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Superovulation and chromosomal aberrations Ma, Sai 1995

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SUPEROVULATION AND CHROMOSOMAL ABERRATIONS by SAIMA M.B., JIXI Medical School, 1977 M.S (Medicine)., Harbin Medical University, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Obstetrics and Gynecology, Reproductive and Developmental Sciences Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 5,1995 © S a i Ma, ] 995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department Of Qh .s l - . f vh r i r ^ a r , vn P r -n1 n a y The University of British Columbia Vancouver, Canada Date At**/ t$ tqfg DE-6 (2/88) ii ABSTRACT In vitro fertilization (IVF) has been achieved in several mammalian species, including humans. Using the technique of IVF and embryo transfer, pregnancies can now be established in infertile women and thousands of live births have occurred. However, many of the embryos transferred to the uterus fail to achieve or sustain implantation. The failure of implantation has raised questions about the possible detrimental effect of superovulation on chromosomal complements. In this study, the hypothesis that chromosomal aberrations exist in mouse preimplantation embryos and human unfertilized oocytes after superovulation, was tested. The incidence of aneuploidy in spontaneously ovulated group (5.6%) was similar to that reported by others. The rate of aneuploidy in the three superovulated groups was 4.4, 5.0 and 7.0%, respectively. It was determined that aneuploidy was not increased as PMSG dose increased. There have been no other studies published on the rate of aneuploidy in 8- 16-cell stage embryos from superovulated mice which could be compared to these results. There was no polyploidy in the spontaneously ovulated group. However, the incidence of polyploidy, recorded for 8- to 16-cell stage embryos was 2.9% and 10.5% in 10 and 15 IU PMSG groups, respectively. It demonstrated a simple dose-response relationship between the PMSG dose and the incidence of polyploidy, including triploidy and tetraploidy. These results confirm the indication given by others that chromosomal anomalies, specifically polyploidy, may be induced by superovulation. The origin of polyploidy was studied in mouse zygote stage embryos. The proportion of polyploid embryos also increased as the dose of PMSG increased as previously observed in 8- 16-cell stage embryos. The frequencies of diploid oocytes fertilized by one haploid sperm were 1.9%, 1.9% and 1.7% in 5, 10 and 15 IU PMSG treated groups, respectively, while the frequencies of haploid oocytes fertilized by two sperms were 2.8% and 4.2% in the 10 and 15 IU PMSG treated groups, respectively. These observations support the hypothesis that both a disturbance at maturation division and an error at fertilization were the causes of polyploidy in CD-I mouse embryo after superovulation. The extent to which the incidence of chromosomal aberrations occur in unfertilized human oocytes after hyperstimulation of ovaries was also studied. The frequency of numerical i i i chromosome abnormalities in metaphase II oocytes from 280 human unfertilized oocytes was determined. Aneuploidy occurred at a frequency of 22.8% in human unfertilized oocytes obtained from stimulated follicles during IVF procedures. The incidence of diploidy in human unfertilized oocytes was found to be 16.8%. Since 9% triploid embryos has been found in this IVF program, diploid oocytes are likely an important source of triploidy as also indicated in the mouse study. Failure to detect a relationship between the frequencies of aneuploidy and dosage of hMG dose not necessarily imply that superovulation does not cause aneuploidy since there is no comparison of oocytes from the natural cycle with those from stimulated cycles. The overall results demonstrated the existence of a dose-response relationship between PMSG dose and the incidence of polyploidy in CD-I mouse embryos after superovulation. Both a disturbance at maturation division and an error at fertilization were the causes of polyploidy. The observation of 16.8% of diploid oocytes in human unfertilized oocytes suggests that the blockage of the meiosis process at the meiosis I level after superovulation may occur in the human as it apparently does in mice. iv TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi List of Figures vii Glossary ix Acknowledgments xi RATIONALE AND OBJECTIVES 1 CHAPTER 1 GENERAL INTRODUCTION I. Physiological Aspects of Oocyte and Fertilization 3 II. Induction of Superovulation 10 III. Superovulation and Chromosomal Aberrations 14 CHAPTER 2 INVESTIGATION OF EFFECTS OF PMSG ON CHROMOSOMAL COMPLEMENT OF CD-I MOUSE EMBRYOS 23 26 35 55 64 CHAPTER 3 CHROMOSOME INVESTIGATION OF UNFERTILIZED HUMAN OOCYTES AFTER SUPEROVULATION I. Introduction 67 n. Materials and Methods 73 HI. Results 77 I. II. m. IV. v. Introduction Materials and Methods Results Discussion Summary IV. Discussion 87 V V. Summary 92 CHAPTER 4 SUMMARY AND CONCLUSION 94 BIBLIOGRAPHY 100 vi LIST OF TABLES Page TABLE 1 The rate of mouse zygotes produced in spontaneously ovulating (SO), 5, 10,15IU PMSG superovulated CD-1 mice. 37 TABLE 2 The rate of normally developed embryos in spontaneously ovulating (SO), 5,10,15 IU PMSG superovulated CD-I mice 38 TABLE 3 Chromosome analysis of mouse 8- to 16-cell stage embryos from spontaneously ovulating (SO), 5,10 and 15 IU PMSG superovulated CD-I mice. 41 TABLE 4 Incidence and origin of polyploidy in mouse zygotes from spontaneously ovulating (SO), 5,10 and 15 IU PMSG superovulated CD-I mice. 48 TABLE 5 Summary of published cytogenetic studies of human unfertilized oocytes. 68 TABLE 6 Cytogenetic study of 280 morphologically unfertilized oocytes. 78 vii LIST OF FIGURES Page FIGURE 1 Life-cycle of the female germ cell 4 FIGURE 2 Illustration of three levels of hormonal feedback proposed for the hypothalarno-pituitary-gonadal axis: (1) long-loop feedback; (2) short-loop feedback; (3) ultra-short-loop feedback. GnRH, gonadotropin releasing hormone; AP, anterior pituitary; PP, posterior pituitary. 6 FIGURE 3 Times of administration of exogenous gonadotropins shown in relation to the 24-hours lighting schedule and the stage of the estrous cycle. 28 FIGURE 4 Schematic illustration of the methodology used for preparation of chromosomes from 8- to 16-cell stage embryos. 32 FIGURE 5a Normal appearing oocyte with the first polar body, x400. 36 FIGURE 5b Fertilized oocytes with two polar bodies, x400. 36 FIGURE 6a Normal appearing 10-cell stage embryo, x400. 39 FIGURE 6b Abnormal embryo with different sizes of fragmented blastomeres, x400. 39 FIGURE 7 A G-banded karyotype obtained from a 8-cell stage embryo, 40, XY. 42 FIGURE 8 Karyotyped mouse 8-cell stage embryo with hypodiploidy, 39, XX, -4. 43 FIGURE 9 Mouse 10-cell stage embryo with hyperdiploidy, 41, XY, +?. 44 FIGURE 10 Karyotyped mouse 8-cell stage embryo with triploidy, 58, XY, -16, -X or-Y? 45 FIGURE 11 Karyotyped mouse 8-cell stage embryo with tetraploidy, 80, XXYY. 46 FIGURE 12 First-cleavage mouse embryo fertilized in vivo showing differential condensation of male and female chromosomes. cr = male chromosomal complement; ? = female chromosomal complement. 49 FIGURE 13 First-cleavage mouse embryo fertilized in vivo showing a triploid chromosomal complement derived from a diploid oocyte and a haploid sperm. 50 FIGURE 14 Fist-cleavage mouse embryo fertilized in vivo showing a triploid chromosome complement derived from dispermy and a haploid oocyte. 51 FIGURE 15 First-cleavage mouse embryo fertilized in vivo showing a tetraploid chromosome complement derived from a diploid oocyte and dispermy. 52 FIGURE 16 Chromosomal complement at higher magnification (xlOOO) from the tetraploid embryo. Each chromosomal set indicated by alphabet vii i corresponding to Figure 15 53 FIGURE 17 Schematic illustration of mechanisms of chromosomal aberrations by superovulation. 65 FIGURE 18 Hypohaploid human oocyte (22, X, -C). 79 FIGURE 19 hyperhaploid human oocyte (25, X, +E?, +E?). 80 FIGURE 20 Diploid human oocyte (46, XX). 81 FIGURE 21 Human oocyte with mitotic hypohaploid complement, 22, X, -G. 83 FIGURE 22 Prematurely condensed sperm chromosomes (large arrowhead) and metaphase II chromosomes from oocyte (small arrowhead). Karyotyped hypohaploid oocyte, 22, X, -C. 84 FIGURE 23 The distribution of chromosome patterns in unfertilized human oocytes according to the doses of hMG (first group: hMG doses from 300 to 3000IU; second group: hMG doses from 3150 to 9000IU) 85 FIGURE 24 The distribution of chromosome patterns in unfertilized human oocytes according to maternal age (first group: women age from 24 to 34; second group: women age from 35 to 41 years old). 86 ix GLOSSARY AP ATP BSA BW cAMP CC CG cm DPBS E2 EGF ET FSH g GnRH GV GVBD HBSS hCG hMG hr IU kg IVF anterior pituitary adenosine triphosphate bovine serum albumin body weight cyclic adenosine monophosphate clomiphene citrate cortical granules centimeter Dulbecco's phosphate buffered saline estradiol-17P epidermal growth factor embryo transfer follicle stimulating hormone gram gonadotropin releasing hormone germinal vesicle germinal vesicle breakdown Hank's balanced salt solution human chorionic gonadotropin human menopausal gonadotropins hour (s) International Units kilogram In vitro fertilization LH luteinizing hormone X LHRH MH mg min ml mm MPF No. PCC PCO pH pmol PMSG PP sec mg mm mM US v/v v/v/v WHO w/v ZP1 ZP2 ZP3 luteinizing hormone releasing hormone meiosis II milligram minute milliliter millimeter metaphase promoting factor number premature chromosome condensation of sperm polycystic ovary -long H + concentration in a solution picomolar pregnant mare serum gonadotropin posterior pituitary second milligram micrometer micromolar ultrasound volume/volume volume/volume/volume world health organization weight/volume zona pellucida 1 zona pellucida 2 zona pellucida 3 xi ACKNOWLEDGMENTS I would like to express my sincere appreciation: To my supervisors, Dr. Moon, YS and Dr. Ho Yuen, B for providing me the opportunity to pursue this study and for their continuous support, patience and encouragement throughout the project; To members of my supervisor committee, Dr. Dill, F., Dr. Juriloff, D and Dr. Kalousek, DK for their valuable advice and guidance; To Dr. Francke, U for helping me to identify the mouse karyotypes. To the staff of the Research Center animal holding facility for the care of mice and students of the Department of Obstetrics and Gynecology for providing an invigorating scientific environment to study; To Mr. Mackinnon, M for his statistical help and Mrs. Rajamahendran, P., Dr. Katagiri, S for their technical assistance. To Dr. Currie, D and Mrs. Barrett, I for tireless critical assistance during the preparation of this thesis. To the Medical Research Council of Canada and the British Columbia Health Care Research Foundation for their financial support. And finally to my parents for their moral support. This work is dedicated to you, Ying Lin Ma, Jie Ying Yin, for you are the truly important part of my life. 1 RATIONALE AND OBJECTIVES Superovulation with exogenous gonadotropins creates a spectrum of pre- or periovulatory hormonal changes with subsequent detrimental effects on oocyte quality, fertilization and embryo development. Early defects may occur during the process of follicular development and oocyte maturation before ovulation by hyperstimulation of the ovary. Premature or atypical ovulations of meiotically aberrant oocytes may be a contributing factor in inferior oocyte quality and reduced fertility. Nuclear and cytoplasmic maturation of oocytes should occur in synchrony for an ordered and undisturbed segregation of chromosomes during meiosis. Asynchrony between nuclear and cytoplasmic maturation of oocytes may result from aberrant signals from surrounding somatic cells. Such signals may be poorly timed or of insufficient strength. The asynchrony of oocytes may result in inhibition of single or multiple chromosomes (hyperhaploidy), or even of all chromosomes (diploidy). Immediately after fertilization, the release of proteinases and glycosidases modifies the zona pellucida, blocking additional sperm binding and inhibiting zona-bound sperm penetration. A mixture of oocytes at different maturation stages is obtained after superovulation. Either immaturity or overmaturity of oocytes may cause an incomplete cortical reaction or polar body retention. These suboptimally matured oocytes may be vulnerable to penetration by more than one sperm. Normal embryonic development requires ordered segregation of chromosomes during meiosis and mitosis. Alteration of the hypothalamic-pituitary-gonadal axis function for endocrine control of follicular function may increase the probability of meiotic and mitotic errors. Thus, superovulation may exert a long-term effect on fertilization and embryonic development in early pregnancy. Knowledge of the exact incidence and type of chromosomal abnormalities in animal models will contribute to an understanding of the mechanisms underlying the detrimental effects of superovulation. Most information on this topic has been obtained by investigation of mouse oocytes and zygotes. However, there appear to be no reports concerning the chromosomal abnormalities and their relationship to superovulation in 8- to 16-cell stage mouse embryos. Analysis of 8- to 16-cell 2 stage mouse embryos may provide information on the mechanism of chromosomal behavior after superovulation and may indicate whether or not superovulation causes chromosomal abnormalities affecting embryo viability. Experimental design used to examine mouse embryos are practical. Examination of human embryos is limited due to technical and ethical difficulties. However, it is possible to examine unfertilized human oocytes from in vitro fertilization programs. Cytogenetic studies of large numbers of unfertilized human oocytes may supply information on (i) chromosomal abnormalities, (ii) maturity of the oocyte and (iii) cytogenetic abnormalities after superovulation. Together, the examination of murine embryos and human oocytes may advance knowledge of the effects of superovulation on chromosome constitution. This information may be utilized to better the future management of superovulatory protocols for human patients. To this end, there were three main objectives for this thesis. 1. To determine whether superovulatory doses of PMSG affects embryo chromosomal complements in CD-I mouse 8- to 16-cell stage embryos in vivo. 2. To define the mechanisms that cause the chromosomal abnormalities after superovulation in CD-1 mouse zygote stage. 3. To estimate the incidence of chromosomal abnormality in unfertilized human oocytes following superovulation. CHAPTER ONE 3 General Introduction I. Physiological aspects of oocyte maturation and fertilization A. Oocyte maturation a. Meiotic process The meiotic process is believed to be similar in all female mammal species (Byskov 1987), although the timing and duration of the various stages differ from one species to another (Peters, 1970). In most species, the oogonia enter meiosis prenatally so that by the time of birth, or shortly thereafter, most oocytes have reached the diplotene (dictyate) stage of meiotic prophase (Borum 1961; Baker 1979). Regardless of the timing of the onset of oogenesis, oocytes of all mammalian species become arrested in their meiotic progression at the dictyate stage. At this point in meiosis, a large, euchromatic spheroid nucleus appears. This stage is termed as the germinal vesicle (GV) stage. This extended period of arrest is species-dependent and terminates sometime after the mammal has reached sexual maturity, when a mature antral follicle is exposed to preovulatory gonadotropin stimulation. The oocyte becomes irreversibly committed to resume meiosis and subsequently undergoes germinal vesicle breakdown (GVBD), followed by changes in chromatin configuration. These changes involve condensation of the chromatin at diakinesis, progression through metaphase I, completion of the first meiotic division, and transformation of the primary oocyte to a mature oocyte at metaphase of the second division (Figure 1). These events, inclusively, are referred to as oocyte meiotic maturation (Donahue, 1972). DEVELOPMENTAL EVENTS STATE OF GERM CELLS Multiplication by mitosis 0 0-o o 2N 0 Primordial germ cells Oogonia Initiation of meiosis Meiotic arrest Birth Puberty Non-growth Full growth Resumption of meiosis Ovulation Sperm penetration Second meiotic division, fertilization and emission of second polar body N Metaphase II Meiotic arrest Resumption of meiosis Meiosis complete Figure 1. Life-cycle of the female germ cell 5 b. Molecular control of meiosis Metaphase-promoting factor (MPF) is known as a regulator of the meiotic cycle. MPF is composed of two cell-cycle-regulating proteins, cdc2 protein kinase and cyclin (Nurse, 1990; Jacobs, 1992). In mammals, the MPF concentration within the ovarian eggs increases after breakdown of the germinal vesicle (Hashimoto and Kishimoto, 1988). MPF disappears temporarily during the first meiotic division, then increases again as oocytes enter the second meiotic division (Hashimoto and Kishimoto, 1988). The reason for mature unfertilized eggs being arrested at metaphase U for many hours is unknown. It was speculated that (i) a cytostatic factor (CSF) stabilizes MPF, directly or indirectly through its action on microtubules, to maintain a high MPF concentration and (ii) inactivation of CSF causes degradation of MPF, allowing the egg to escape from metaphase arrest. In the mouse, both MPF and CSF disappear after fertilization. Transient Ca+2 elevation upon fertilization may trigger the degradation of these proteins (Longo, 1987). Besides the nuclear changes that accompany meiotic maturation, cytoplasmic maturation renders the oocyte competent to undergo normal fertilization and embryonic development (Chang, 1955a,b; Thibault et al., 1987). These changes are under hormonal influence. c. Hormonal regulation The systemic factors controlling oocyte maturation are gonadotropins, i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin releasing hormone (GnRH) from the hypothalamus stimulates FSH and LH secretion from gonadotropes in the anterior pituitary gland. Steroids and other factors secreted by the ovary in turn feed back on the hypothalamus and pituitary to regulate the secretion of gonadotropins (Austin and Short, 1984) (Figure 2). The response of the ovary to gonadotropins depends on the presence of specific receptors on the surface of the different ovarian cell types; granulosa and theca cells. Granulosa cells are the cells of the epithelial lining of a follicle, while theca cells are the layer of dense stroma located immediately outside the granulosa cells. The actions of FSH are restricted to the granulosa cells. In contrast, LH actions are exerted on granulosa and theca cells. The hormones appear to induce a Figure 2. Illustration of three levels of hormonal feedback proposed for the hypothalamo-pituitary-gonadal axis: (1) long-loop feedback; (2) short-loop feedback; (3) ultra-short-loop feedback. GnRH, gonadotrophin releasing hormone; AP, anterior pituitary; PP, posterior pituitary. 