Genes, Chromosomes, and Reproductive Failure

FERTILITY AND STERILITY Copyright ~ 1980 The American Fertility Society

Vol. 33, No.2, February 1980 Printed in U.SA.

GENES, CHROMOSOMES, AND REPRODUCTIVE FAILURE

JOE LEIGH SIMPSON, M.D. Section of Human Genetics, Department of Obstetrics and Gynecology, Prentice Women's Hospital and Maternity Center of Northwestern Memorial Hospital, Chicago, Illinois 60611

In this presentation three major problems of recent concern and immediate clinical relevance are covered: (1) control of sex differentiation, (2) causes of ovarian failure, and (3) causes for spontaneous abortion. As a prelude to this discussion, * it seems instructive to return to the founding of The American Fertility Society-1944-in order to recall how meager was the genetic knowledge in the three areas cited. In fact, in each area, the basic understanding was almost diametrically opposite our current beliefs. To start with the problem of sex determination, in 1944, the number of human chromosomes was still believed to be 48. The X and Y chromosomes had been identified in humans, but their specific functions had not yet been deduced. The prevalent theory at that time was that sex determination in humans was analogous to that in the fruit fly Drosophila. Since experiments by Bridges! in the 1920s, it had been known that flies with a diploid set of autosomes and two X chromosomes were female. Those with a diploid set of autosomes, one X, and one Y, were male; those with three haploid sets and three X chromosomes were female, but those with three haploid sets and only two X chromosomes were intersexes. From these data it could be deduced that the Y chromosome played no important role in sex differentiation in Drosophila. Instead, autosomes were male-determining, whereas X chromosomes were female-determining. The sexual phenotype thus depended upon a balance between autosomes and X chromosomes. With respect to the causes of ovarian failure, virtually nothing was known. In 1938, 6 years prior to the founding of The American Fertility Society, Turner2 described seven patients with

sexual infantilism and short stature. However, Turner believed that the sexual infantilism was due to pituitary hypofunction. The ovarian basis of this disorder did not become apparent until several years later, and not until 1944 did Wilkins and Fleischmann3 visualize streak gonads in a patient with Turner stigmata. Not surprisingly, genetics and chromosomes did not figure prominently in speculation concerning the etiology of ovarian failure; most texts hinted that failure resulted from environmental or "idiopathic" factors. The causes for spontaneous abortion were likewise obscure. Recall that 1944 was an era in which pregnant women were advised not to undertake long train or car rides for fear of disrupting the embryo. Indeed, the equivalent of "old wives" tales played as prominent a role in both the medical and lay conception of spontaneous abortion as did knowledge of endocrine or the yet-unknown genetic factors. This lack of knowledge unfortunately fostered therapeutic regimens devoid of scientific basis. GENETIC ADVANCES

In the 35 years since the founding of The American Fertility Society, genetics advanced rapidly. In 1944, Avery et a1. 4 showed that DNA was the hereditary material in all living organisms with the exception of certain viruses. In 1953, Watson and Crick5 showed that DNA existed in the form of a double helix, a configuration that helped to explain structurally how DNA could replicate itself. In the mid-1950s, other advances allowed in vitro techniques to be performed by many laboratories; formerly, studies were conducted only by the relatively few investigators who seemingly possessed the black magic required to culture cells. Other techniques facilitated chromosomal *Upjohn Lecture. Presented at Thirty-Fifth Annual Meeting analysis. In 1956, Tjio and Levan6 reported that of The American Fertility Society, February 3 to 7,1979, San Francisco, Calif. the human chromosome number was not 48, but 107

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rather 46. Thereafter, clinical advances were rapid. By 1959, the chromosomal basis of the Turner syndrome 7 and Klinefelter syndrome8 became known. In the subsequent decade, chromosomal studies of spontaneous abortuses were reported, and chromosomal banding techniques developed. With these new genetic tools, more specific information could therefore be obtained. The problems designated at the beginning of this presentation could therefore be investigated.

In the absence of these hormones, female genital and ductal differentiation occurs. Location of Testicular Determinants. More precise data concerning testicular determinant(s) were later deduced, both by myself and by other cytogeneticists. A study of individuals with structurally abnormal Y chromosomes led to the conclusions that not the entire Y chromosome but rather only specific loci on the Yare responsible for permitting testicular differentiation. 9 , 10 Testicular determinants appear localized near the centromere of the Y chromosome. Such a deduction follows from observations that individuals with tiny ring Y chromosomes can show normal male development. (Mosaicism was excluded so far as is possible.) Similarly, other investigators showed that an isochromosome for the long arm of the Y was associated with a female phenotype and bilateral streak gonads. Because an isochromosome for Yq is associated with complete deficiency for Yp, testicular determinants must not be present in the Y long arms. There are possible exceptions, but I personally consider the short arm near the centromere to be the location for the testicular determinants.

