium, decidual cells migrate to the site of implantation and are reported to induce the shedding phenomenon. The rodent blastocyst has been described to lack invasive behavior until its surrounding epithelium is removed away.

Further, the basal lamina is denuded so that the stromal invasion can occur for reorganization of stromal tissue surrounding the blastocyst. In essence, trophoblast penetration through the epithelium is not involved in rodents, since LE is removed away before the blastocyst commences stromal cells migration and rearrangement to from the implantation chamber (Carson et al., 2000). Such type of implantation is called displacement implantation.

In primates, implantation is intrusive. Invasion proceeds between epithelial cells without destroying them. In humans, thin folds of trophoblast cells form invadopodia that help in intrusive invasion to reach the underlying basement membrane (fig. 2). After destroying basement membrane, trophoblast cells reach the stromal compartment. During progression, some trophoblast cells fuse to form a syncytium that proliferates and invades the endometrial stroma. Thereafter, the blastocyst is completely embedded in the uterine stroma and the site of entry is covered by fibrin, over which the uterine epithelial cells grow and hide the blastocyst (Bischoff and Campana, 1996).

Fig. 2. (a) Implantation of the human blastocyst step by step.

Step 1: transport. The blastocyst arrives in the uterus 132–144 h after fertilization (Findlay, 1984). Step 2: orientation. The inner cell mass is orientated towards the endometrial epithelial lining. Step 3: hatching. The zona pellucida dissolves perhaps because of the secretion of proteases by trophectodermal cells. Step 4: apposition. The blastocyst is now in close contact with the endometrial lining but no connections have been established. The embryo can still be dislodged by washing. Step 5: adhesion. Connections of an unknown nature are established between the embryo and the endometrial epithelium. The embryo can no longer be dislodged. (b) Implantation of the human blastocyst step by step. Step 6: invasion. Thin folds of trophectodermal cells intrude between the endometrial epithelial cells. Step 7: syncytialization. Some trophectodermal cells fuse to form syncytia. These syncytia proliferate and invade the endometrial extracellular matrix. Step 8: villous formation. The former trophectodermal cells, now called cytotrophoblastic cells, migrate between the syncytia followed by the fetal stroma. This will lead to the formation of the placental villi (Bischof and Campana, 1996).

(B) Embryo position

Based on the position of embryo in the uterus, implantation can be classified into three categories: centric, eccentric and interstitial (fig. 3). In centric type, blastocyst grows to a larger size and remains in the centre of uterine lumen for further growth and development (e.g. ruminants, and pigs). The eccentric type of implantation is observed in rodents. In these

chorionic villi. These villi further branch extensively and by day 21 a definite structure of placenta becomes apparent. Each villus is composed of outer STB covering the monolayer of CTB. Gradually, STB disappears from the tip of villus following apoptosis and thus, allowing the underlying CTB to proliferate. The CTB invade the decidua and remodel the spiral arteries so that fresh blood from mother begins to flow into the intervillous space and bath the embryo (Bischoff and Campana, 1996). Further, extraembryonic connective tissue underlying the trophoblast penetrates the cytotrophoblasts strands. This is closely followed by the invasion of blood vessels that are extension and ramifications of the allantoic blood vessels.

TYPES OF PLACENTA

Although the placenta of all eutherian mammals shares common structural and functional features, the contact sites between allantochorion and endometrium vary considerably. In horse and pig, the entire surface of the allantochorion is in contact with endometrium to form the placenta. This type of placenta is known as diffuse placenta. However, in ruminants, instead of entire surface of allantochorion, some discrete sites referred as cotyledon make contact with the patches of the endometrium to form the cotyledonary placenta. In case of zonary placenta in dog, cat, bear, and elephant, the contact sites between allantochorion and endometrium is in the form of a distinct band or ring. Placenta in primates and rodents is discoidal, as only some parts of allantochorion in the form of a disk interact with the endometrium to form the placenta (fig. 4).

Fig. 4. Types of placenta depending on contact sites between allantochorion and endometrium

The other way of classifying placenta is the number of layers separating the fetal and maternal blood supply. In fact, there are three maternal tissue layers (endothelium, connective tissue and endometrial epithelium) and equal number of fetal tissue layers (fetal chorionic epithelium, connective and endothelial tissue) that separate the fetal blood from the maternal blood (fig. 5). In horses and ruminants, all the six layers are present. Here, uterine endometrial epithelium remains in close association with fetal chorion, and therefore, the placenta is referred as epitheliochorial type. In contrast, in case of humans, chorionic villi have eroded through maternal endothelium, resulting in the direct contact of maternal blood with fetal chorion. This type of placenta is called as hemochorial placenta. In third type of placenta, the endothelium of uterine blood vessels is retained. It prevents the direct contact of maternal blood with chorioallantois. This category of placenta, endotheliochorial, is observed in dog and cat.