7 response in the cell through a common mechanism involving adenylate cyclase, the enzyme responsible for stimulating the production of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). The elevated cAMP enters the oocyte through gap junctions and inhibits meiotic maturation. In general, any molecule that directly modulates cAMP levels in the oocyte could be involved in regulating meiotic arrest and maturation. (Kaji et al., 1987). In addition, LH may stimulate the follicle to synthesize an oocyte maturation promoter that overrides follicular arrest. The maturation promoters may include epidermal growth factor (EGF) and other growth factors, i.e., transforming growth factor (TGF-P), insulin-like growth factors (IGF-1 and IGF-II), and erythroid differentiation factor (activin-A) (Dekel and Sherizly, 1985; Feng et al., 1987; Ueno et al., 1988; Downs, 1989). Estradiol, a product of gonadotropin action on theca and granulosa cells, appears to play a key role in determining the ability of a given follicle to respond to FSH and ovulate in response to LH. The ability of granulosa cells to respond to estradiol appears to determine whether a given follicle becomes atretic or ovulates. Regulation of estradiol production seems to be controlled by 2 specific mechanisms: 1) androgen production by theca cells in response to LH and 2) aromatization of androgens to estrogen by granulosa cells in response to FSH. Atresia occurs when FSH levels are insufficient, possibly because of an increase in the androgen to estrogen concentration ratio; androgen may antagonize the actions of estradiol (Richards, 1978). Alternatively, atresia might occur if LH concentrations were insufficient since both androgen and estradiol concentrations would fall. Therefore, androgen and estradiol as well as LH and FSH regulation of ovarian cell function are essential for follicular and oocyte maturation. The LH surge triggers the final maturation of the oocyte by inducing cytoplasmic and nuclear changes (resumption of meiosis), which prepare the oocyte for fertilization at the time of rupture of the follicle (Moor and Warnes, 1979). d. Pheromone regulation The ovarian cycle is a temporal event defined by the interval between successive ovulations. Therefore, chemosignals that alter cycle length will alter the rate of ovulation. Pheromones are chemical substances produced by one individual that affect the behavior or 8 physiology of another. Different pheromones are produced at different phases of the ovarian cycle. These pheromones can either shorten or lengthen the ovarian cycle, possibly by changing the duration of follicular development, the time of the gonadotropin surge or the life span of the corpus luteum. These changes can either enhance or suppress ovarian cycles of other females in a social group (McClintock, 1983) Female mice produce pheromones to lengthen the cycle, and males produce pheromones to shorten the cycle. Pheromones in the female may play a role in negative feedback from the ovary to the hypothalamic-hypophyseal system. Male murine pheromones may act as a positive stimulus to the female gonadal system. There is evidence that FSH secretion in the female is depressed by a primer pheromone originating from other females, and stimulated by one from males (Whitten, 1966). The function of pheromones in the estrous cycle is also recognized in rats (Mora and Cabrera, 1994). Female rats appear to produce two pheromones; one to enhance and another to suppress the cycle. It was demonstrated that women who live together tend to have menstrual synchrony (McClintock, 1971; Quadagno et al., 1981). It was proposed that the effect was mediated by pheromones and a common environment (Weller and Weller, 1993). There is no direct evidence that pheromonal communication exists between humans. B. Fertilization Fertilization is not a single event but a series of interactions between the egg and sperm leading to their activation, fusion and eventual joining of nuclear materials to establish the hereditary constitution of a new individual (Longo, 1987). a. Sperm capacitation In order to fertilize an oocyte, spermatozoa must first undergo capacitation before approaching the ovulated oocyte in the oviduct (Moore and Bedford, 1983). Capacitation involves a number of intracellular changes as well as alterations of the sperm surface proteins, mainly in the 9 acrosomal region. A progressive destabilization of the cellular membrane leads to an increased permeability to calcium ions and results in an increased intracellular calcium concentration (Clegg, 1983). As a consequence of these modifications, spermatozoa exhibit a change in their movement pattern. Once capacitated, spermatozoa move through the enveloping cumulus oophorus, which is a projecting mass of granulosa cells that bears the developing ovum in a follicle (Longo, 1987). b. Gamete interaction After passing through the cumulus mass, capacitated spermatozoa bind to the zona pellucida that surrounds the mammalian oocyte (Hartmann et al., 1972; Saling et al., 1979). The mouse and human zona pellucida are composed of three major glycoproteins, zona pellucida 1 (ZP1), zona pellucida 2 (ZP2), and zona pellucida 3 (ZP3). This sperm-receptor activity of the zona has been ascribed to a class of O-linked oligosaccharides on ZP3 (Florman and Wassarman, 1985). ZP2 has been implicated as a secondary sperm receptor that binds sperm only after the induction of the sperm acrosome reaction. Immediately after fertilization, the release of proteinase and glycosidases from cortical granules (CG) modifies the zona pellucida. This reaction blocks binding of additional sperm and inhibits penetration of already zona-bound sperm (Hartmann et al., 1972; Sato, 1979). Both ZP2 and ZP3 are modified by the zona reaction: ZP2 undergoes a proteolytic cleavage that is associated with the block to polyspermy (Bleil et al., 1981), and ZP3 loses its ability to induce the acrosome reaction and its sperm receptor activity (Bleil and Wassarman, 1983; Bleil et al., 1988). c. Sperm penetration and pronuclear growth After penetrating the ooplasm, the sperm head decondenses because of the action of a male pronucleus growth factor (Longo, 1987) and forms the male pronucleus. Gamete fusion triggers oocyte activation. Female meiosis ends with the extrusion of the second polar body containing half of the chromosomes while the remaining chromosomes in the oocyte cytoplasm decondense and form the female pronucleus (Longo, 1973). Meanwhile the centrosomal apparatus becomes much more conspicuous. As it approaches the female pronucleus, the male pronucleus with its associated centrosome rotates so that the 10 centrosome moves ahead of the chromatin material. During this activity the female pronucleus has moved toward the center of the ovum from the peripheral position it occupied when the second polar body was extruded. By the time the two pronuclei are close to each other, the centrosome has divided and formed a mitotic spindle on which the chromosomes contributed by the sperm and ovum aggregate. Fertilization can be regarded as complete when the maternal and paternal chromosomes are positioned for the impending first cell division in the life of the new individual. d. Metabolic energy and DNA and protein synthesis during pronuclei development Numerous physiological changes occur at fertilization that profoundly affect the activity of the oocyte, e.g. changes in permeability of small molecules, oxygen uptake, carbohydrate metabolism and synthesis of DNA, RNA and protein. These changes occur not only at fertilization but throughout embryogenesis (Longo, 1987). Like other cells, the zygote uses ATP as the metabolic energy for mitotic division and expulsion of polar bodies (Longo, 1987). ATP hydrolysis is essential for spindle elongation (anaphase B). An ATPase is involved in spindle elongation (Cande, 1982) During normal fertilization, DNA synthesis begins almost synchronously in the sperm and egg pronuclei (Krishna and Generoso, 1977; Howlett and Bolton, 1985). Pronuclear eggs have translation activity and synthesize new proteins perhaps using stored maternal mRNA (Cullen et al., 1980). At least five new proteins are synthesized during the pronuclear stage (Howlett and Bolton, 1985). The nature and function of these proteins are not clear, but some could be mitosis-triggering proteins (Howlett, 1986) stockpiled during the pronuclear stage for subsequent cleavages. II. Induction of superovulation A. History of superovulation The awareness of the importance of the pituitary gland and its gonadotropins led to the use of pituitary extracts to stimulate the growth of many follicles during a single estrus cycle in several mammalian species. Superovulation and estrus were induced in mice, rats and rabbits by Smith 11 and Engle (1927), Cole (1936) and Runner and Palm (1953). Smith and Engle (1927) used a preparation of pregnant mare's serum gonadotropin (PMSG) as a rich source of FSH activity, followed a few days later by injections of human chorionic gonadotropin (hCG) to induce ovulation. However, dogma insisted that only immature animals would respond to exogenous gonadotropins; mature females were refractory because their own cycle caused upsets in follicular growth induced by exogenous hormones. The situation changed in 1957, when Fowler and Edwards (1957) obtained estrus and superovulation in mature female mice using PMSG and hCG. Ovulation and mating occurred in the majority of females with large numbers of oocytes being ovulated and litters of almost 40 fetuses plus many resorbing embryos recorded just before parturition. Exact control could be imposed on folliculogenesis, oocyte maturation and ovulation, so that the maturational stages from diakinesis to metaphase of the second meiotic division and extrusion of the first polar body could be recorded (Edwards and Gates, 1959). Gemzell et al. (1958) successfully introduced superovulation in amenorrheic women, using pituitary extracts. Lunenfeld (1963) introduced preparations of human menopausal gonadotropins (hMG) as follicular stimulants. Recently, superovulation with hMG has been used for the treatment of ovarian dysfunction (Wyshak, 1978; Hack and Lunenfeld, 1979) and for in vitro fertilization (IVF) and embryo transfer (ET) (Lopata et al., 1978; Edwards, 1981; Moon et al., 1985). B. Mechanism of superovulation A principal action of exogenous gonadotropins in the induction of superovulation is now known to be recruitment of a non-growing pool of primordial follicles into a more mature-size class (Chatterjee et al., 1977; Hirshfield, 1989) and /or rescue of follicles at an early stage of atresia (Peters, 1979; Braw and Tsafriri, 1980). The commercially available gonadotropins PMSG and hMG are used widely for superovulation in animals and humans, respectively. Administration of exogenous gonadotropin stimulates steroidogenesis (Aguado and Ojeda, 1985; Johnson and Griswold, 1984) and mitogenesis in granulosa cells (Peluso et al., 1977; Vidyashankar and Moudgal, 1984; Hirshfield, 12 1985; Hirshifield and Schmidt, 1987). In response to gonadotropins, follicles at different developmental stages can undergo rapid growth and reach preovulatory size (Peluso et al., 1977; Orly, 1989). Exogenous gonadotropins elevate estradiol, androgens and progesterone (Sorrentino et al., 1972; Wilson et al., 1974; Miller and Armstrong, 1981a; Yun et al., 1987). Estradiol and progesterone act via positive feedback at the hypothalamus and pituitary to trigger gonadotropin surge (Zarrow and Gallo, 1969; Hagino and Goldzieher, 1970; Zarrow and Dinius, 1971; Wilson et al., 1974). Administration of a second gonadotropin, called ovulatory hormone, is used to rupture mature follicles. Any preparation that is high in LH activity may be used, the most convenient because of its commercial availability is hCG. The time interval between the injection of the priming and the ovulatory hormones varies between species, according to the natural timing of the gonadotropin surge (Gates, 1971). C. Source and biological activity of pregnant mare serum gonadotropin (PMSG) PMSG was one of the first commercially available gonadotropins. It has been widely used to superovulate domestic animals and for reproductive studies (Allen and Stewart, 1978; Papkoff, 1981; Reeves, 1987). PMSG is a glycoprotein synthesized and secreted by trophoblast cells of the endometrial cups (McDonald, 1977; Papkoff, 1981). Serum levels of this protein are detectable in pregnant mares by day 40, peak between days 60 to 80, and then decline to undetectable levels by day 140 of gestation (McDonald, 1977). PMSG is composed of noncovalently bound alpha and beta subunits. The alpha subunit consists of 96 amino acids and is identical for LH and FSH. The beta subunit is comprised of 149 amino acids and is identical to that of LH beta subunit. The differences between LH and PMSG appear to be due to different glycoslylation. PMSG is the most heavily glycoslyated of all known mammalian glycoprotein hormones, with a sugar content of about 45% of the total molecular mass. 13 a. Alpha-subunit PMSG alpha-subunit is consistent with the structures of the alpha-subunit of other mammalian glycoprotein hormones, and which was confirmed by the elucidation of the nucleotide sequence encoding for the PMSG alpha-subunit (Stewart et al., 1987). The amino acid sequence is highly conserved across a wide range of species. PMSG shows about 80% homology with that of most other mammals but differs from other species by a unique transposition of tyrosine and histidine at positions 87 and 93, respectively. Together with some nonconservative substitutions of amino acids 33, 70, and 96 this transposition has been reported to alter the hydrophobicity relative to that of other mammalian alpha-subunit (Murphy and Martinuk, 1991). b. Beta-subunit PMSG beta-subunit differs from other mammalian LH beta-subunit by a 30 amino acid C-terminal extension, the biological significance of which is still unclear. Part of the pronounced heterogeneity of PMSG has been attributed to differences in the N-terminal amino acid composition, which became apparent when PMSG from different plasma pools (Aggarwal et al., 1980a) or from trophoblast cell cultures were examined (Aggarwal et al., 1980b). N-terminal variability has also been observed in other glycoprotein hormones and may be important for their biological activity (Stockwell-Hartree, 1989). PMSG has 12 cystine half residues which are identical for all gonadotropin beta-subunits. c. Glycosylation PMSG contains the highest amount of carbohydrate of all the mammalian pituitary or placental glycoprotein hormones. Approximately 40% to 45% of its mass is attributable to sugars consisting of L-fucose, D-mannose, D-galactose, D-(N-acetyl) gluscosamine, D-(N-acetyl) galactosamine and sialic acids (Christakos and Bahl, 1979). The quality and quantity of sialinisation appears to be unique for PMSG. Recent studies show that the alpha-subunit carries approximately 20% carbohydrate by weight, whereas the beta-subunit contains more then 50% (Kamering et al., 1990). 14 One of the unique features of PMSG is its dual LH- and FSH-like activity in species other than the horse. Most striking is the enormous potency of PMSG as an FSH-agonist both in vivo and in vitro in a wide range of mammals. This high intrinsic FSH activity, the very long plasma half life (up to 60 hr in ewes, up to 120 hr in cows), and the convenient low price have led to the wide spread use of PMSG for the induction of superovulation in laboratory and farm animals. D. Detrimental effects of superovulation Despite widespread use of the superovulation technique in research, commercial and clinical fields, superovulation has been shown to adversely affect fertility in rats and mice (Miller and Armstrong, 1981b; Fossum et al., 1989; Ertzeid et al., 1993). It was demonstrated that the proportion of abnormal preimplantation embryos and postimplantation mortality were increased by superovulation. In human IVF, the live birth rate per embryo transferred is about 13% (Canada, Royal Commission on New Reproductive Technologies). A number of investigators have ascribed the reduced fertility to abnormalities in the process of fertilization, abnormal development of preimplantation embryos, implantation failure and fetal mortality (McLaren and Michie, 1959; Beaumont and Smith, 1975; Miller and Armstrong, 1981b; Ertzeid and Storeng, 1992). It appears that the detrimental effects and their associated mechanisms may be multifactorial, including genetic deficiencies in superovulated oocytes/embryos and/or hostile factors in the maternal environment such as an insufficient progesterone or excess estrogen stimulation of the reproductive tract leading to asynchrony between embryonic and uterine development (Moon et al., 1990). Embryonic chromosomal aberrations are recognized as one of the primary causes of reproductive failure (Bou6 et al., 1975). However, it has not yet been determined if superovulation adversely affects the chromosome constitution of either released oocytes or embryos (Fujimoto et al., 1974; Hansmann and El-Nahass, 1979; Moon et al., 1990; Hansmann and Pabst, 1992). III. Superovulation and chromosomal aberrations A. Asynchrony of oocyte maturation 15 Despite many attempts to superovulate oocytes capable of normal embryonic development, ovulation time remains unpredictable, resulting in heterologous aging of oocytes at fertilization. During ovulation induction, especially by administration of gonadotropins, multiple waves of premature or asynchronous ovulations are commonly observed in cattle (Callesen et al., 1987), humans (Navot et al., 1984), hamsters, mice and rat (Walton et al., 1983; Stern and Schuetz 1970; Sato and Marrs, 1986; Yun, 1989). Superovulation in immature rats induces a biphasic ovulatory response (De La Lastra et al., 1972; Kostyk et al., 1978; Miller and Armstrong, 1981a; Walton et al., 1983; Yun et al., 1987) which coincides temporally with the two peaks of LH activity (Yun, 1989). The first wave of ovulation occurs directly as a result of intrinsic LH activity of exogenous gonadotropins and the second wave indirectly via positive steroid hormone feedback at the hypothalamic-pituitary axis and an endogenous gonadotropin surge (McCormack and Meyer, 1963; Ying and Meyer, 1969a,b; Ying and Meyer, 1973; De La Lastra et al., 1972; Sorrentino et al., 1972). If more than one ovulation wave occurs with gonadotropin stimulation, aging and asynchronous nuclear maturation of oocytes may increase the incidence of abnormal embryos (Moon et al., 1990). Deterioration of oocytes or oocyte aging reduces the chance of embryonic survival. The longer the time interval between ovulation and fertilization, the greater the likelihood of a developmental anomaly (Braden, 1954; Britton, 1991; Santalo et al., 1992). In particular, fertilization of aged oocytes leads to an increased incidence of chromosomal anomalies in amphibians, rabbits and pigs (Vickers, 1969). Aged rabbit oocytes demonstrate ultrastructural abnormalities such as chromosomal pyknosis, coalescence or scattering (Austin, 1967). Furthermore, the chromosomal abnormalities produced by disintegration of the second meiotic spindle have been observed in aged oocytes (Austin, 1967). Oocyte degeneration was particularly evident in animals with precocious ovulation. In superovulated cows, a very low fertilization rate has been related to embryo donors resulting from precocious ovulation (Greve et al., 1983). A study of bovine oocytes from precocious ovulation showed that almost 50% of oocytes degenerated in culture and were unsuitable for in vitro fertilization (IVF) (Greve et al., 1984). Callesen et al. (1987) suggested that it was the inherent LH activity of PMSG that directly induced premature ovulations, produced abnormal endocrine 16 profiles and caused disturbances in oocyte meiosis. Furthermore, PMSG has been reported to cause premature activation of oocyte maturation in sheep and goats, possibly leading to ovulation of aged or abnormal oocytes (Moor et al., 1985; Kumar et al., 1990). In rats, premature ovulation and degeneration of oocytes and reduced fertilization rates were associated with superovulation with large doses of PMSG (Evans and Armstrong 1984; Yun et al., 1987). In addition, Kumar et al. (1990) suggested that the increased incidence of premature ovulation associated with superovulation was associated with premature activation of the initial stages of nuclear maturation in oocytes. B. Failure of endocrine control of meiosis The final step of oocyte maturation in vivo depends on the preovulatory gonadotropin surge. The steroid pattern in follicular fluid during the regular cycle is characterized by a shift from estrogen-dominance before the LH surge to increasing progesterone-dominance after the surge, as observed in cattle (Fortune and Hansel, 1985; Hyttel et al., 1986), sheep (Moor et al., 1973) and humans (McNatty et al., 1979; Testart et al., 1983; Moon et al., 1985). The altered steroidogenic patterns associated with the preovulatory and post-ovulatory peak of LH depend on the size of ovarian follicles, degree of atresia and number of LH receptors on granulosa and theca cells (Fortune and Hansel, 1985; Hyttel et al., 1986; Ireland and Roche, 1982; McNatty et al., 1984). Resumption of meiosis in oocytes is under hormonal influence. Changing the steroid hormone levels during the normal cycle not only triggers resumption of meiosis in the oocyte, but also controls the rate of meiosis via the prevailing steroid microenvironment in the follicle (Crowley and Gulati, 1979). Therefore, it seems likely that the nature of follicular growth in response to various ovulation-induction agents may influence the final oocyte meiotic maturation. It has been demonstrated in several mammalian species that cyclic adenosine monophosphate (cAMP) plays a dominant role in maintaining the G2-arrested stage. The transition from the G2 to the M phase is associated with a drop in the intracellular concentration of cAMP and the preovulatory LH surge (Aberdam et al., 1987; Dekel, 1987; Schultz et al., 1983). It was suggested that LH-induced oocyte maturation is a cAMP-mediated response (Dekel 1988). The 17 cAMP-regulated step in the G2 to M phase transition in the meiotic cell cycle is the protein dephosphorylation associated with MPF activation (Choi et al., 1991). Activated MPF triggers breakdown of the nuclear envelope and allows the cell to advance to the M-phase (Hashimoto and Kishimoto, 1988). It appears that oocyte maturation is a cascade event. Any disturbance in these events may cause meiotic errors. It was reported that at supra-physiological concentrations, testosterone, progesterone, pregnenolone, as well as other steroids, inhibit meiotic maturation, either alone or in conjunction with dibutyryl cAMP (dbcAMP) or agents that affect intracellular cAMP levels (Smith and Tenney, 1980; Rice and McGaughey, 1981; Racowsky and McGaughey, 1982; Moor et al., 1981; Freter and Schultz, 1984; Eppig and Koide, 1978). In mammals, increased circulating estradiol and androgen concentrations were observed after superovulation (Farooki, 1981; Murphy et al., 1984; Yun et al., 1987). The changes of hormonal concentrations may alter the molecular control of meiosis, e.g., they may directly or indirectly change cAMP or MPF levels, resulting in a disturbance of meiosis. In particular, androgens seem to play an important role in the events preceding non-disjunction because the administration of dihydrotestosterone in gonadotropin-primed females increased the incidence of aneuploidy in Djungarian hamster oocytes (Hansmann and Jenderny, 1983). Thus, alteration of the endocrine control of follicular function may increase the probability of meiotic non-disjunction (Hansmann, 1983). a. Non-disjunction Non-disjunction results in additional or missing chromosome in the female pronucleus. According to the theory of endocrine control of meiosis (Hansmann, 1983), alteration of hormonal levels may cause non-disjunction. An increase in aneuploid oocytes with hyperhaploid chromosome complement was observed in Djungarian hamsters after superovulation (Hansmann and Jenderny, 1980). Aneuploidy was direcdy proportional to PMSG dose injected, being lowest at 1.25 IU with 2.0% and highest at 20IU with 17%. Aneuploidy was not affected by exogenous gonadotropins in mouse, Chinese and Syrian hamsters oocytes (Hansmann and El-Nahass, 1979; Hansmann and Probeck, 1979; Golbus, 1981; Martin-Deleon and Boice, 1983; Catala et al., 1988; Badenas et al., 1989). However, these studies cannot exclude the possibility of an influence of 18 artificially stimulated ovulation on later division, i.e., the second meiotic or even post-meiotic division. Induced ovulation has also been suspected to increase the frequency of chromosomal abnormalities in human abortuses (Boue- and Boue\ 1973). This hypothesis has not been confirmed by direct cytogenetic analysis of human oocytes or embryos. No correlation has been found between the incidence of aneuploidy and different modes of follicular stimulation (Plachot et al., 1988b; Van Blerkom and Henry, 1988; Pellestor et al., 1989). However, Wramsby et al. (1987) suggested that ovarian stimulation could also induce maturation of abnormal oocytes, which would normally be destined for the atretic course. Due to a lack of comparable data from natural human menstrual cycles, there has been little information on whether the high proportion of aneuploidy in human unfertilized oocytes (24%) (Pellestor, 1991) was partly induced by hyperstimulation of ovary, or whether the human germ cell has a higher incidence of non-disjunction than other mammalian germ cells. b. Arrest of meiosis I or II Oocyte maturation can be associated with lack of extrusion of the 1st or 2nd polar body (Plachot et al., 1987). As a result of arrest of meiosis I or II, the chromosomes do not segregate into two groups, resulting in a diploid oocyte. Hansmann and El-Nahass (1979) reported on one mouse strain (NMRI/Han) that responded to gonadotropins by ovulating significant numbers (10%) of diploid oocytes which were most often arrested at metaphase I. However, other strains of mice did not have the same response after superovulation, suggesting that strain differences in the primary gonadotropin response, i.e. in the ovulation of diploid oocytes, are genetically determined (Hansmann and Jenderny, 1983). In humans, diploid oocytes were observed after ovarian hyperstimulation in several studies, ranging from 5% to 10% (Veiga et al., 1987; Macas et al., 1990; Tarin and Pellicer, 1990; Selva et al., 1991; Zenzes et al., 1992). These observations suggest that meiosis continued, without extrusion of the polar body. This arrest of meiosis at metaphase I may be caused by interference with the spindle apparatus in the superovulation regimen (Hansmann, 1983). 19 As a result of studies with oocytes from Djungarian hamsters and mice of the NMRI/Han strain, Hansmann (1983) proposed a model in which failed endocrine or paracrine control of germ cell maturation and meiosis alters development and regulation of meiotic cell division. Ordered segregation requires distinct processes of differentiation within the germ cell for segregation of homologous chromosomes/chromatids. These include synchronous maturation of the nucleus and cytoplasm, and chromosome assembly at the metaphase plate and movement within the spindle. All of these processes may be under direct/indirect regulatory control by the surrounding somatic compartments. Interference, e.g., by hormonal alterations at any of these steps, may alter the normal program of differentiation., and thus may increase the risk of chromosomal malsegregation. Altered follicular maturation may cause asynchronous maturation of the nucleus and cytoplasm in the oocyte, resulting in aneuploidy or diploidy (Hansmann et al., 1985). C. Errors occurring at fertilization As a cornerstone of sexual reproduction in mammals, fertilization activates the oocyte and re-establishes the diploid chromosome complement needed for normal development of the embryo. Errors of fertilization can result in polyploidy. Polyploidy is a haploid oocyte fertilized by more than one spermatozoon or by diploid sperm, or a diploid oocyte fertilized by a haploid sperm. Human and mouse studies have demonstrated that polyploidy is correlated to either immaturity or overmaturity of oocytes which cause incomplete cortical reaction or polar body retention, respectively (Maudlin and Fraser, 1977; Badenas et al., 1989). These phenomena may correspond with asynchronous ovulation by administration of gonadotropins (Austin, 1967). Takagi and Sasaki (1976) studied pronuclear, morula-blastocyst and postimplantation stage embryos from A/He strain mice. It was observed that the frequency of normal diploid embryos was significantly lower in the superovulated group than in the control group. The difference was ascribed to the fivefold increase in the incidence of digynic triploidy which was most likely due to a defect of the second meiotic division. Maudlin and Fraser (1977) demonstrated a dose-response relationship between PMSG and the incidence of polyploidy detected in first cleavage oocytes 20 fertilized in vitro from TO mice. Their data indicated that the incidence of polyploidy did not vary with PMSG dose for in vivo embryos. The polyploid embryos in their in vitro study were all polyandric. Using CBA/Ca x C57M/6J hybrid mice after administration of 5 IU PMSG, Badenas et al. (1989) studied the influence of the degree of oocyte maturation (either immaturity or overmaturity) on the chromosome characteristics of embryos at the first-cleavage division. An increased incidence of polyploidy was detected in immature (31.2%), and overmature oocytes (31.6%) groups when compared to controls (14%). In human IVF studies (Acosta et al., 1984; Rudak et al., 1985; Van der Ven et al., 1985), a relationship between the stage of oocyte maturation and percentage of polyploid embryos has been reported. The origin of these polyploid embryos has been ascribed to polyspermic fertilization. Trounson et al. (1982) and Van der Ven et al. (1985) found a high level of polyspermy (30%) when immature oocytes were fertilized. In immature oocytes, the cortical granules are still migrating to the vicinity of the plasma membrane and the cortical reaction fails after penetration of the first spermatozoon. Szollosi (1975) found the cortical granules moving toward the center of aged mouse oocyte in vivo. This migration of the cortical granules might result in failure of the cortical reaction, in turn resulting in a less effective mechanism for prevention of polyspermy. Alternatively, an alteration of the ability of the cortical granules to fuse with the vitelline membrane can impair the prevention of polyspermy (Van der Ven et al., 1985). Thus, it is presumed that asynchronous ovulation after superovulation may cause polyploid embryos due to immature or overmature oocytes (Plachot et al., 1987; Santalo et al., 1992). Lastly, some polyploidies may be due to different protocols of superovulation. Two percent of polyploidy have been recorded after clomiphene citrate stimulation and 12% after hMG stimulation (Wentz et al., 1983). A high incidence of diploid oocytes (7%) were recruited from GnRH treatment, which when fertilized may yield a high rate of polyploid zygotes (Tarin and Pellicer, 1990). It has also been suggested that the side effect of the different stimulation protocols is the production of some eggs of suboptimal maturity which are more vulnerable to the penetration by more than one sperm. D. Postzygotic stage 21 The first cleavage division of the zygote takes place shortly after DNA synthesis (Longo, 1987). Subsequent cleavage divisions of the zygote take place at intervals of about 12 hr or more. The earliest stages of embryogenesis are regulated by maternally inherited components stored within the oocyte. As development proceeds and maternally inherited information molecules decay, early embryogenesis becomes dependent on expression of genetic information derived from the embryonic genome (Telford et al., 1990). In the mouse, the transition from maternal control to zygotic control of transcription begins during the early and mid two-cell stage, while in human embryos, the transition time starts in the 4- to 8- cell stage (Telford et al. 1990). DNA damage and its repair or misrepair have been suggested to cause chromosomal abnormalities during early embryonic cell division (Savage, 1990). Chromosomal abnormalities observed in early embryos have at least 3 origins: meiotic errors, fertilization errors and cleavage errors. In general terms: meiotic errors give rise to karyotypes with numerical or structural changes, fertilization errors produce polyploid changes and cleavage errors produce either mosaic embryos or tetraploid embryos (Vickers, 1969). Fujimoto et al. (1974) studied rabbit embryos produced by fertilization of superovulated oocytes. Approximately, 8.3% of blastocysts showed chromosomal abnormalities, including mosaics, aneuploidy and triploidy. No abnormalities were detected in 36 control blastocysts. In contrast, Sengoku and Dukelow (1988) did not find a difference in the incidence of chromosomal abnormalities (aneuploidy) in 8- to 16-cell stage hamster embryos produced by fertilization of superovulated oocytes and naturally ovulated oocytes (2.2 vs 1.2%). Moreover, a variation of the incidence of aneuploidy from early implanted embryos was noted between different strains of superovulated mice; higher in inbred strains than in random-bred mice (Luckett and Mukherjee, 1986). An increased frequency of sister chromatid exchange was shown in preimplantation and postimplantation embryos in mice after superovulation (Elbling and Colot, 1985,1987). In humans, there are few data on the chromosomal complement of fragmented embryos after superovulation. Bongso et al. (1991) analyzed human preimplantation embryos and found that 20% of fragmented embryos displayed mosaicism (diploid/haploid, diploid/triploid, 22 diploid/aneuploid). In addition, cytogenetic analysis on 118 poor quality embryos (unequally sized blastomeres, granular cytoplasm and the presence of numerous fragments) by Pellestor et al. (1993) revealed that only 12 embryos (10%) displayed normal diploid metaphase. All others (90%) showed abnormal or aberrant chromosome complements, including 48% aneuploid, 12% haploid, 37% mosaic (2n/3n, n/2n), polyploidy (3n to 7n) or fragmented chromosome sets. The development of non-isotopic fluorescence in situ hybridization techniques (FISH) (Pinkel et al., 1988) highlighted the study of preimplantation embryos. Specific numerical and structural rearrangements can be detected in interphase nuclei using this molecular cytogenetic technique (Zahed et al., 1991). A recent publication demonstrated the feasibility of detecting specific chromosomal material in interphase nuclei of preimplantation embryos by in situ hybridization using chromosome probes (Ma et al., 1995a). Munne" et al. (1993) showed the presence of 70% aneuploidy in the blastomeres of 10 normally developing human embryos using five chromosomal probes (X, Y, 18 and 13/21), simutaneously. FISH is a useful technique and will enhance the future study for the detection of chromosomal abnormalities in preimplantation embryos. So far, little is known about the chromosomal complement in normal human preimplantation embryos from either normal menstrual cycles or superovulation. However, data from Bongso et al. (1991), Pellestor et al. (1993) and data obtained following spontaneous abortions (Boue- et al., 1975) suggested that chromosomal abnormalities (aneuploidy, structural abnormalities, polyploidy and mosaicism) may be a major causes of early embryonic loss. Poor quality embryos could result from severely unbalanced chromosomal complements. It is plausible that chromosome imbalance might affect early embryonic development. A direct cytogenetic study of preimplantation embryos after superovulation might demonstrate the higher incidence of chromosomal destruction. However, the ability to determine chromosomal behavior in early mammalian embryos after superovulation is limited. Most of the studies above were based on small sample size or did not provide details of chromosomal abnormalities (Fujimoto et al., 1974; King, 1985; Murray et al., 1986; Luckett and Mukherjee, 1986; Elbling and Colot, 1985, 1987; Sengoku and Dukelow 1988). 23 CHAPTER TWO Investigation of Effects of PMSG on the Chromosomal Complement of CD-I Mouse Embryos I. Introduction A. Background Perturbation of chromosomal segregation during gametogenesis or in the early embryo leads to abnormal embryonic development. The process involves complex interactions of a variety of functions in somatic and germ cells, is poorly understood, and presumably may be affected by a variety of genetic and environmental factors. The chromosomal abnormalities observed in oocytes and embryos of different species may result from non-disjunction at meiosis I or meiosis II. Chromosomal malsegregation occurring during postzygotic divisions in embryos may also contribute to chromosomal abnormalities. An experimental approach for the analysis of genetic factors, especially chromosomal complement, is to the measure chromosomal distribution pattern in early stage embryos. Mice represent the most suitable model system because of the availability of many strains, their well-defined reproductive behavior, as well as the relatively inexpensive cost. Furthermore, the rapidly-advancing state of molecular genetic knowledge, including mutagenesis (Rossant et al., 1993), allows investigators to culture and manipulate early mouse embryos to identify factors that may control early developmental events. This experimental system permits the manipulation of embryos and offers the possibility to study a large number of embryos to increase the statistical validity of the test. Furthermore, the mouse model has been used widely to study the effects of different conditions on IVF, on mammalian preimplantation embryo culture, and also as a quality control for human IVF-ET programs (Fraser and Maudlin, 1979; Trotnow, 1982; Ackerman et al., 1984). B. Reproductive cycle of female mice 24 The mouse reproductive pattern is characterized by an estrous cycle. It consists of proestrus, estrus, metestrus and diestrus, which can be determined by a vaginal smear analysis. The major normal stages and their characteristics are as follows: 1) proestrus - anabolic, active growth in the genital tract (1-1.5 days); 2) estrus - or heat, anabolic, active growth in the genital tract (1-3 days); 3) metestrus - catabolic, degenerative changes in the genital tract (1-5 days); 4) diestrus - quiescent period of slow growth of epithelium (2-4 days). The murine estrous cycle is roughly analogous to the follicular phase of the human cycle (Hogan et al., 1986). Mouse follicular growth and development leading to ovulation are regulated by a highly integrated sequence of hormonal events. Circulatory 17pV-estradiol (estradiol) levels commence to rise on the afternoon of diestrus, peak on the morning of proestrus and then decline. The decline of estradiol is followed by the gonadotropin surge, characterized by rapidly rising and declining levels of LH and FSH, respectively. These events occur on the noon of proestrus and lead to a transient elevation of progesterone and testosterone. The endogenous preovulatory gonadotropin surge occurs as a result of the circadian rhythm. A second FSH surge occurs in the early morning of estrus (Kovacic and Parlow, 1972). This pattern of hormone release leads to ovulation. The cyclic surges of LH and FSH ensure the maturation of 10-12 follicles every 4 days. This appears to be the number of follicles required for synthesis of adequate estradiol for the gonadotropin release. At estrus, a group of follicles exists on the ovary which have completed gonadotropin-independent development and require high levels of FSH for continued growth, without which they will undergo atresia. It has been suggested that the secondary FSH release following the gonadotropin surge selects the cohort of follicles which will ovulate at the next estrus. C. Superovulation in mice PMSG has been used to superovulate mice (Fowler and Edwards, 1957; Gates, 1971; Hogan et al., 1986). PMSG exerts LH and FSH bioactivity as it contains peptide sequences in its 25 beta-subunit which are homologous to those in the murine pituitary gonadotropins (Speroff et al, 1994). The ovulatory effect of PMSG is mediated by estradiol. Increasing estradiol levels stimulate LH release and induce ovulation. The marked elevation in both estradiol and androgen levels is attributed to the large number of follicles recruited by the high dose of PMSG. Human chorionic gonadotropin (hCG) is the second gonadotropin that is administered to induce superovulation. It serves as a substitute for LH and is required for rupture of the matured follicles. A 42- to 48-hour interval between the PMSG injection and the hCG injection has been found to be optimal in terms of egg yield. Ovulation takes place approximately 10 to 13 hr after injection of hCG (Hogan et al., 1986). The number of oocytes ovulated in PMSG induced mice is dose related. While a low dose of PMSG (1-3 IU) induces ovulation of 8-12 oocytes, higher doses (5-10 IU) induce 30-50 oocytes (Edwards and Wilson, 1963). Under optimal conditions, 80 or more ova can be obtained from prepubertal animals (Gates, 1971) and 30 to 40 from mature mice (Fowler and Edwards, 1957; Biggers and Whittingham, 