Fig. 5. Types of placenta depending on the number of layers separating the fetal and maternal blood supply

PREGNANCY RECOGNITION AND MAINTENANCE The maternal recognition of pregnancy reflects the various ways in which the mother responds to the presence of conceptus within her reproductive tract. The conceptus acquires some measures to regulate the function of corpus luteum, uterine blood supply and maternal physiology including immune responses. The embryo signals its presence to maternal system and thereby prolongs the life span of corpus luteum (CL) (Niswender et al., 2000). CL majorly produces the pregnancy maintenance hormone, progesterone, which makes the uterine environment conducive for embryonic development. However, if embryo fails to signal its presence to the mother, CL regresses. In most of the mammals, CL regression and degeneration (luteolysis) is mediated by prostaglandin F2α (PGF2α) (Roberts et al., 1996).

In man and anthropoid apes, the chorionic gonadotropin (CG) which is functionally comparable to LH, is primarily involved in maintenance of pregnancy. The immunization of marmoset monkeys against the CG results in luteolysis and termination of pregnancy. CG abrogates the luteolytic effect of PGF2α of ovarian origin (Roberts et al., 1996). During first eight week of pregnancy, CG regulates the progesterone production from CL (Niswender et al., 2000). Thereafter, progesterone from STB takes over the function of CL since ovariectomy after eight week of gestation fails to influence the pregnancy (Niswender et al., 2000). In mice, instead of CG, prolactin secreted from the pituitary in response to neural- reflex activated by mating maintains the early phase of pregnancy upto 10-11 dpc by keeping the CL functional. After that, trophoblastic giant cells start producing prolactin-like lactogens known as placental lactogens. This takes over the function of pituitary prolactin because hypophysectomy after embryonic day 11 does not terminate the pregnancy (Malassine et al., 2003). In cattle and sheep, CL regression is mediated by the pulsatile release of PGF2α, secreted from the uterine endometrium. To maintain the prolonged functionality of CL, the secretion of PGF2α is inhibited by interferon-tau (IFN-τ) (Roberts et al., 1996). This antiluteolytic factor IFN-τ is secreted by mononucleated trophoblasts.

MATERNAL ADAPTATIONS DURING PREGNANCY

A plethora of changes occur in maternal respiratory, cardiovascular and renal systems during pregnancy to facilitate the fetal growth (Norwitz et al., 2005). Placental progesterone stimulates the respiratory centre in brain. This causes hyperventilation to rapidly remove the CO2 from the maternal blood. Consequently, maternal plasma CO2 is lowered down to less than the fetus. This difference facilitates the effective diffusion of CO2 from fetus to maternal circulation. In addition, differences between the oxyhemoglobin dissociation curves of fetal hemoglobin and adult hemoglobin helps the fetus to effectively extract the oxygen from the

ectopic pregnancy. It might lead to haemorrhage in pregnancy and also maternal deaths during first trimester. Ectopic pregnancy can be categorized into two types:

1. Tubal pregnancy: In this case, the fertilized ovum fails to enter into the uterine cavity. herefore, the implantation of embryo occurs in fimbriae, ampulla or isthumus rather than the uterine cavity.
2. Abdominal pregnancy: It is characterized by the development of zygote in the peritoneal cavity. The fertilized ovum either enters the peritoneal cavity and becomes attached to the mesentery or abdominal viscera, or expelled in the peritoneal cavity due to rupturing of oviduct or uterus.

(B) Molar pregnancy

It is a kind of genetic disorder due to chromosomal imbalance at the time of conception. The normal fertilized egg contains 23 chromosomes from the father and 23 from the mother. Any chromosomal imbalance such as the loss of maternal chromosomes or an excess copy of paternal chromosomes in the fertilized egg leads to the development of a malformed placenta known as "mole". Such type of placenta is clinically known as hydatidiform mole. The mole is further classified in two types:

1. Complete Mole: It is a pathological condition in which the loss or inactivation of ovum’s nuclei occur. The fertilized egg has none of the mother’s chromosomes. In order to compensate, the father’s chromosomes are doubled. This causes the development of an abnormal placenta which looks like a cluster of grapes that occupy the entire uterine cavity and prevents the formation of the fetus.
2. Partial Mole: A partial mole is formed when an egg is fertilized by two sperms. As a result, the zygote contains 69 chromosomes (23 from mother and 46 from father) instead of 46. In this case along with cluster of abnormal cells, partial development of normal placental tissue occurs. Also, the fetus development does occur in the uterus. However, due to extra chromosomes of father, the embryo suffers severe birth defects and dies in the uterus.