1971). D. Detrimental effects of PMSG in mice Data from the literature have consistently shown that pre- and postnatal mortality is higher in immature and mature mice which have superovulated than in mature control mice which ovulated spontaneously. Death most commonly occurs during cleavage (Allen and McLaren, 1971), about the time of implantation, at mid-pregnancy or at, or shortly after, parturition (Edwards and Fowler, 1959; McLaren and Michie, 1959). Furthermore, in hormone-treated mice, intrauterine growth retardation appeared to occur irrespective of the number of implantation. Superovulation in mice has been found to induce malformations such as forelimb defects, to a lesser extent central nervous system anomalies, as well as delayed skeletal ossification (Elbling, 1973; Ertzeid and Storeng, 1992). It is not clear to what extent oocyte or maternal factors are responsible for the detrimental effects observed. Superovulation may affect oocyte maturation and chromosomal abnormalities may be responsible for impaired oocyte quality (Elbling and Colot, 26 1985, 1987). Treating mice with gonadotropins results in higher levels of circulating ovarian steroids, which may cause a disordered interaction between somatic cells and the preovulatory oocyte, resulting in chromosomal abnormalities of the ovulated oocyte. Takagi (1970) highlighted the possible detrimental effect of superovulation on female germ cells in mice. He found that the incidence of triploidy increased considerably among zygotes obtained through superovulation followed by natural mating in A-strain mice. Later, he studied pronuclear embryos from A/He strain mice and observed 19.2% digynic triploidy in the 10 IU PMSG treated group, while digynic triploidy was 3.7% in the natural cycle control group (Takagi and Sasaki, 1976). Maudlin and Fraser (1977) demonstrated a dose-response relationship between PMSG and the incidence of polyploidy detected in first cleavage oocytes fertilized in vitro, with the level of polyploidy rising from 8.0% with a PMSG dose of 1.5 IU to 20.8% with 10 IU. These observations raised the question of whether the incidence of chromosomal abnormalities in preimplantation embryos are the same as indicated at the zygote stage as observed by others (Takagi and Sasaki, 1976; Maudlin and Fraser, 1977). Chromosomal analyses of 8- to 16-cell stage mouse embryos should provide the baseline data to investigate this fundamental question. This study was undertaken to examine the hypothesis that superovulation can cause chromosomal aberrations in mouse embryos. Using a mouse model, the effect of exogenous gonadotropin on the chromosome complement of the embryos was assessed. II. Materials and Methods A. Mice Mature female and fertility-proven, mature male CD-I mice (6- to 8-weeks) were obtained from Charle's River Canada Inc (St. Constant, Quebec). These mice have been used for other studies of development and have no unusual incidence of malformations. The mice were maintained for one week after delivery prior to commencement of the study. 27 a. General care The mice were housed in the Animal unit at Children's Hospital Research Center, University of British Columbia (Vancouver, B.C). The mice were kept at 20-25°C on a 12 hr light: 12 hr dark cycle and were fed standard mouse laboratory chow and water ad libitum and housed in polycarbonate cages with wood shaving bedding. Animals were housed together with no more than three animals per cage. Males used for matings were housed individually in cages with a maximum of 2 females per night. b. Superovulation with pregnant mare's serum gonadotropin (PMSG) Vaginal smears were taken daily until at least three consecutive four day estrus cycles had been observed. These mice were then selected for the study. Between 1300 and 1400 hr on the first day of metestrus, experimental animals were given an intraperitoneal injection of 5,10 and 15 IU PMSG (Equinex, Ayerst, Montreal, Quebec) respectively, followed 48 hr later by 5 IU hCG (Serono Canada Inc., Ontario, Mississauga). The hCG was given at the expected time of the LH surge, estimated to occur 15 to 20 hr after the mid-point of the second period of darkness following the administration of PMSG (Gates, 1971; Figure 3). c. Time of pregnancy Timed pregnancies were obtained as follows: females were placed in cages with single males from approximately 1700 until 0900 hr the following morning, when females were checked for vaginal plugs. The presence of plugs was defined as first day of pregnancy and these females were caged separately. d. Embryo retrieval Mice were sacrificed by cervical dislocation. Following sacrifice, the oviducts and uterus were removed from each mouse and placed in Hank's Balanced Salt Solution (HBSS) (Life Technologies, Inc. Grand Island, NY). The oviducts and uterine horns were separated at the uterotubal junction. The oviducts obtained from each female were placed in a few drops of Ham's 28 Hours 12:00 24:00 12:00 24:00 12:00 24:00 12:00 • Endogenous LH 'surge' W, Ovulation Vaginal plug found Figure 3 Times of adminstration of exogenous gonadotropins shown in relation to the 24-hours lighting schedule and the stage of the estrous cycle (Gates, 1971). 29 F-10 medium (Gibco, Grand Island, NY) in a 10 x 35 millimeter (mm) petri dish (Falcon, Oxnard, CA). With the aid of a dissecting microscope, the embryos were flushed from either oviduct or uterus by inserting a blunt 30 gauge needle into the infundibulum and flushing with 0.3-0.5 ml Ham's F-10 medium containing 1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) into a 10 x 35 mm petri dish. e. General treatment of zygotes When necessary, zygotes from day 1 of pregnancy were transferred to Ham's F-10 culture medium (Gibco, Grand Island, NY) containing 0.2% hyaluronidase (Ovine Type II, Sigma Chemical Co) for 2 min, to remove cumulus cells still attached to the zona pellucida. Zygotes were washed twice in Ham's F-10 medium, transferred to droplets of culture medium containing 10~5 mM-vinblastine sulphate (Sigma Chemical Co). The medium used to culture the zygotes was Ham's F-10 medium supplemented with L-glutamine and 10% calf serum (Gibco, Grand Island, NY) and 100 IU/ml penicillin (Gibco, Grand Island, NY). The culture system consisted of microdroplets of medium under sterile, equilibrated mineral oil (Aldrich Chemical Company, Milwaukee, WS) in a 10 x 35 mm petri dish. The mineral oil was equilibrated with the culture medium. Between 5 and 10 zygotes were injected into each microdroplet. The zygotes were cultured in a humidified 5% CO2 incubator at 37°C overnight. f . Control group Spontaneously ovulated mice served as control. A vaginal smear was taken daily from each mature mouse between 0800 and 1300 hr to establish the stage of the estrous cycle. Mature mice with three consecutive four day estrous cycles were selected for the study. Between 1700 and 1800 hr on the day of proestrus, control mice were housed with males overnight. The females were removed between 0800 and 0900 hr on the following day (day one of pregnancy) and checked for the presence of a vaginal plug. The treatment of embryos was the same as in step 5 above. 30 B. Experimental Design a. Investigation of dose effects of PMSG on 1) ovulatory response and embryo quality; 2). chromosomal normality of zygote. 1. Ovulatory response and embryo quality At age 7- to 9-weeks, CD-I mature female mice were injected intraperitoneally with 5IU, 10 IU, or 15 IU of PMSG and, approximately 48 hr later, with 5 IU hCG. The number of embryos retrieved from day 1 was recorded and embryos were assessed for stage of development under a dissecting microscope at x80 magnification. Oocytes were considered to be fertilized if a second polar body was present in the perivitelline space. A zygote was considered to be fragmented if there were two or more cell fragments, both smaller than the original cell, with a large perivitelline space. If the single cell was reduced in size with a correspondingly large perivitelline space, or if the cell had ill-defined cell borders with opaque cytoplasm, it was considered to be degenerated. 2. Chromosomal normality of zygotes 1) Chromosome preparation After 20-24 hr in culture, the zygotes were transferred to a petri dish containing hypotonic solution (1% sodium citrate, w/v in H2O) (BDH Inc., Toronto, Ontario) for 10 min. The number of zygotes with a polar body was recorded. A maximum of 5 zygotes were placed on each slide. The zygotes were placed in the center of a clean slide and the excess hypotonic solution was removed. Two drops of a 3:1 mixture of ethanol-glacial acetic acid were gently dropped with a Pasteur pipette onto the area of the slide where the zygote had been placed. After 15-30 sec, 2 more drops of the same fixative were added. After about 1 min, the slide was dried on a slide warmer. The slide was immersed in fresh fixative for 1 min in order to clear the cytoplasm, was then gently blown dry (Tarkowski, 1966), and stained with 5% (v/v) Giemsa (Gurr's R66 Improved, BDH Inc., Toronto, Ontario) in phosphate buffer (pH 6.8) ( BDH Inc., Toronto, Ontario) for 5 min. 31 2) Chromosome analysis Only oocytes possessing a second polar body were considered to be fertilized oocytes and were defined to be zygotes. At this stage, paternally and maternally derived chromosomes can be distinguished by their degree of condensation, which allows assignment of gender of origin to chromosome abnormalities. The chromosome number was determined for both maternally and paternally derived sets of chromosomes using xlOOO magnification (Nikon photomicroscope). All zygotes with an abnormal number of chromosomes were photographed. b. Investigation of dose effects of PMSG on embryo quality and chromosomal complement of embryos at the 8- to 16-cell stage. On day 3 of pregnancy, treated and control females were injected subcutaneously with 3 i^g/g body weight of vinblastine sulfate 4-8 hr prior to sacrifice to obtain metaphase in 8- to 16-cell stage embryos. Embryos were considered to be of good quality if the blastomeres occupied most of the space within the zona pellucida and were of approximately equal size, with well demarcated cell borders, finely granular cytoplasm. Embryos were considered to be of poor quality if there was variation in the size of cells, if there were one or more opaque, poorly defined cell within the cell mass or if there was accumulation of cell debris and small fragments in the perivitelline space. Embryos were considered fragmented if they consisted of multiple cell fragments with marked variation in size and shape. 1. Chromosome preparation (Figure 4) 1) Digestion of zona pellucida Eight to sixteen cell stage embryos were recovered 4 hr after injection of vinblastine by flushing the oviduct and uterus with Ham's F-10 culture medium (Gibco, Grand Island, NY). Morphological criteria were used for assessing the embryonic development, and for chromosome preparation only the embryos displaying normal appearance and development to the 8- to 16-cell stage were used. Zona pellucida was removed by incubation in 0.5% Pronase (Bacchus and 32 >c~-X5j£ Embryo retrieval Vinblastine treatment in vivo to arrest mitosis at metaphase 8-16 cell stage embryo /I ^ \ 7 J-V Pronase removal of zona pellucida Pass through several rinses of medium Hypotonic citrate Transfer embryo to slide in small drop of hypotonic solution Add fixative drop-wise Air dry G-band Figure 4. Schematic illustration of the methodology used for preparation of chromosomes from 8-16 cell stage embryos 33 Buselmaier, 1988) (Sigma Chemical Co., St. Louis, MO) in Ham's F-10 culture medium for 5 min and mechanical disruption by repeated withdrawal through a fine glass micropipette. 2) Hypotonic treatment When the zona pellucida was completely digested, embryos were removed individually using a finely drawn out Pasteur pipette (internal diameter of approx. 0.2 mm) and transferred into a microdroplet containing 50 jil of 1% sodium citrate. Blastomeres were separated mechanically by aspiration with a finely drawn Pasteur pipette during the hypotonic treatment. 3) Fixation. During hypotonic treatment, fresh fixatives were prepared: fixative A consisting of distilled water, acetic acid, and ethanol v/v/v (5:1:4); and fixative B consisting of ethanol and acetic acid v/v (3:1). The blastomeres from each embryo were drawn up in a minimal amount of hypotonic solution into a fine glass Pasteur pipette containing fixative solution A in the tip of the pipette. The pipette was left on an angle of ~ 45° for 10 sec to allow the blastomeres to gradually accumulate at the tip of the pipette. The contents of the pipette were expelled drop-wise onto clean, dry slides from a height of ~ 2.0 cm. Then three drops of fixative B were expelled onto the blastomeres and this fixative was evaporated off by placing the slide on a warming plate at 37°C. The slide, with the blastomeres, was then immersed in fresh fixative for 2 min before air drying and scanning. A maximum of 5 embryos was placed on the slide and the blastomeres from each embryo were separated on different areas of a slide. 4) G-Banding. The slides were incubated overnight at 60°C. G-bands were produced by treating with 2x SSC (Saline sodium citrate; 0.3 M NaCl, 0.03 M tri-sodium citrate) at 60°C for 2 hr, followed by 0.25% (w/v) trypsin (Gibco, Laboratories, Life Technologies, Inc, NY) at 4°C for 2 min. The slides were then stained with 5% (v/v) Giemsa (Gurr's R66 Improved, BDH Chemicals, UK) for 2 min (Roberts and O'Neill, 1988). 34 2. Chromosome analysis All slides were coded and then examined in a blind fashion. The metaphase spreads were counted and examined for chromosomal abnormalities at xlOOO magnification. Only embryos with an unambiguous number of chromosomes were used for calculations of frequency of chromosome abnormalities. One or two cells in metaphase were available for analysis from each embryo. Many embryos were discarded because the chromosomes were overlapped, too contracted, or too widely spread. Embryos with chromosome numbers less than 38 were discarded since these might have arisen through faulty preparation. The metaphase spreads with abnormal chromosome numbers or structural abnormalities were photographed. A system of nomenclature for mouse chromosome banding described by Nesbitt and Francke (1973) was used for the mouse karyotype standard in this study. c. Statistical analysis The dose response of normally developing embryos or chromosome abnormality to PMSG was examined using the F exact trend test (Cochrane Amitage trend test in the exact form, Westfall and Young, 1993). This test is designed to identify a possible overall linear increase or decrease in the abnormality response and is more powerful than making pairwise comparisons which only utilize a fraction of the data. Similarly the dose response of oocytes or the embryos per mouse retrieved to PMSG was examined using a linear trend test. In contrast to the proportion data of the chromosome abnormality this data is continuous and so a linear contrast from analysis of variance was used. Where subanalyses were required, a Bonferroni correction was applied to minimize the occurrence of type I error. This was done by multiplying the p value in the subanalysis by the number of comparisons in the analysis, thus yielding a corrected p value. Differences were considered significant at p<0.05. III. Results 35 A. Morphological investigation of the effects of different doses of PMSG on embryo quality a. Oocyte collection The findings demonstrated that the treatment regimen used stimulated superovulation in 6-8 week CD-I mice; the mean number of oocytes in treated animals was 56.8, 33.0 and 21.6 in 5,10 and 15 IU PMSG treated groups, respectively (Figure 5a, b). Spontaneously ovulating controls had a mean number of 10.5 oocytes per mouse (Table I). The greatest superovulatory response was obtained when 5 IU was injected (56.8 ± 7.9/female; range 27-70 oocytes/female). The number of oocytes decreased significantly as the dose increased (P<0.01). Abnormal oocytes with various types of degeneration were recovered from all groups. However, the percentage of abnormal oocytes generally increased in a dose-dependent manner in the PMSG-superovulated mice when compared with the control group. The maximum proportion of degenerated oocytes (23.4%) was observed in 15 IU PMSG-treated mice (Table I). b. Eight- to 16-cell embryos The number of embryos per mouse was increased by 5 IU PMSG decreased by 10 IU, with a further decrease at 15 IU PMSG (Table II) (Figure 6 a, b). The percentage of normally developing embryos decreased as the PMSG-dose increased. The embryos from the spontaneously ovulated group, 89.1% were morphologically normal. The frequency of normal embryos derived from the 5 IU PMSG treated group was 87.4%, while the frequency of morphologically normal embryos decreased further to 64.0% and 54.1%, in the 10 and 15 IU groups, respectively (Table II). There was a significant response in terms of the incidence of morphologically abnormal embryos related to PMSG dose (p<0.01) in the PMSG groups as compared with spontaneously ovulated group. It appears that at this early stage of development, embryos from higher doses (10 and 15 IU PMSG) of PMSG are less viable than those from the spontaneously ovulating group. 36 ;# P P* »> Figure 5. a. Normal appearing oocyte with the first polar body, x400. b. Fertilized oocytes with two polar bodies, x400. 37 Table I. The Rate of Mouse Zygotes Produced in Spontaneously Ovulating (SO), 5,10 and 15IU PMSG Superovulated CD-I Mice Group SO 5IU 10 IU 15 IU No. (%)ofMice Pregnant/No. of Mice Treated 20/-5/10 (50.0) 8/15 (53.3) 17/50 (34.0) No. of Oocytes Per Mouse* (range) 10.5 ± 0.5* (7-13) 56.8 ± 7.9 (27-70) 33.0 ±7.1 (18-75) 21.6 ±3.0 (8 - 62) No. (%) of Zygotes^/No. of Oocvtes 205/210 (97.6) 272/284 (95.8) 234/264 (88.6) 281/367 (76.6) * Mean ± SE 1. The average number of oocytes retrieved per mouse from control were compared with those from different PMSG treated groups. A linear trend test, p<0.01. 2. Number of zygotes from control were compared with those from PMSG treated groups. F exact trend test, p<0.01. 38 Table II. The Rate of Normally Developed 8- to 16- Stage Embryos in Spontaneously Ovulating (SO), 5,10 and 15 IU PMSG Superovulated CD-I Mice No. of Pregnant Mice Group /No. of Treated Mice No. of Embryos Per Mouse 1 (Range) No. of Normally Developed Embryos^ (%) SO 5IU 10 IU 15 IU 205/-92/185 (49.7) 106/265 (40.0) 72/240 (30.0) 9.3 ± 0.2* (8-12) 30.0+1.4 (10 - 70) 24.3 ± 1.5 (9 - 72) 19.6 ±1.8 (5-45) 1700/1907 (89.1) 2413/2760 (87.4) 1649/2575 (64.0) 764/1411 (54.1) * Mean ± SE 1. The average embryos per mouse from control group were compared with those from different PMSG treated groups. A linear trend test, p<0.01. 2. Number of normally developed embryos from control were compared with those from different PMSG treated groups. F exact trend test, p<0.01. ^'"T" * s • r - * A ,*% 4 * % Figure 6. a. Normal appearing 10-cell stage embryo, x400. b. Abnormal embryo with different sizes of fragmented blastomeres, x400. 40 B. Investigation of the effects of different doses of PMSG on chromosomal complement of embryos at 8- to 16-cell stage The results of chromosome analysis of mouse embryos at the 8- to 16-cell stage are presented in Table HI and Figure 7. Aneuploidy. The frequency of hyperdiploid embryos was 2.8, 2.2, 2.5 and 3.5% of the analyzed embryos in the control, 5,10 and 15 IU PMSG treated groups, respectively (Figure 8, 9). Theoretically, non-disjunction should produce an equal number of embryos with 39 and 41 chromosomes. The numbers of embryos with 39 chromosomes exceeded the numbers of embryos with 41 chromosomes (Table HI). Since it is not possible for an accurate estimate of the frequency of hypodiploid embryos due to non-disjunction, a conservative estimate of the non-disjunction rate can be obtained by doubling the hyperdiploid rate, namely, 5.6, 4.4, 5.0, and 7.0% in the four groups. There was no significant effect of PMSG-dose in the levels of hyperdiploidy among the four groups when applying exact trend test (p>0.05). Polyploidy. There was no polyploidy found in the control or 5 IU PMSG treated groups. However, in the 10 IU PMSG treated group, 9 (2.9%) of 317 embryos were polyploidy, including 4 (1.3%) triploid (Figure 10) and 5 (1.6%) tetraploid (Figure 11). In the 15 IU PMSG treated group, 15 (10.5%) polyploid embryos were found in 144 analyzed embryos, including 6 (4.2%) triploidy and 9 (6.3%) tetraploidy. The proportion of polyploid embryos increased as the dose of PMSG increased (p<0.01). Structural abnormalities. A chromosome marker of unknown origin was observed in 2 (1%) and 5 (1.6%) embryos from spontaneously ovulating and 10 IU PMSG treated groups, respectively. 41 Table HI. Chromosome Analysis of 8- to 16-cell Stage Mouse Embryos from Spontaneously Ovulating (SO), 5,10 and 15IU PMSG Superovulated CD-I Mice Groups SO 5IU 10 IU 15 IU Diploidy 187/211 (88.6%) 199/225 (88.4%) 264/317 (83.2%) 108/144 (75%) F exact trend test !p>0.05; 2p<o.01 Aneuploidv1 Hyper Hypo 6 16 (2.8%) (7.6%) 5 21 (2.2%) (9.3%) 8 31 (2.5%) (9.8%) 5 16 (3.5%) (11.1%) Polyploidy2 Tri Tetra 0 0 0 0 4 5 (1.3%) (1.6%) 6 9 (4.2%) (6.3%) Hyper=hyperdiploidy; Hypo=hypodiploidy; Tetra=tetraploidy. Structural Abnormalities Marker 2 (1%) 0 5 (1.6%) 0 Tri=triploidy 42 / r l y > ~ . ? (? )« H >/ ;f ft H <t -><-6 7 8 9 ' n P H )! )) 11 12 13 14 15 <f »»' ft u I , 16 17 18 19 SEX Figure 7. A G-banded karyotype obtained from a 8-cell stage embryo, 40, XY. 43 *' ?-i m . • » 10 t| f I 1 1 12 13 14 15 16 17 18 if 19 SEX Figure 8. Karyotyped mouse 8-cell stage embryo with hypodiploidy, 39, XX, -4. 44 .-#* i f * % III M li H is l i ** if O H ll II • • )* SEX Figure 9. Mouse 10-cell stage embryo with hypeidiploidy, 41, XY, +?. * * \ • ^ Via * * V* « 6 * i * 4 % t I 1 1 16 • t* * 2 ?*P 12 17 ill ir 10 iit m iv 13 14 i 5 #*f» 19 SEX Figure 10. Karyotyped mouse 8-cell stage embryo with triploidy 58, XY, -16, -X or -Y? « • - f * » , > Figure 11. I t i f MM lift P P w M p i **§ SEX Karyotyped mouse 8-cell stage embryo with tetraploidy 80, XXYY. 47 C. Investigation of the effects of different doses of PMSG on chromosomal complement at the zygote stage The number of zygotes available for cytogenetic analysis were 126, 108, 106 and 120 in the spontaneously ovulating, 5,10, and 15IU PMSG treated female mice, respectively (Table IV). Aneuploidy and structural abnormalities were difficult to judge because of overlapping chromosomes prepared from the zygotes. Polyploidy was the most frequently detected anomaly in this study. The best choice to determine the origin of chromosome set is to identify sex chromosome, specific Y chromosome. However, it is difficult to recognize the Y chromosome due to the extension of chromosomes in male pronucleus. The origin and gametic source of the polyploid embryos was determined on the basis of differential condensation of maternally and paternally derived chromosomes. Male pronuclei showed longer chromosomes with a stronger tendency to overlap, whereas female chromosomes were shorter and usually showed less overlap (Figure 12). This finding was consistent with observations by Santalo et al. (1986). No polyploidy was found in 126 embryos examined in the untreated group. In comparison, 15 zygotes (4.5%) from the superovulated groups were found to be polyploid (14 with triploidy and 1 with tetraploidy). The proportion of polyploid embryos increased as the dose of PMSG increased (p<0.01). In the 5 IU PMSG treated group, 2 of 108 embryos were polyploid and were of maternal origin (Figure 13). In the 10 IU PMSG treated group, 5 polyploidy out of 106 embryos were present with 2 derived from maternal and 3 of paternal origin (Figure 14). A total of 8 polyploid embryos were found from 120 embryos in the 15 IU PMSG group, including 2 triploidy of maternal origin, 5 triploidy of paternal origin and 1 tetraploid embryo. The tetraploid embryo showed four sets of haploid chromosomes which was qualified as "presumptive diploid oocyte fertilized by two haploid sperms" due to the differential condensation of female and male chromosomes (Figure 15, 16). The number of diandric triploid and digynic triploid embryos in each group was too low to allow for statistical analysis of PMSG dose response. Polyspermic fertilization. The frequencies of embryos fertilized simultaneously by two sperms were 3 (2.8%) and 5 (4.2%) in the 10 and 15 IU PMSG treated groups, respectively. 48 Table IV. Incidence and Origin of Polyploidy in Mouse Zygotes from Spontaneously Ovulating (SO), 5,10 and 15IU PMSG Superovulated CD-I Mice Groups SO 5 IU 10 IU 15 IU Total No. of Embryos Examined 126 108 106 120 460 TriDloidv Diploid Oocyte 0 2 (1.9) 2 (1.9) 2 (1.7) 6 (1.3) Dispermy ( % • ) 0 0 3 (2.8) 5 (4.2) 8 (1.8) Tetraploidy 0 0 0 1 (D £>)* (0.8) 1 (0.2) Totall 0 2 (1.9) 5 (4.7) 8 (6.7) 15 (3.3) *D£> = diploid oocyte, dispermy I F exact trend test p<0.01 49 I t ? 12. First-cleavage mouse embryo fertilized in vivo showing differential condensation of male and female chromosomes. Due to marked contraction of chromosomes in female pronucleus and extension of chromosome in male pronucleus, the sex chromosome identification can not be done. & = male chromosomal complement; $p «• female chromosomal complement. 50 6 ? * Figure 13. First-cleavage mouse embryo fertilized in vivo showing a triploid chromosomal complement derived from "a presumptive diploid oocyte" and a haploid sperm. Qiromosomal complement identified according to the differential condensation of female and male chromosome. 51 (j 1%. 0 Figure 14. First-cleavage mouse embryo fertilized in vivo showing a triploid chromosome complement derived from dispermy and a haploid oocyte. 52 C ^W^^ * . f • ' # • * • d «* ^ r\ * Figure 15. First-cleavage mouse embryo fertilized in vivo showing a tetraploid chromosome complement derived from "a presumptive diploid oocyte" and dispermy. of or #^» J* % ki ^ ^ * \ %r » % _ • * • * c o • • « • ? F Figure 16. chromosomal complements at xlOOO magnification from the tetraploid embryo. Each chromosomal set indicated by alphabet corresponding to Figure 15. 54 Poly gynic fertilizations. The frequencies of diploid oocytes fertilized by one haploid sperm were 2 (1.9), 2 (1.9) and 2 (1.7%) in 5, 10 and 15 IU PMSG treated groups, respectively. VI. Discussion 55 A. Ovulation response This study showed that PMSG dose had an effect on the number of 8- to 16-cell stage embryos and the number of zygotes recovered. The mean number of zygotes or 8- to 16-cell stage embryos found in all superovulated group of mice was higher than in the control group. The maximum ovulation response was observed at 5 IU PMSG. However, the proportion of normal embryos was lower than in the control group. The percentage of mice with fertilized oocytes and 8- to 16-cell stage embryos decreased as PMSG dose increased (p<0.01). The number of degenerated oocytes and 8- to 16-cell stage embryos seems to be homogeneous among the two experiments performed (Table I and II). This is in agreement with findings by Walton et al. (1983) and Sato and Marrs (1986), who found that although the number of ovulating oocytes increased in mice and rats superovulated with increasing doses of PMSG, the fertilization rate of the oocytes decreased. Circulating levels of LH are essential for the production of steroid hormones that regulate the timing of ovulation and target tissue response. The literature suggests that elevated levels of serum LH during the follicular phase are unnecessary for follicular maturation and deleterious to normal reproductive processes (Chappel and Howies, 1991). This would suggest that the best results for ovulation induction would be expected with purified follicle-stimulating hormone (FSH). However, the PMSG preparation used in this study contained equal amounts of FSH and LH activities (Allen and Stewart, 1993). Elevated levels of LH during the follicular phase may result in premature resumption of meiosis, leading to ovulation of post-mature oocytes with low fertilization rates and poor preimplantation embryo quality (Kumar et al., 1990). The degenerated oocytes and embryos from superovulated groups in this study may have resulted from LH bioactivity of the PMSG preparation. This may be confirmed by future studies using FSH alone. B. Abnormal embryonic development 56 A progressive increase in the number of abnormal embryos was observed from day 1 to 3 in superovulated immature rats by Sherman et al. (1982) and Leveille and Armstrong (1989). Similarly, an increase in poor quality embryos was observed from day 1 to 3 of pregnancy in the present study. The increase in poor quality embryos after PMSG suggests that there was an adverse effect of PMSG on embryo quality. Aging of oocytes has been postulated as a teratogenetic factor for poor embryo quality observed after superovulation (Yanagimachi and Chang, 1961; Marston and Chang, 1964; Maurer et al., 1969; Thompson and Zamboni, 1975; Usui and Yanagimachi, 1976; Gianfortoni and Gulyas, 1985; Vickers, 1969; Saito et al., 1993). In mice, superovulation resulted in two different sets of oocytes; an initial set ovulated within 29 hours after the PMSG injection and a second set ovulated in response to the administration of hCG 48 hr after the PMSG injection (Stern and Schuetz, 1970). The normal fertilization time of the mouse oocyte at metaphase II after it has entered the fallopian tube is estimated to be 15 hr (Marston and Chang, 1964). If fertilization of the initial set of oocytes occurs at about 70 hr, abnormal embryonic development may result (Moon et al., 1990). With increasing post-ovulatory age, oocytes progressively lose the potential for normal and advanced embryonic development (Yanagimachi and Chang, 1961; Marston and Chang, 1964; Maurer et al., 1969; Thompson and Zamboni, 1975; Usui and Yanagimachi, 1976; Gianfortoni and Gulyas, 1985; Vickers, 1969; Saito et al., 1993). Therefore, a proportion of oocytes from the asynchronic ovulation in mice following superovulation may be responsible for the abnormal embryonic development observed in this study. The elevated steroid hormone levels in superovulated mice may also have an adverse effect on embryonic development (Yun et al., 1988). It was observed that elevated estradiol levels during early pregnancy cause fragmentation and degeneration of embryos in mice and rabbits (McGaughey and Daniel, 1966; Kirkpatrick, 1971). This appears to be the result of prolonged exposure to estrogen, since elevation of estrogen for 15 hr on day 1 of pregnancy has no effect on embryo quality (Ortiz et al., 1979). However, embryos cultured in fluid from estrogen-dominated donors were significantly less able to develop to morula (65%) or blastocyst (14%) than were 57 those cultured in progesterone-dominated fluid (87% and 36%). A high protein concentration was observed in estrogen-dominated fluid. It appears that adverse effect of estrogen on embryonic development may be mediated by an alteration of oviductal fluid protein content (Cline et al., 1977; Stone et al., 1977). To date, the identity of this protein factor(s) has not been elucidated. An increase in abnormal and degenerated embryos was observed in superovulated immature rats and mice (Walton and Armstrong, 1981; Miller and Armstrong, 1981a; Sherman et al., 1982; Yun et al., 1988; Leveille and Armstrong, 1989; Ertzeid and Storeng, 1992). In rats, improvement in embryo viability is noted when androgen and estradiol levels are reduced by ovariectomy, anti-PMSG or administration of the antiandrogen, flutamide (Walton and Armstrong, 1981; Yun et al., 1988). Lowering the LH activity of the superovulatory regime also reduced estradiol and androgen levels and significantly improved embryo viability (Leveille and Armstrong, 1989). In the present study, degenerative changes were most commonly observed in 8- to 16-cell stage embryos. C. Chromosome analysis of 8- to 16-cell stage embryos It appears that superovulation has a detrimental effect on fertilization and embryo quality in mice. This leads to the question of whether superovulation has an adverse effect on chromosomal complement in early stage embryos. The present study has provided important data on the incidence of chromosomal abnormalities in CD-I mouse preimplantation embryos after superovulation. With regard to the failure of endocrine control of meiosis theory (Chapter 1), there are two major theoretical assumptions: first, the process of non-disjunction may produce a high incidence of aneuploidy in preimplantation embryos and, second, fertilization of suboptimally matured oocytes after superovulation may result in an increased incidence of polyploidy. The results from this study only support the second assumption. a. Aneuploidy A higher percentage of hypodiploidy (37.8%) than hyperdiploidy (11%) was detected in this study. The greater frequency of hypodiploid embryos may be due to multiple causes, such as 58 displacement of chromosomes from the metaphase plate (Ford and Lester, 1982), anaphase lag (Martin, 1984) or alterations in the cytoskeletal function (Eichenlaub-Ritter et al., 1988). Artifactual chromosome loss should also be taken into account. In the present study, the hyperdiploid rate was doubled according to a 1:1 segregation ratio for non-disjunction. The calculated incidence of aneuploidy in the spontaneously ovulated group (5.6%) was similar to that reported by Catala et al. (1988). The 4.4% incidence of aneuploidy in oocytes from the 5 IU PMSG treated mouse group was consistent with a 3.6% incidence of aneuploidy in mice treated with the same dose of PMSG observed by Martin-Deleon and Boice (1983). Here, the calculated rate of aneuploidy in the three superovulated groups was 4.4, 5.0 and 7.0%, respectively, and there was no statistically significant correlation with the PMSG doses (P>0.05). In hamsters, aneuploidy in oocytes is directly correlated with PMSG dose injected (Hansmann et al., 1980). Hansmann (1983) proposed that failure of endocrine control of oocyte maturation alters meiotic division. However, this hypothesis was not confirmed by this or other studies of mice (Maudlin and Fraser, 1977; Golbus, 1981; Hansmann and Jenderny, 1983). The inducibility of aneuploidy by gonadotropins is a genetically determined species and strain specific phenomenon (Hansmann and Jenderny, 1983). The reasons for this specificity and the mechanisms leading to aneuploidy have not been elucidated. Catala et al. (1988) used CBA/Ca x C57B1/6J hybrid mice to study the age effect on the incidence of aneuploidy in immature, young and older mice in zygote. Using a 5 IU PMSG superovulation regimen for all mice studied, they found that there was a greater incidence of aneuploidy (10.0%) in immature mice than in mature mice (3.4%). Since the control of the hypothalamic-pituitary-gonadal axis might be subject to irregularities in prepubertal females, the effect of exogenous gonadotropins might be more pronounced, and, consequently, might induce failures in the endocrine control of meiosis at this stage of animal maturation. Catala et al. (1988) suggested that immature or prepubertal female mice may be more sensitive to gonadotropin stimulation than mature mice. 59 b. Polyploidy The incidence of triploidy in CD-I mouse zygotes was observed to be about 1% in the natural estrous cycle (Martin-Deleon and Boice, 1983). Tetraploidy was found to be a rare event in mammalian embryogenesis affecting only 0.1% of preimplantation mouse embryos (Beatty and Fischberg, 1951, 1952). In this study, no polyploidy was found in spontaneously ovulated oocytes. The incidence of polyploidy recorded for 8- to 16-cell stage embryos was 2.9% and 10.5% in 10 and 15 IU PMSG groups, respectively showing a simple dose-response relationship between PMSG and the incidence of polyploidy in CD-I mice, 8- to 16-cell stage embryos in vivo . These results confirm the suggestion by Bou6 and Boue" (1973) and Maudlin and Fraser (1977) that polyploidy may be induced by superovulation. During superovulation by gonadotropins, multiple waves of premature or asynchronous ovulations are often observed in several species. If more than one ovulation wave occurs with gonadotropin stimulation, asynchronous nuclear maturation of oocytes may occur. This may result in the production of oocytes at various stages of maturity, including immaturity and overmaturity, which in turn may be a source of the chromosome imbalance (Badenas et al., 1989). Polyspermy and digyny were reported to be increased in occurrences of delayed fertilization in rabbits (Shaver and Carr, 1967,1969; Chang and Hunt, 1968) and mice (Marston and Chang, 1964; Vickers, 1969). Superovulation of mice (Chang, 1977; Maudlin and Fraser, 1977) and hamsters (Sengoku and Dukelow, 1988) with PMSG increased triploidy which was also ascribed to delayed fertilization (Chang, 1977). In addition, an increased incidence of polyspermy in association with delayed fertilization has been reported in rats (Odor and Blandau, 1956). Therefore, in superovulated mice, where fertilization of oocytes is delayed, an increased incidence of polyspermy and digyny may occur. An increase in the rate of polyspermy in embryos derived from immature oocytes compared to the controls was observed in mice. A significant increase in polyspermy was also observed at one superovulatory PMSG dose during in vitro fertilization in immature rats (Evans and Armstrong, 1984). In human studies, Trounson et al. (1982) and Van der Ven et al. (1985) found a high level of polyspermy (about 30%) when immature oocytes were fertilized. Polyspermy in immature oocytes can be explained by a failure in cortical reaction, since it has been shown that 60 there is an incomplete migration of cortical granules to the vicinity of the plasma membrane (Badenas et al., 1989). In humans, most tripronuclear oocytes have altered cleavage patterns and various types of mosaicism (Ma et al., 1990). Ma et al. (1995b) studied the chromosomal complement of 72 embryos derived from tripronuclear zygotes using both traditional cytogenetic analysis and fluorescence in situ hybridization with chromosome 1,16 and X centromeric probes. Cytogenetic information could be obtained from only 25 of 50 tripronuclear embryos, whereas 18 of 22 embryos could be evaluated by fluorescence in situ hybridization. They detected 61% of embryos with mosaicism from FISH study, 36% of embryos from traditional cytogenetic analysis, only 28% of embryos with triploidy in both groups. The results demonstrate the usefulness of FISH for the detection of the accurate picture of triploid embryos. Unbalanced chromosome complements could lead to anomalous cleavage and poor quality embryos and may be an important factor in early embryonic loss. In keeping with this suggestion, it is plausible that the extent of chromosome imbalance might have a direct effect on early embryonic development. One might speculate that the greater the imbalance of the active genome, the earlier the manifestations of developmental disturbance. Therefore, polyploidy may be partly responsible for embryo degeneration at the preimplantation stage. Polyploid embryos can be maternal and paternal in origin. In the literature, some authors suggest that immature oocytes can be fertilized by more than one sperm, whereas overmaturation increases the incidence of diploid oocytes due to non-extrusion of the second polar body (Marston and Chang, 1964; Trounson et al., 1982; Rudak et al., 1985; Webster, 1985; Santalo et al., 1992). However, other studies of mice, pigs and humans have shown increased polyspermy related to delayed fertilization (Veeck et al., 1983; Rudak et al., 1984; Vickers, 1969; Meyer and Longo, 1979). It appears that the degree of oocyte maturation is associated with the incidence of polyploidy. Polyploidy found in this study was of both maternal and paternal origin, likely due to asynchrony of oocytes produced by superovulation. C. Chromosomal analysis of zygote stage embryos 61 In this study, the proportion of polyploid embryos increased as PMSG dose increased as previously observed in 8- 16-cell stage embryos. This result confirms that superovulation can cause chromosome abnormalities, mainly polyploidy. Although no polyploid embryos were found in 8- to 16-cell stage embryos from the 5 IU PMSG treated group, two polyploid embryos appeared in the 5 IU PMSG treated group examined at the zygote stage. It may be that early lethality of triploid embryos occurs at around the time of fertilization. The results of this investigation yield insight into the cause of polyploid