PREGNANCY TEST

(A) hCG assay: The chorionic gonadotropin (hCG) assay in urine sample is the most common test used for detecting human pregnancy. hCG is the heterodimeric glycoprotein hormone. It is composed of dissimilar α and β -subunits that are held noncovalently. It is the unique β-subunit that confers the biological and immunological specificity to the hCG molecule. hCG produced by syncytiotrophoblast is detectable as early as seven days after ovulation and is considered the earliest hormonal signal of pregnancy. The level of hCG rises very rapidly after implantation. Peak levels occur at approximately 9-10 weeks of gestation. After that the levels fall slowly, reach to a nadir by 17-18th week of gestation, and remain low for the remainder of pregnancy. The β- hCG ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay (Ozturk et al., 1987). The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the β -subunit of the hCG molecule. Mouse monoclonal anti-β -hCG antibody is used for solid phase immobilization on microtiter wells. The test samples of urine are added to the monoclonal anti-β -hCG coated microtiter wells and incubated at 37°C. The hCG present in sample binds to the antibody coated on wells. After 30 min, the wells are repeatedly washed to remove unbound hCG in sample. Thereafter, rabbit anti-β -hCG antibody conjugated with enzyme horseradish peroxidase is supplemented to microtiter

wells. As a result, β -hCG molecule is sandwiched between the solid phase and enzyme- linked antibodies. The wells are washed with deionized water to remove unbound antibody. A solution containing 3, 3’, 5, 5’ tetramethylbenzidine (TMB) and hydrogen peroxide is added and incubated for 20 min to develop the blue color. The color development is stopped with the addition of stop solution (1N HCl) changing the color to yellow. Absorbance is measured spectrophotometrically at 450 nm. The concentration of β -hCG is directly proportional to the color intensity of the test sample.

(B) Ultrasound

The high frequency sound waves are extensively used in medical field to localize the size and shape of any organ in the body, and also for imaging the fetus and female pelvic organs during pregnancy. Obstetrical ultrasound can be performed trans-vaginally or trans-abdominally. Trans- vaginal ultrasound is used commonly in the first trimester, while trans-abdominal ultrasound is most useful in the second and third trimesters. In addition to pregnancy test, healthcare provider uses ultrasound to look for growth of baby, rule out any pathological condition, evaluate the volume of amniotic fluid, diagnose multiple pregnancy, and to confirm the expected date of baby’s birth. It is to be noted that identification of sex following ultrasound is illegal and amounts to punishment.

CONCLUSION

Pregnancy is a complex sequential process that begins with fertilization and ends with parturition. Fertilization occurs in the ampullary region of the oviduct. Fusion of male and female gametes leads to the formation of zygote that undergoes cleavage to form the morula. With the formation of a fluid-filled cavity, morula is transformed into the blastula. The various developmental processes leading to blastula formation is controlled by up/down regulation of different gene products such as IQGAP, rac-1, Cdc 42, MAPK, actin filaments and Oct-4. The trophoectodermal cells of blastula acquire the competence to attach to the receptive endometrium. The process of implantation completes in three steps, namely, apposition, adhesion and penetration. During apposition, trophoblasts come close to the endometrium. The decrease of MUC-1 and increase of integrins, heparan sulphate, lacto-N- fucopentose 1, trophonin and tastin facilitate the adherence of blastocyst to the endometrium. After that, blastocyst penetration takes place. The degree of penetration varies from species to species. The penetration is facilitated by the expression of matrix metalloproteinases, serine proteases and plasmins on the surface of trophoblasts. The blastocyst penetration is accompanied by decidualization of stromal cells. The decidual cells secrete a number of factors that helps in nourishing the developing embryo and regulates the invasion of trophoblasts. The preovulatory E2 and luteal P4 primarily control the implantation. Moreover, the E2 of luteal phase is responsible for the receptivity of endometrium. However, the blastocyst-derived CG is reported to be mandatory in baboon to induce the uterine receptivity and decidualization. In the absence of uterine receptivity due to lack of luteal phase E2, the implantation does not occur and the blastocyst remain in dormant stage for a certain period. This is called the delayed implantation. Embryo develops a transient organ called placenta for deriving nutrients and oxygen from the mother. It also helps in excreting metabolic wastes in the maternal vascular system during placentation. The ICM-derived extraembryonic mesoderm together with trophoectodermal cells forms the allantochorion that associate with endometrium or uterine blood vessels. The different types of placenta are described based on the intensity of contact between allantochorion and endometrium or number of maternal tissue layers separating the fetus from the maternal blood. Progesterone is the key player in maintenance of pregnancy. The high level of P4 down regulates the

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