embryos in CD-I mice following superovulation. Using mouse zygotes as an experimental model system, it was observed that the extra chromosomal set in polyploidy embryos originated by both (1) fertilization of a diploid oocyte and (2) dispermy. a. Digyny The extra chromosome set of digynic triploidy has a maternal origin and may come from fertilization of an egg with a non-reduced (diploid) chromosome number by a normal (haploid) spermatozoon. Diploid oocytes may arise through two mechanisms (Beatty, 1957; Austin, 1960). Firstly, a diploid oocyte may result from a meiotic block operative at the anaphase stage of the first maturation division. Secondly, failure at anaphase of the second meiotic division prevents extrusion of the second polar body (Dyban and Baranov, 1987). The increase in digyny after superovulation could be due to suppression of either the first or second polar body. One mouse strain (NMR/Han) responds to gonadotropins by ovulating a significant number of diploid oocytes most often arrested at metaphase I (Hansmann and El-Nahass 1979; Hansmann and Jenderny, 1983). In a A/He mouse strain, the frequency of digynic triploid embryos was significantly higher in the superovulated group than in the control group (Takagi and Sasaki, 1976). The major cause of this digyny was a defect of the second meiotic division. It was reported that the fertilization of a diploid oocyte that resulted from a failure of first meiotic division displayed two separated haploid sets of chromosomes of maternal origin and one haploid set from paternal origin (Dyban and Baranov, 1987). Thus, the observation in this study 62 of a distinct separate haploid set of maternal origin was indicative of failure of polar body extrusion leading to binucleated oocyte with both nuclei being haploid. The arrest of meiosis either at metaphase I or II in the superovulation regimen may be caused by interference of the spindle apparatus or asynchronous chromosome separation of all bivalents in oocyte. The mechanism for the arrest of meiosis may also relate to the molecular pathway in the superovulation regimen. Oocyte maturation is under the influence of MPF (Masui and Markert, 1971). MPF is considered a cell cycle regulator, ubiquitously present in cells undergoing mitotic and meiotic divisions. MPF functions as an oscillator responsible for the transition from G2- to M-phase (Hashimoto and Kishimoto 1988; Choi et al., 1991). A high MPF activity precedes GVBD and persists up to metaphase I. During the segregation phase, MPF activity drops below detectable levels and is only measurable again when the oocytes reach metaphase II (Hashimoto and Kishimoto 1988). Possibly, altered hormonal profiles after superovulation could change the MPF activity within oocytes resulting in the arrest of meiosis. One interesting aspect for understanding the role of MPF in generating diploid oocytes, would be the analysis of meiotic rate. The increase or decrease of MPF level may alter the meiotic rate and could be detected by manipulative MPF activity within oocytes. Future molecular analysis of oocyte maturation in an animal model will contribute to understand the mechanism for the formation of diploidy. b. Diandry The extra chromosome set in triploidy may have resulted from dispermic or diploid spermatozoon fertilization. The block to polyspermy in mammalian species involves modifications of 2 primary egg surface structures, the zona pellucida and the egg plasma membrane. The zona pellucida (ZP) block to polyspermy occurs as a result of the zona reaction, a change in the zona that causes it to become refractory to sperm binding or penetration (Wolf, 1981; Schmell et al., 1983). In the mouse, these changes in the zona pellucida constitute a block to polyspermy (Wassarman, 1990). The mouse ZP is composed of three glycoproteins, ZP1, ZP2, and ZP3. ZP3 binds acrosome-intact sperm and induces the acrosome reaction in bound sperm. ZP3 is then modified to a form called ZP3f after fertilization and loses both of these biological properties. ZP2, which 63 binds acrosome-reacted sperm, is modified to a form called ZP2f after fertilization (Bleil et al., 1981) and no longer binds to acrosome-reacted sperm. The modification of ZP2 to ZP2f is most likely due to the release of a CG-associated protease. These changes result in the inability of sperm to penetrate the ZP and constitute the ZP block to polyspermy (Ducibella et al., 1993). Thus, the ability of eggs to undergo CG exocytosis is critical for the establishment of the zona block to polyspermy. Fertilization of germinal vesicle (GV)-intact oocytes is frequently associated with polyspermy, which may be due to its inability to undergo CG exocytosis and mount a zona block (Ducibella et al., 1990). Consistent with this observation is that calcium ionophore, A23187, treatment of metaphase II eggs results in a mean CG loss of 71% and the ZP modification (Ducibella et a., 1990; Moller and Wassarman, 1989); the extent of these changes is similar to that which normally occur in response to sperm. In contrast, A23187 treatment of fully grown GV-intact mouse oocytes does not result in a detectable loss of CGs (Ducibella et al., 1990). Treatment of metaphase I (MI) eggs with A23187 results in a mean CG loss of 28% (Ducibella et al., 1990). Changes in CG distribution during aging have been observed in eggs of mice and many other species. These results suggest that meiotic maturation is accompanied by the development of competence to undergo CG exocytosis. The response to PMSG demonstrated here by an increase in diandric polyploidy suggests that the hormone not only affects the oocyte nuclear events but also extra-nuclear events. These might involve incompetence to undergo CG exocytosis as observed in A23187 treated metaphase I oocytes (Ducibella et al., 1990). Human and mouse studies have demonstrated that polyploid fertilization is correlated to either immature or overmaturity of oocytes (Marston and Chang, 1964; Vickers, 1969; Chang, 1977; Maudlin and Fraser, 1977; Trounson et al. 1982; Van der Ven et al., 1985). Therefore, the effect of the different stimulation regimes is the production of some oocytes of suboptimal maturity which are more vulnerable to penetration by more than one sperm. c. Tetraploidy Tetraploidy is a rare event in mammalian embryogenesis. Among 921 human abortuses with an abnormal karyotype 6.2% were tetraploid (Bou6 and Boue\ 1974, 1976; Boue" et al., 1975). In preimplantation mouse embryos 0.1% were tetraploid (Beatty and Fischberg, 1951, 64 1952). In the present study, only one tetraploid zygote was found which had resulted from a digynic ovum fertilized by two normal haploid sperms. This finding suggests that some of the diploid oocytes obtained by superovulation were immature and therefore resulted in polyspermy. The initially haploid paternal and maternal pronuclei in mammals have to double their DNA content, and the zygote containing a tetraploid amount of DNA then undergoes the first cleavage division, with two diploid blastomeres formed. The complete block of the first cleavage division results in a tetraploid zygote which, if it proceeds to the next cycle, gives rise to two tetraploid blastomeres (Dyban and Baranov, 1987). Endoreduplication may be another cause of tetraploidy by which chromosomes replicate two times without mitosis, resulting in a tetraploid zygote (Therman et al., 1983). It has been proposed as a mechanism by which haploid parthenogenotes make a step-wise progression from haploid to polyploid (King et al., 1988). This mechanism has also been implicated in ploidy increase associated with tumorogenesis. Since only one tetraploidy was found in zygote stage, it was proposed that tetraploidy found in 8- to 16-cell stage embryos may be caused by a complete block of the first cleavage division in a diploid zygote or by endoreduplication. Both mechanisms could result in a tetraploid zygote. The mechanism of the first mitotic arrest after superovulation is unclear and needs further investigation. The results obtained from zygote stage suggest that the detrimental effect of superovulation on chromosomal complement is due to alteration of hormonal levels which lead to a failure of endocrine control of meiosis and mitosis resulting in polyploidy (Figure 16). IV. Summary Embryonic development, investigated extensively in mice following induced ovulation, has revealed that the occurrence of substantial mortality results during cleavage, at implantation, midpregnancy and parturition. The extent to which chromosome imbalances are related to embryonic mortality has not been explored. Observations here suggest that triploidy constitute an 65 SUPEROVULATION I Failure of endocrine control of meiosis and mitosis y \ Arrest of mitosis Immature or aging \ \ Tetraploidy Polyspermy \ Polyploidy Arrest of MI or MII I Digyny I Failure of implantation or spontaneous abortion Figure 17. Schematic illustration of mechanisms of chromosomal aberrations by superovulation 66 appreciable portion of preimplantation losses in CD-I mice. The following are the final conclusions from this study. 1) A positive dose-response relationship between PMSG dose and the incidence of polyploidy was detected in CD-I mouse 8-16 cell stage embryos developed in vivo. 2) Polyploidy, especially, triploidy, was derived from both digyny and diandry. The dose-response relationship between PMSG and the incidence of polyploidy may be caused by either suppression of meiotic division or alteration of the zona pellucida during oocyte maturation. These events may be related to asynchrony of oocyte maturation by exogenous gonadotropins. 3) PMSG used for stimulation of ovulation has no effect on segregation of individual chromosome during the meiotic and mitotic division that would lead to aneuploidy in CD-I mouse embryos. 67 CHAPTER THREE Chromosome Investigation of Unfertilized Human Oocytes after Superovulation I. Introduction A. Background For many years, cytogenetic analysis of female gametes was impossible due to technical and ethical reasons. Consequently, little data was collected on the chromosomal complement of human oocytes (Edwards, 1968; Jagiello et al., 1976). The introduction and development of in vitro fertilization (IVF) techniques provided a unique opportunity to perform extensive cytogenetic investigations of human oocytes. Indeed, 30-50% of oocytes recovered during IVF procedures fail to be fertilized and can, therefore, be used for chromosomal analysis. Wramsby and Liedholm (1984) were the first to analyze unfertilized oocytes from IVF programs. They demonstrated that 25% of unfertilized oocytes had chromosomal abnormalities. Since then, a number of laboratories around the world have reported cytogenetic analyses of human oocytes, albeit based on small sample numbers. A summary of the frequency and types of chromosome abnormalities observed in the oocytes is presented in Table V. A definitive determination of the background frequency of chromosomal abnormalities in unfertilized, meiotically mature human oocytes would be expected to have a major impact on our understanding of the causes of early developmental failure and embryo wastage in humans. Such information on whether current protocols of follicular stimulation and initiation of preovulatory oocytes generate a higher incidence of chromosomally aberrant oocytes would also benefit IVF programs. B. Relationship of certain factors with incidence of chromosomal anomalies Studies of cytogenetic abnormalities of human unfertilized oocytes in IVF have tried to Table V. Summary of Published Cytogenetic Studies of Human Unfertilized Oocytes Reference Mode of Age of No. of Frequency of Frequency of Frequency of stimulation women metaphases aneuploidy (%) diploidy(%) structural (years) analysed abnormalities (%) Wramsby and Liedholm (1984) Michelmann and Metder (1985) Martin etal. (1986) Wramsby and Fredga (1987) Veiga etal. (1987) Wramsby etal. (1987) Plachot etal. (1988b) Bongso etal. (1988) Van Blerkom and Henry (1988) Djalali etal. (1988) Papadopoulos et al. (1989) Pieters et al (1989) Pellesor etal. (1988; 1989) Ma etal. (1989; 1995c) Tarin et al. (1990) Delhanty and Penketh (1990) Macas etal. (1990) Michaelietal. (1990) Selva etal. (1991) Angelletal. (1991) Sutter etal. (1991) Zenzes etal. (1992) CC ? CC-HMG CC-HMG CC-HMG CC CC-HMG HMG/FSH LHRH-HMG FSH-HMG CC/CC-HMG CC/CC-HMG HMG ? CC/HMG CC-HMG CC-HMG LHRH-HMG LHRH-HMG CC-HMG LHRH-HMG CC-HMG LHRH-HMG CC-HMG LHRH-HMG CC-HMG LHRH-HMG CC/HMG FSH/HMG ? 2242 24-35 22-38 7 25-38 25-42 2742 3240 24-39 ? ? 2240 24-41 ? 24-39 24-38 2741 27-37 24-36 ? 2543 8 33 33 52 115 21 316 251 135 96 25 28 377 227 168 155 57 67 198 135 87 161 25.0 3.0 30.0 50.0 10.8 57.1 24.0 21.1 8.1 27.1 24.0 21.4 27.0 26.0 3.6 5.2 18.0 46.7 28.0 13.0 39.0 27.4 / / / / 7.0 / 1.8 2.0 / / / / 2.9 16.7 7.1 8.4 5.0 3.3 10.8 1.5 2.3 8.1 / / 4.0 1.9 4.9 / / 0.4 / / 24.0 / 0.5 / / / 1.8 / / / / / Total 2745 22.1 5 1 oo 69 answer the basic questions: why were the oocytes not fertilized and did the method of ovulation induction have any influence on the cytogenetic findings? Several theories have been proposed to elucidate the effects stimulated cycles might have on chromosomal abnormalities. These theories can be divided into those that have their basis in the protocol of follicular stimulation and those that take into the consideration other influencing factors such as advanced maternal age, in-vitro aging of oocytes and the effect of a specific population of patients. a. Association of chromosomal abnormalities with the method of follicular stimulation A variety of follicular stimulation protocols are used in different IVF clinics. They are clomiphene citrate (CC)/human menopausal gonadotropin (hMG), gonadotropin-releasing hormone (GnRH)/hMG, FSH and hMG. Most commonly, exogenous gonadotropins are employed to recruit and develop ovarian follicles for the generation of multiple oocytes. The question arises if hyperstimulation of the ovary with exogenous gonadotropins generates aneuploidy in oocytes. Some studies showed no correlation between treatment and frequency of aneuploidy (Van Blerkom and Henry, 1988; Plachot et al., 1988b; Pellestor, 1989; Pieters et al., 1991). Wramsby et al. (1987) suggested that ovarian stimulation could induce maturation of abnormal oocytes, which would become atretic without stimulation. In a recent study, Tarin and Pellicer (1990) found that excessive ovarian response to gonadotropins resulted in both a significant increase of diploid oocytes without extrusion of the first polar body and a higher incidence of premature condensation of sperm chromosomes (p<0.05). Furthermore, Pieters et al. (1991) noted that after CC/hMG stimulation, 34% of unfertilized oocytes were immature compared with only 18% after GnRH/hMG stimulation (p<0.05). The frequency of aneuploidy was the same for both groups. It appeared that different ovarian stimulations may not increase aneuploidy. However, an accurate evaluation of the effects of follicular stimulation would require comparison of aneuploidy rates in oocytes recovered in stimulated cycles versus oocytes recovered in spontaneous cycles. However, such an analysis is impractical since IVF is rarely performed on unstimulated women. 70 b. Association of increased frequency of chromosome abnormalities with maternal age The effect of maternal age on fertility and the frequency of chromosome aneuploidy is documented. The mean age of women attending the in vitro fertilization program at the University of British Columbia was 34.5 years compared to an average age of 27 years conceiving in the British Columbia Provincial population (Province of British Columbia, Ministry of Health, 1987). The influence of maternal age on the frequency of chromosome abnormalities of oocytes has been established. Epidemiological studies have indicated that an increase in the frequency of specific autosomal trisomies correlates with an increase in maternal age (Ferguson-Smith and Yates, 1984; Hassold and Chiu, 1985). The large majority of trisomies are the result of maternal meiotic errors but the cause of age-related aneuploidy still remains obscure. Henderson and Edwards (1968) found a significant decrease in the chiasma frequency in the oocytes of old female mice. They proposed that univalents were produced in the older females during the prolonged dictyotene stage of meiosis. This hypothesis assumes that the oocytes formed late in a woman's fetal development to be both the most susceptible to trisomy and the last to be ovulated. Other hypotheses state that the occurrence of abnormalities is linked to physiological aging of the reproductive system, delayed ovulation and hormonal imbalances (Page et al., 1983). In addition, a few authors have suggested that the incidence of embryonic abnormalities could be due to reduced intrauterine selection, not advanced maternal age (Ayme and Lippman-Hand, 1982). Brook et al. (1984) proposed a physiological hypothesis in which increased age-dependent aneuploidy would be determined not by chronological age but by biological age as well. They used a CBA female mouse model to manipulate the normal pattern of physiological aging of the reproductive system by unilateral ovariectomy. Unilateral ovariectomy induced earlier onset of acyclic estrus cycles in female mice. It also induced an earlier rise in aneuploidy levels, as compared to sham-operated animals. These results suggested that abnormal segregation of meiotic chromosomes is a phenomenon of physiological aging of the reproductive system. Only results from cytogenetic studies of human oocytes can provide the information essential to resolve these conflicting hypotheses. Early data from the studies of Van Blerkom and Henry (1988) and Pellestor and Sele (1988) suggested that maternal age does not contribute to the occurrence of aneuploidy. This conclusion must be considered tentative since older women (> 35) 71 represented less than 10% of the patients included in the report of Van Blerkom and Henry (1988). The data derived from a multicenter study of Plachot et al. (1988b) demonstrated an almost 14% increase in aneuploidy when the chromosomal status of 224 oocytes from women 35 and younger (24%) were compared with that of 69 oocytes from women over 35 years of age (38%). The frequency of aneuploidy in oocytes from women greater than 35 years of age was significantly higher than those in younger women. However, only 69 oocytes were obtained from women over 35 and the number of oocytes in this sample from women over 39 was not indicated. While it might be expected that increasing maternal age is accompanied by an elevated frequency of chromosomal aberrations in oocytes, a definitive conclusion with respect to increase in maternal age and induced ovulation will require a larger and more specific data base than currently exists. c. In-vitro aging of oocytes Another factor that should be taken into account in chromosomal analysis of human oocytes is the in-vitro aging of oocytes. Even if in vitro culture conditions for oocytes (pH, temperature, % CO2, composition of media) are of comparable methodology, the duration of culture before oocytes can be considered unfertilized varies between published protocols from 40 to 60 hr. Less is known about the effect of several hours of in vitro culture on chromosome characteristics of oocytes (Spielman et al., 1985). Plachot et al. (1988a) reported an 87% incidence of chromosome abnormalities in human embryos (23 embryos) after 20 hr delayed fertilization, compared with an incidence of only 20% when fertilization took place immediately after insemination (252 embryos). Including the results reported by Plachot et al. (1988a), there exist insufficient data to determine whether the delayed fertilization has an impact on the chromosomal complements in human oocytes. In contrast, some investigations have demonstrated adverse effects on the morphological (Ortiz et al., 1982) and biochemical (Gifford et al., 1987) integrity of human oocytes cultured for 24 to 60 hr post-recovery. It must be noted that Martin et al. (1986) and Wramsby and Fredga (1987) analyzed non-inseminated oocytes immediately after recovery from an in vivo state and reported incidence of aneuploidy similar to those resulting from a study of in-vitro unfertilized oocytes cultured for 48 hr. Thus, the effect of incubation times used in IVF on chromosomal complements in oocytes is not clear. 72 d. Chromosome abnormalities due to patient-specific causes rather than as a direct consequence of ovarian stimulation Van Blerkom and Henry (1988) reported that a significant fraction of aneuploid and potentially aneuploid oocytes was derived from a very small number of patients. For example, approximately 27% of the hypohaploid oocytes, and 33% of the oocytes that exhibited chromosomes not associated with the Mil spindle were derived from two patients. Zenzes et al. (1992) found that patients who were previously parous produced significantly reduced numbers of aneuploid oocytes, compared with the nonparous group. An excess (p=0.01) of patients had multiple oocytes all alike (all haploid or all aneuploid), showing a correlation among multiple oocytes of a patient and specific chromosome status. Van Blerkom and Henry (1988) suggested that chromosomal perturbation is more likely patient-specific than due to ovarian stimulation. This patient variation may contribute to the widely differing frequencies of chromosomally abnormal oocytes that have been reported (Table V). Clearly, a much greater data base than that which currently exists will be required to determine unambiguously whether or not a relatively small population of women account for a disproportionate number of chromosomally aberrant oocytes. In summary, chromosomal abnormalities occur at a frequency of about 28.1% in meiotically mature oocytes obtained from stimulated follicles during IVF procedures (Table V). The chromosomal abnormalities observed are 1) aneuploidy (22.1%), due to non-disjunction; 2) diploidy (5%) caused by suppression of polar body extrusion and 3) structural abnormalities (1%). This high frequency of chromosomal abnormalities after stimulated cycles in humans may be directly related to several factors such as: 1) hyperstimulation; 2) maternal age or 3) in vitro fertilization. With regard to the occurrence of aneuploidy, these data agree with results from epidemiological surveys of liveborns and spontaneous abortions which have indicated the strong predominance of maternal origin of the extra chromosomes in automosomal trisomies (Juberg and Mowrey, 1983; Gait et al., 1989). These observations lead to the question of whether the chromosomal abnormalities might be directly related to ovarian hyperstimulation or to an aging population of women or to oocytes prone to have chromosomal abnormalities after ovarian 73 hyperstimulation. Analysis of a larger sample of human unfertilized oocytes from in vitro fertilization will provide important baseline data to investigate this fundamental question. In this Chapter, the incidence of chromosomal abnormalities in human unfertilized oocytes obtained by superovulation is investigated. II. Materials and Methods A. Patients This study included 194 couples from the IVF program at the University of British Columbia (UBC) who were enrolled because of infertility. The indications for IVF were tubal factor (55%), endometriosis (17%), ovulatory dysfunction (12%), and male factor (16%). The female mean age was 33.6 with a range of 24-41 years. The male mean age was 36.3 with a range of 25-45 years. a. Hormonal treatment Oocytes available for this study came from a clomiphene citrate (CC; Serophene, Serono, Mississauga, Ontario)-human menopausal gonadotropin (hMG, Pergonal, Serono) stimulation regimen (Messinis et al., 1985). Follicular development was induced with CC 100 mg/day on cycle days 3 to 7, and hMG was administered from day 6, dosage dependent on individual response. Follicular growth was monitored daily by serum 17|3-estradiol levels and frequent ultrasound scanning. Final follicle maturation was induced with 10,000 IU of hCG, (Profasi, Serono or Organon, Scarborough, Ontario) when the leading follicle attained a diameter of £16-mm and when the 17p%estradiol reached 700-1500 pmol/l/preovulatory follicle. Follicles were aspirated 34-36 hr after hCG administration, using transvaginal ultrasound guidance and local anesthesia. b. Sperm preparation After semen sample liquefaction, volume, sperm concentration, per cent motile spermatozoa and per cent normally formed spermatozoa were assessed according to WHO 74 guidelines (Belsey et al., 1980). The semen sample was prepared by the 'swim-up* technique. The semen was transferred to a tube to which was added a similar amount of Ham's F-10 medium (Gibco, Grand Island, NY) with 7.5% maternal serum (Ashwood-Smith et al., 1989). The tube was immediately incubated at a 45° angle for 1 hr at 37°C and 5% CO2. After this time, a small fraction of the top layer was reassessed for semen quality. The remainder of the top layer, containing highly motile spermatozoa, was transferred to another tube and incubated 3 hr at 37°C and 5% CO2 to allow for capacitation. Then the semen sample was used for insemination. c. Oocyte culture and in vitro fertilization The cumulus masses were identified in the culture room (adjacent to the operating room). Oocytes with expanded corona radiata were recorded as mature and those with discernible compactness of the corona radiata cumulus cells were recorded as immature (Veeck, 1988). Oocytes were rinsed in phosphate-buffered saline solution (PBS) (BDH Inc, Toronto) (Dandekar and Quigley, 1984) and then in Ham's F-10 medium (Gibco, Grand Island, NY) supplemented with 0.5% bovine serum albumin (BSA) (Sigma, St. Louis, MO). After rinsing, oocytes were transferred to approximately 1 ml Ham's F-10 medium (Lopata et al., 1980) with 7.5% maternal serum (Ashwood-Smith et al., 1989) at 37°C in an atmosphere of 5% O2, 5% CO2 and 90% N2. Following a preinsemination incubation period ranged from 5 to 7 hr, the oocytes were inseminated with about 50,000 highly motile spermatozoa. At 12 to 16 hr after insemination, the oocytes were transferred to growth medium (15% maternal serum in Ham's F-10 medium) and examined for fertilization by analysis of pronuclei. Normal postfertilization development was assessed using morphologic criteria, namely, the observation of two polar bodies, two pronuclei, equal-size blastomeres, and first and second cleavage at regular intervals. Twenty-seven to 44 hr after insemination, embryos at the 2- to 8-cell stage that had cleaved regularly were transferred to the patient's uterine cavity. Seven hundred and forty-one oocytes were selected as 'presumed to be unfertilized' by the criteria of the absence of two pronuclei and lack of cleavage after 48 hr of insemination. Because of some degeneration, only 488 of 741 unfertilized oocytes were processed individually according 75 to the modified method of Tarkowski (1966) (Wramsby and Liedholm, 1984) for cytogenetic analysis. d. Chromosome preparation After unfertilized oocytes were selected from IVF, they were placed in 1% sodium citrate for 10 min at room temperature. Each oocyte was transferred in a minimal amount of hypotonic fluid to freshly prepared fixative A (water, acetic acid, methanol; v/v/v (5:1:4)) for 30 to 40 sec. Then, the oocyte was moved to a grease-free glass microscope slide. Three drops of freshly prepared fixative B (methanol, acetic acid; v/v (3:1)) were placed onto the oocyte and the oocyte flattened onto the slide. The preparations were allowed to dry on a warming plate (37°C) to enhance chromosome spreading and stained for 2 min with 10% (v/v) Giemsa (Gurr's R66 Improved, BDH Chemicals, UK) in phosphate buffer (pH 6.8) (BDH Inc, Toronto). The slides were then examined at xlOOO magnification under a Nikon microscope. Karyotypes were prepared from photographic prints and analyzed for numerical and structural abnormalities. e. Chromosome analysis The oocyte analysis was based on the following cytogenetic criteria: 1. Oocyte meiotic chromosomes. Oocyte chromosomes in metaphase II are highly condensed and appear rather shortened, curly; the chromatids are frequently separated. Such limitations in the karyotyping of metaphase II cells make the identification of structural rearrangements difficult 2. Prematurely condensed sperm chromosome (PCC). Beside oocyte metaphase chromosomes, there appear chromosomes up to two times the length of oocyte metaphase chromosomes. These chromosomes were considered as Gl-phase prematurely condensed sperm chromosomes (PCC), as described by Schmiady et al. (1986). 3. Mitotic chromosomes. These chromosomes are not of meiotic morphology but most likely represent an mitotic metaphase. They are much longer than oocyte metaphase chromosomes, and chromatids are frequently not separated. 76 The oocytes displaying 18 to 22 chromosomes were regarded as hypohaploidy, while oocytes exhibiting 24 to 25 chromosomes were defined as hyperhaploidy. Those showing <18 chromosomes were discarded because of the possibility of artifactual chromosome loss, and those with >28 chromosomes were scored as hypodiploid metaphase II (Plachot et al„ 1988b). All oocytes with an abnormal number of chromosomes were photographed. Only oocytes with an unambiguous number of chromosomes were used for calculations of chromosomal abnormality frequencies. Two hundred and eighty oocytes with good chromosome morphology were cytogenetically analyzed. Chromosomes were arranged in groups according to Denver classification (ISCN, 1985). The oocytes were classified into 6 categories based on chromosome number: haploidy (23,X), hypohaploidy, hyperhaploidy, diploidy, hypo and hyperdiploidy. Two hundred and eight oocytes were unanalyzable due to clumped chromosomes or excessively spread chromosomes. B. Correlations Correlations between clinical parameters and the rate of chromosome abnormalities were analyzed. The parameters were: a. Dosage of gonadotropin. The dosage of hMG used for each patient varies from 300IU hMG to 9000 IU hMG, depending on ovarian response. Patients with 300 IU hMG to 3000 IU hMG were considered as lower dose of hMG group and 3150 to 9000 IU hMG were defined as high dose hMG group according to the criteria used in a multicenter study by Plachot et al.(1988b). The distribution of chromosomal patterns from the two groups were compared. b. Maternal age. Two groups were considered, aged < 35 years or > 35 years. The ages of patients ranged from 24 to 41 (mean = 33.6 years). C. Statistical analysis Statistical analysis was carried out using chi-square test. Differences were considered significant at p<0.05. III. Results 77 Four hundred eighty-eight oocytes were prepared for cytogenetic investigation and chromosome analysis was performed on 280 oocytes. The remaining 208 oocytes were unsuitable for analysis due to inadequately spread chromosomes or technical reasons. The chromosomal complement of the 280 analyzed oocytes is given in Table VI. A. Haploid Mil, 23,X One hundred and forty-six oocytes were included into this group. B. Haploid abnormal This group contained 77 oocytes including hyperhaploid and hypohaploid oocytes, (i) Hyperhaploidy with one or two extra whole chromosomes was observed in 32 oocytes, the extra chromosome being of A, B, C, D, E, G groups, respectively (Figure 18). (ii) Hypohaploidy with 18- to 22-chromosomes was observed in 45 oocyte, having a missing chromosome in a A, B, C, D, E, F, G group (Figure 19). To calculate the incidence of aneuploidy, the rate of hyperhaploidy was doubled (22.8%). This method eliminates the possibility that aneuploidy arose through technical losses. C. Diploid oocytes Diploidy was observed in 47 oocytes, 29 were 46, XX (Figure 20); 14 were hypodiploid with chromosome numbers ranging from 33 to 44; four were hyperdiploid having chromosome numbers ranging from 47 to 53. D. Fertilized oocytes Among 280 oocytes, 10 displayed mitotic chromosomes, with 7 oocytes showing 17 to 50 mitotic chromosomes and another three polyploidy with 76- to 94-chromosomes (Figure 21). Table VI. Cytogenetic Study of 280 Morphologically Unfertilized oocytes* Haploidy 23, X (146)§ 146 (52.1%) Hypohaploidy 22, XX, -C (3) 22, X, -E, -F (3) 22, X, -E (3) 22, X, -19 (3) 22, X, -F (3) 22,X, -F 21, -C, -X? (3) 21, X, -C, -C (3) 20, X, -C, -E, -F (5) 20, X, -D, -E, -F (4) Meiosis Hyperhaploidy 24, X, +B (2) 24, X, +A (2) 24, X, +G (10) 24, X, + E (2) 24, X, +D (10) 25, X, +G, +Frag 25, X, +E?, +E? 25, X, +D, +G (2) 25, X, +B, +C (2) 19, X, -B, -C, -D, -D(9) 18, X, -D, -D, -E, -E, -G (3) 18, X, -A, -B, -E, -F, -G (2) 45 (16%) 32 (11.4%) Diploidy 46.XX (29) 29 (10.4%) Hypo & 33 (2), 36, 43 (3), 44 53 18 (6.4%) Hyperdiploidy , 38 (3), (2), 47, 40(3), 48 (2), Mitosis 17 22.X 30, XX 43, XX 46, XX 47, XX 50, XX 76, XX 84, XX 94, XX 10 (3.6%) *The part of data is the continuation of previous study and 215 human unfertilized oocytes have been done before the registration of graduate study. § Number in parentheses are the number of oocytes 00 79 * > * • * ' • » k* * * « M * 4 A * It t « t «W A *f * * D * * « * * Figure 18. Hypohaploid human oocyte (22, X, -C). 80 # • / ^ * i f « * i 1 % ** - » D # Figure 19. Hyperhaploid human oocyte (25, X, +E?,+E?). 81 it » • # * * *> ^ J1 * * * Jtf nn * * * * * * * » H4 am # l * km> M * • # • A * * € ft* AH. D 9 c* «*» •» «»•« * j * Figure 20. Diploid human oocyte (46, XX). 82 V it k 1 * & I 1 i I I 'm * * A I D C G Figure 21. Human oocyte with mitotic hypohaploid complement, 22, X, -G. 83 Considering the mitotic appearance of their chromosomes, these oocytes must have been fertilized, even though two pronuclei had not been observed. E. Gl-prematurely condensed sperm chromosomes (PCC) Fifty-two of 280 oocytes characterized by metaphase II chromosomes showed prematurely condensed sperm chromosomes (Gl-PCC), besides the maternal metaphase chromosome (Figure 22), including 30 haploidy, 6 hypohaploidy and 2 hyperhaploidy, 14 diploid range. F. Group comparison 1. No correlation was found between the dose of gonadotropins administered and chromosomal abnormalities (p>0.05) (Figure 23). 2. When the overall rate of chromosomal aneuploidy in unfertilized oocytes was compared between two donor age groups; a nearly uniform rate of aneuploidy was found (p>0.05) (Figure 24). m€ * * # * $, 4% W D G -'**/ - ^|f J l v-: — « «* *#* n f * # *t Figure 22. Prematurely condensed sperm chromosomes (large arrowhead) and metaphase II chromosomes from oocyte (small arrowhead). Karyotyped hypohaploid oocyte, 22, X, 85 Distribution of chromosomal patterns according to doses of hMG 60n Haploidy Hypohaploidy Hyperhaploidy Diploidy p>0.05 Figure 23. The distribution of chromosome patterns in unfertilized human oocytes according to the doses of hMG (first group: hMG doses from 300 to 3000IU; second group: hMG doses from 3150 to 9000 IU). 86 Distribution of chromosomal patterns according to maternal age p>0.05 Figure 24. The distribution of chromosome patterns in unfertilized human oocytes according to maternal age (first group: women age from 24 to 34; second group: women age from 35 to 41 years old). IV. Discussion 87 The incidence of aneuploidy (22.8%) in 280 oocytes in this study was similar to the 22.1% incidence of aneuploidy reported in the literature (refer to Table V). However, if we compare this figure (22.8%) with other mammalian oocytes (1-10% anomalies) (Bond and Chandley, 1983), the rate of chromosomal anomalies in human unfertilized oocytes seems to be very high. The high rate of aneuploidy observed in human oocytes leads to the question: Is there a relationship between superovulation treatment and incidence of chromosomal abnormalities? Ideally to answer this question the rate of chromosomal aneuploidy in naturally ovulated oocytes should be compared to that in oocytes obtained after superovulation. Unfortunately, data on the incidence of chromosomal aneuploidy in human oocytes of non-stimulated cycles are almost impossible to obtain. To date, the only practical way to assess the effect of superovulation on chromosomal complement is to compare the cytogenetic results from different protocols used in IVF. Plachot (1988b) first used a large sample size (316 oocytes) to examine the effect of hormonal induction on non-disjunction. They divided karyotypes into four groups according to the hormonal protocol used (CC/hMG, hMG alone, pure FSH, LHRH/hMG). There was no relationship found between the mode of stimulation and the frequencies of aneuploidy. Furthermore, similar studies by Van Blerkom and Henry (1988) and Pellestor et al. (1989) also found that there was no significant differences in the aneuploidy rate among the different protocols used. Evidence to date indicate that the induction protocol itself has no tendency to increase the rate of aneuploidy. Human menopausal gonadotropin (hMG) is a widely used exogenous gonadotropin for the treatment of infertility. In most IVF clinics, hMG is used in combination with clomiphene and GnRH analog. Depending on the ovarian response, the dosage of hMG used in each patient differs. This raises a question whether a high concentration of exogenous gonadotropin has an effect on chromosomal complements. This study compared the incidence of aneuploidy to that obtained after superovulation using two range of hMG dosage (group one: 300-3000 IU hMG; group two: 3150-9000 IU hMG). There was no significant difference between the two groups (p>0.05), consistent with the findings of Plachot et al (1988b) whose results similarly failed to 88 demonstrate an effect of hMG dosage on incidence of aneuploidy. The data in this study together with published data suggest that the incidence of chromosomal aneuploidy in human oocytes is not affected by the mode of hyperstimulation protocols or the dosage of exogenous gonadotropin. A recent study by Gras et al. (1992) using 20 oocytes from non stimulated cycles, reported 20% aneuploidy which is close to the incidence of aneuploidy in human unfertilized oocytes from IVF. Since the sample size is small, it is not possible to draw definitive conclusions on whether superovulation has an effect on chromosomal complement in human oocytes. It also remains unclear whether a higher baseline rate of aneuploidy exists in humans compared to other mammals. The process of meiotic nondisjunction should produce an equal number of n+1 and n-1 gametes. In published data, the proportion of hypohaploidy and hyperhaploidy is not equal; hypohaploid gametes occur approximately three times as often as hyperhaploid gametes (Pellestor, 1991). Also in the present study, hypohaploidy (16%) was in excess of hyperhaploidy (11.4%) and did not seem to fall into the expected 1:1 ratios. While it is possible that hypohaploidy due to anaphase lag may occur in addition to non-disjunction, it is impossible to distinguish these from technical artifacts (Martin et al., 1986). Thus, in this study, the incidence of aneuploidy was calculated by doubling the hyperhaploidy figure. From data on sperm combined with information on the relative frequencies of maternal and paternal errors in the etiology of trisomies from induced and spontaneous abortions, a minimum estimate of 20% aneuploidy from meiotic errors can be obtained (Jacobs, 1990; Burgoyne et al., 1991). This figure closely approximates 22.8% obtained following follicular stimulation in this study. As in previous reports, the majority of the oocytes examined cytogenetically revealed a haploid set of second meiotic metaphase chromosomes. The one major difference between results here and those cited (Table V) has been with respect to frequency of diploid oocytes. Diploid oocytes in which the first polar body has not been extruded, thus giving rise to 46 instead of 23 meiosis II chromosomes, have been reported with different frequencies in various surveys. This is to some extent a reflection of the selection criteria of the oocytes. Pellestor and Sele (1988) included only oocytes considered to be mature on the basis of first polar body exclusion. Martin et al. (1986) used only morphologically normal oocytes. Both situations effectively select against diploid oocytes which were not reported in either study. The frequency of 16.8% diploidy 89 observed here is higher than observed by Delhanty and Penketh (1990) (8.4%) and Selva et al. (1991) (10.8%). Such variability may reflect sampling error or, alternatively, may be associated with different ovulation induction regimens. Diploid oocytes have been studied in other mammalian species and mice. Hansmann and El-Nahass (1979) showed that significantly more diploid oocytes were found in hormonally stimulated mice than in non-stimulated mice. This mouse study (Chapter 2) and others (Takagi and Sasaki, 1976; Kaufman, 1991) showed that diploid oocytes from hormonally stimulated animals can undergo fertilization, and the resulting triploid embryos have the potential to develop up to or even beyond the post-implantation stage. In humans, diploid oocytes are also not an impairment to fertilization since in some triploid spontaneous abortions an error at the first meiotic division on the maternal side has been reported (Jacobs et al., 1978). Oocyte maturation is under precise hormonal control and the ultimate site for hormonal influence on the oocyte is the hormonal microenvironment in the follicles. It has been suggested that formation of diploid oocytes may result from a disturbed hormonal microenvironment of the follicles arising from hyperstimulation (Hansmann et al., 1980). Tarin and Pellicer (1990) found that peripheral 17P-estradiol per follicle visualized was lower in the group of patients with an increased incidence of diploidy compared to those with lower incidence of diploidy, thus suggesting that some changes may occur in those follicles. Evidence from rodent studies suggests that there is a dose-dependent relationship between the presence of diploid oocytes and the amount of gonadotropins used to induce follicular development and ovulation (Hansmann et al., 1980). The results from mouse (Chapter 2) showed that superovulation caused triploidy, partially due to diploid oocytes fertilized by one sperm. In the present study a greater dosage of hMG was not related to a higher incidence of diploidy. The dose of gonadotropins is individualized in women on a day-to-day basis. The ovarian response is increased when other factors such as the age (young women), body mass (low), or endocrine status are present. Thus, the response to gonadotropins in humans should be regarded as a reflection of the sensitivity of the ovary to superovulation rather than simply the dose of medicine used. 90 These cytogenetic findings have enhanced our understanding of the origin of the multifollicular response to the stimulation protocols in IVF. The cohort of follicles recruited by gonadotropins in a given IVF cycle is theoretically accompanied, in cases of excessive response, by a recruitment of "younger" cohorts or recruited atretic follicles (Pellicer et al., 1988). Based on the present study, the probable origin of some oocytes is from those follicles in early stages of development. These oocytes did not extrude the first polar body, suggesting an incomplete development of competence to resume meiosis. Another important factor that may have exerted an impact on the chromosomal complement of the oocytes studied is the maternal age. The influence of maternal age on meiotic nondisjunction, leading to an increased incidence of chromosome abnormalities in spontaneous abortions and newborns, has been demonstrated (Boue et al., 1975; Hook, 1981). Hassold and Chiu (1985) found a positive correlation between increasing maternal age and the incidence of trisomies. This was most evident at maternal age > 35 years and, by extrapolation, they suggested that a large number of oocytes from women > 40 years of age are aneuploid. A significant increase of nondisjunction associated with maternal age was also reported in mice (Gosden, 1973). The frequency of nondisjunction is almost nil in young female mice (Martin et al., 1976). In the present study, no correlation (p>0.05) was found between the incidence of overall aneuploidies and maternal age. Numerical abnormalities were equally observed in all maternal age groups from 24 to 41 years. This result is similar to that reported by Pellestor and Sele (1988) who did not observe an increase of aneuploidy with maternal age. They suggested that there could be decreased maternal selection against affected conceptuses with advanced maternal age. However, Plachot et al. (1988b) reported that maternal age significantly increased the rate of aneuploidy in oocytes. Later, Zenzes et al. (1992) found that aneuploidy, most frequently involving group G chromosomes, appeared to be associated with advanced maternal age. They suggested that maternal age affects the reproductive performance and is related to specific chromosomal aneuploidy. To date, the data from human unfertilized oocytes do not provide conclusive evidence for a contribution of maternal age to the increased incidence of nondisjunction. Thus, investigation of the relationship between aneuploidy and maternal age should continue. 91 After sperm penetration, the appearance of male and female pronuclei indicates fertilization. The observation of mitotic metaphase chromosomes indicates that fertilization had indeed occurred in 10 oocytes (3.6%) although no pronuclei had been observed on inspection of the oocytes in culture. Mitotic metaphase in unfertilized oocytes has also been found by other investigators. Mettler and Michelmann (1985) showed that 3 out of 49 unfertilized oocytes were polyploid (6%). Pieters et al. (1989) observed that 7 out of 150 unfertilized oocytes showed one set of mitotic chromosomes (5%). These results are similar to findings in this study of 3.6% of mitotic metaphase in unfertilized oocytes. The majority of the fertilized oocytes in this and other studies showed numerical abnormalities. Thus, post-fertilization failure and developmental-arrest of zygotes seem to be the first steps of natural selection against chromosomally abnormal zygotes before implantation. Penetration of a sperm into an oocyte can be demonstrated by the presence of prematurely condensed chromatids of the Gl-phase (PCC) separated from the oocyte chromosome. A clear explanation of PCC in human oocytes was first given by Schmiady et al. (1986), who pointed out that the situation was dependent on the permanent arrest of the oocytes at metaphase II after sperm penetration. It was suggested that altered levels of maturation promoting factor (MPF) may induce PCC (Zenzes et al., 1990; Calafell et al., 1991). In a normal meiotic division, MPF levels increase during MI and Mil and decreases immediately after fertilization as observed in Xenopus oocytes (Minshull, 1989; Murray and Kirschner, 1989). In mammalian oocytes, the MPF probably follows a similar cycle, such that if non-activated (cytoplasmically immature) oocytes are fertilized, premature chromosome condensation of the male pronuclei may occur due to the presence of MPF in the oocyte cytoplasm (Calafell et al., 1991). In human IVF, the presence of an additional set of PCC with metaphase II oocyte has been widely described by different authors. The frequency of haploid meiosis II metaphase oocytes with PCC has been reported as 3% to 28% (Schmiady et al., 1986,1989; Pieters et al., 1989; Macas et al., 1990; Zenzes et al., 1990). In this study, PCC was observed in 18.6% oocytes which is similar to the study by Pieters et al. (15.4%) (1989). In 47 diploid oocytes, 14 showed PCC, an accepted characteristic of fertilization of immature oocytes (Badenas et al., 1989). Conaghan et al. (1989) used LHRH/hMG protocol and delayed oocyte recovery by one day. They found 92 fertilization rates and clinical pregnancy rates were significantly higher in the "delayed group" than in the standard regime. They suggested that oocytes maturing longer in vivo are more competent to undergo normal fertilization and development. Angell et al. (1991) found similar results when they used the modified protocol of Conaghan. Also, they observed that the rate of PCC decreased as the rate of pregnancy increased. They suggested that the less time the oocyte matures in vivo, the greater the likelihood of abnormal fertilization in which sperm PCC are formed. The fact that high percentage of PCC occurring in mouse immature oocytes as observed by Calafell et al. (1991) support this idea. V. Summary In the previous chapter, evidence was presented for the existence of polyploid embryos after superovulation. In this chapter, the study focused on cytogenetic analysis of human unfertilized oocytes. The frequency of numerical chromosome abnormalities in metaphase II oocytes from 280 human unfertilized oocytes was determined. Aneuploidy was found to occur at a frequency of 22.8% in meiotically mature oocytes obtained from stimulated follicles during IVF procedures and did not vary with the dose of hMG or maternal age. The observation of 16.8% of diploid oocytes suggests that blockage of the meiotic process at meiosis I level after superovulation may occur in the humans as it does in the mouse. Human triploid embryos account for up to about 15% of all first trimester spontaneous abortions. One percent of all fertilized oocytes are estimated to be polyploid in vivo (Jacobs et al., 1982) and 10% triploid embryos were found in IVF (Pieters et al., 1992). The distribution of digyny and diandry in tripronuclear embryos after IVF is unknown. In the present study, the observation of diploid oocytes may provide evidence that some triploidies in IVF may be derived from fertilization of a diploid oocyte as the mouse study indicated. The high percentage of diploidy may relate to the induction regimen used. Considering the asynchronous maturity of oocytes resulting from superovulation, it appears that oocytes matured longer in vivo are less prone to show Gl-PCC. 93 In conclusion, human unfertilized oocytes had an incidence of numerical chromosome abnormalities of 39.6%, including 22.8% aneuploidy and 16.8% diploidy. This high frequency of chromosome abnormalities in unfertilized oocytes may explain the low pregnancy rate in IVF programs suggesting that natural selection against chromosome abnormalities may occur even prior to fertilization. 94 CHAPTER FOUR Summary and Conclusion Mouse studies Considerable evidence suggests that elevated androgen and estrogen levels after superovulation cause degeneration of oocytes and developmentally retarded embryos by disturbing the specific follicular endocrine microenvironment. Since resumption of meiosis in oocytes is under hormonal influence, the alteration of hormonal levels may cause meiotic chromosomal abnormalities in the ovum. As a result, after fertilization, the embryos may contain an abnormal chromosomal complement. In the present study, using a CD-I mouse model, the chromosomal characteristics of the embryos obtained by superovulation were assessed. The cytogenetic analysis of 8- to 16-cell stage mouse embryos to evaluate chromosomal normality after superovulation was performed. A technique to obtain analyzable metaphases from individual blastomeres was developed and used to determine the cytogenetic composition of 8- to 16-cell stage mouse embryos. The hypothesis that superovulation affects the chromosomal complement in CD-I mouse embryos was tested. It was shown that the number of oocytes and embryos retrieved was increased by 5 IU PMSG and decreased at a dose of 10 IU, with a further decrease at a dose of 15 IU PMSG. These changes were similar to those observed in other species. It was postulated that increased PMSG dose gave rise to higher frequency of abnormal embryonic development likely associated with an increased frequency of chromosomal abnormalities. Therefore, two experiments were performed to determine whether superovulation of mature mice with PMSG leads to chromosomal abnormalities in mouse embryos. 1) 8- to 16-cell stage embryos Three types of chromosomal abnormalities were detected in this experiment; aneuploidy, polyploidy and structural abnormalities. The incidence of aneuploidy in the spontaneously ovulated group (5.6%) was similar to that reported by others. The aneuploidy rate in the three superovulated 95 groups was 4.4,5.0 and 7.0%, respectively and therefore did not increase with PMSG dose. Even though the increases do not reach statistical significance, it is worth noting that there is an imperical increase from low to high dose. A large sample size might lead to statistically detectable differences. No polyploidy was found in the spontaneously ovulated group or in the 5 IU PMSG superovulated group. The incidence of polyploidy, recorded for 8- to 16-cell stage embryos was 2.9% and 10.5% in the 10 and 15 IU PMSG group, respectively. A linear dose-response relationship has been demonstrated for PMSG dose and the incidence of polyploidy, including triploidy and tetraploidy. These results suggest that polyploidy induced by superovulation may be responsible for the increased lethality of preimplantation embryos. During ovulation induction, multiple waves of premature or asynchronous ovulations occur and the increase in polyploid embryos may be due to both immaturity and overmaturity of oocytes. It may also result from an increased frequency of polyspermy or a reduction in polar body extrusion. 2) Mouse zygote studies The origin and gametic source of the polyploid embryos was determined on the basis of differential condensation of maternally and paternally derived chromosomes. No polyploidy was found in the spontaneously ovulated group. The frequencies of diploid oocytes fertilized by one haploid sperm were 1.9, 1.9 and 1.7% in the 5, 10 and 15 IU PMSG treated groups, respectively. The diploid chromosomal set from an oocyte recognized in the zygote stage was identified as a failure of first meiotic division. Due to immaturity of the cytoplasm, diploid oocytes may be more susceptible to penetration by multiple sperms. One tetraploidy derived from a diploid oocyte from the 15 IU PMSG treated group fertilized by two sperms was found in the zygote stage. The frequencies of haploid oocytes fertilized by two sperms were 2.8 and 4.2% in the 10 and 15 IU PMSG treated groups, respectively. The extra chromosome set in these triploid zygotes 96 were of paternal origin. The changes in the zona pellucida reaction to fertilization may lead to polyspermy after superovulation. The above described observation support the hypothesis that disturbances at maturation division and/or an error at fertilization cause polyploidy observed in 8- to 16-cell CD-I mouse embryos after superovulation. Since only one tetraploidy was found in the zygote stage, it is possible that the most of the tetraploidy found in 8- to 16-cell stage embryos may be caused by a complete block of the first cleavage division in a diploid zygote. The mechanism of the first mitotic arrest after superovulation is unclear and needs further investigation. The study from mouse zygotes and 8- to 16-cell embryos and zygote reveals a number of interesting findings: (1) A positive dose-response relationship between PMSG dose and the incidence of polyploidy was detected in CD-I mouse 8-16 cell stage embryos developed in vivo. 2) Polyploidy, especially, triploidy, was derived from both digyny and diandry. The dose-response relationship between PMSG and the incidence of polyploidy may be caused by either suppression of meiotic division or alteration of the zona pellucida during oocyte maturation. These events may be related to asynchrony of oocyte maturation by exogenous gonadotropins. 3) PMSG used for stimulation of ovulation has no effect on segregation of individual chromosome during the meiotic and mitotic divisions that would lead to aneuploidy in CD-I mouse embryos. Human unfertilized oocyte studies Studies on the incidence of chromosome abnormalities of unfertilized human oocytes from IVF reveal a high incidence of chromosomal abnormalities (3-57%) in the literature. This variation in incidence of chromosomal abnormalities in human oocytes may be associated with differences in superovulation techniques utilized in IVF. However, the production of human embryos for experimental purposes is generally unacceptable; this makes the design of an experimental procedure to assess the influence of superovulation on the chromosomal complements of the embryo almost impossible. The only source of human materials in which to study the effects of 97 superovulation on meiotic division is unfertilized human oocytes from IVF programs. This study was undertaken to estimate the incidence of chromosomal abnormalities in a large sample of unfertilized human oocytes following superovulation and to evaluate the effects of exogenous gonadotropin and maternal age on the chromosomal abnormalities. Aneuploid was found to occur at a frequency of 22.8% in 280 unfertilized human oocytes obtained from stimulated follicles during IVF procedures. This incidence of aneuploidy was similar to the 22.1% incidence of aneuploidy in the literature (Table V). The dosage of hMG used in 194 infertile women for this study differed from 300 IU to 9000 IU. According to Plachot's criteria (1988b), the patients were divided into two different hMG dosage groups (300-3000 IU PMSG vs 3150-9000 IU). There was no significant difference between the two groups, consistent with the findings of Plachot (1988b) whose results similarly failed to demonstrate an effect of hMG dosage on incidence of aneuploidy. A positive correlation has been shown between increasing maternal age and the incidence of trisomies. It was most evident at maternal age >35 years (Hossold and Chiu, 1985). Therefore, two different age groups (< 35 vs >35) were divided in this study. No correlation was found between the incidence of overall aneuploidies and maternal age. Numerical abnormalities were equally observed in all maternal age groups from 24 to 41 years. Since there is no access to unstimulated human oocytes, it is impossible to compare the incidence of aneuploidy in oocytes from the natural cycle with those from a stimulated cycle. Therefore, whether the high incidence of aneuploidy detected in human unfertilized oocytes can be attributed to superovulation or to humans have a higher baseline rate of aneuploidy compared to other mammals remains unclear. The one major difference between results from this study and those from literature has been with respect to frequency of diploid oocytes. The observation of 16.8% of diploid oocytes in unfertilized oocytes was higher than observed by other reports. This result may relate to the induction regimen used and suggests that the induction of oocyte maturation may cause asynchrony between nuclear and cytoplasmic maturation. This asynchrony within the oocyte may inhibit of segregation of all chromosomes (diploidy). It is possible that oocytes matured longer in 98 vivo are less prone to have a diploid complement and Gl-PCC. The diploid oocytes are likely an important source of triploidy. The incidence of numerical chromosome abnormalities (including 22.8% of aneuploidy and 16.8% of diploidy) was 39.6% in unfertilized human oocytes. Numerical chromosomal abnormalities in oocyte may be partially responsible for the low pregnancy rate in IVF. Conclusion The most significant finding here is the demonstration of a dose-response relationship between PMSG and the incidence of polyploidy in CD-I mouse embryos. Both a disturbance at maturation division and an error at fertilization were shown to cause polyploidy. The other major finding is the observation of 16.8% of diploidy in unfertilized human oocytes. This result suggests that a block during meiosis I after superovulation may occur in humans and mouse oocytes. This information may be important in the future management of superovulatory protocol in humans. A model of diploidy is presented in which altered hormonal levels can lead to diploidy, via an underlying mechanisms of suppression of meiosis I or II. Evidence supporting this view is drawn from mouse and human data. The results obtained in the course of this work raise many new questions and suggest further experiments designed to investigate the molecular mechanism for the blockage of meiosis I after superovulation. The most critical question has to deal with the molecular nature of the signal triggering GVBD and the resumption of meiosis. MPF plays an important role in the cell cycles control in oocytes (Masui and Marker, 1971). A high MPF activity is preceding GVDB and persists up to metaphase I. During the segregation phase activity drops drastically below a detectable level and is only measurable again when oocytes reached metaphase II (Hishimoto and Kishimoto, 1988). 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