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CONTROL OF SEX DETERMINATION AND TESTICULAR DIFFERENTIATION

Let us first consider the genetics of sex determination. Human chromosomal studies in the late 1950s and early 1960s showed that the Y chromosome in humans plays the pivotal role in gonadal differentiation. Individuals with a Y chromosome underwent testicular differentiation, despite the presence of four or five X chromosomes. Conversely, individuals lacking a Y chromosome invariably showed a female phenotype. The sex-determining mechanism in humans, then, was fundamentally different from that in Drosophila. In mammals, testicular differentiation is controlled by the Y chromosome, which directs the indifferent gonad toward testicular differentiation. The testes elaborate both the Mullerian inhibitory factor and testosterone, the latter of which is converted to dihydrotestosterone by the enzyme 5a-reductase. Testosterone, dihydrotestosterone, and the Mullerian inhibitory factor direct male embryonic differentiation (Fig. 1).

Testosterone Olffl.n St.bilizotion Persistence of Semi nol Vesicles Vasa Deferentia Epididymides

~

MUllerian-Inhibitory Foctor

Mullerian Inhibition Regression of Uterus, Fallopian Tubes, and Upper Vagina

5a-Reductose¢ Oihydrotestosterone Genital Virilizotion¢Penis Scrotum Labioscrotal Fusion

FIG. 1. Diagram of normal male differentiation. The Y chromosome elaborates factors responsible for transformation of indifferent gonad into a testis. Hormones synthesized by fetal testes accomplish subsequent steps.
H-Y Antigen and Mechanism of Testicular Differentiation. Knowing the location of the testicular determinant not only allows one to make some phenotypic-karyotypic predictions, but more importantly permits testing of postulates concerning the mechanism of action of the Y testicular determinants. Two general hypotheses concerning action of the Y testicular determinants can be envisioned. The determinants could act in (1) regulatory fashion or (2) structural fashion. Determination in regulatory fashion assumes that the Y chromosome contains a regulatory gene which activates, or derepresses, a locus on an autosome or X chromosome which then permits testicular differentiation. Nature works very often through regulatory genes, and it would not be surprising if testicular differentiation were controlled in this fashion. Lending support for this assumption, but not proving it, is evidence that factors on both autosomes and the X chromosome influence testicular differentiation. Witness the heritable nature of such disorders as agonadia,lo a disorder characterized by absence of both gonadal and ductal elements. One important disorder that must be explained before understanding the genetic control of sex determination is XY gonadal dysgenesis. Affected individuals are 46,XY, yet show bilateral streak gonads indistinguishable from those of normal females. 10, 12, 13 In a female recently

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•

GENES, CHROMOSOMES, AND REPRODUCTIVE FAILURE

studied by German and colleagues,13 the disorder segregated in X-linked recessive fashion. No monosomic cells were detected in analysis ofmultiple tissues. Such families suggest that testicular differentiation IS not assured despite a functioning Y chromosome. Instead, another locus, presumably on the X, can intercept or suppress the message for testicular differentiation. If an X-linked or autosomal gene essential for testicular differentiation exists, it is reasonable to wonder whether such a locus might ordinarily be activated by the Y to permit normal testicular development. On the other hand, it would perhaps be more parsimonious to assume that the Y testicular determinant is a structural gene capable of coding for a compound that directs the indifferent gonad into a testicular pathway. Wachtel and others l4. 16 have proposed that H-Y antigen, a cell surface antigen, is the gene product of the testicular determinant. The importance of this hypothesis dictates a brief digression to explain the observations that led Wachtel and others to postulate an integral relationship between H-Y and testicular differentiation. In rodents, brother-sister matings can be performed for 20 generations. At the end of this span all mice in a given inbred strain are homozygous at almost all loci. Reciprocal skin grafts can, predict. ably, be transferred from animal to animal, with one exception-grafts from a male transferred to a female are rejected. Because all autosomal loci are identical, only the Y chromosome could contain histocompatibility loci responsible for the rejection. Histocompatibility loci must, therefore, exist on the Y chromosome, and these loci were called H-Y antigen. H-Y is assayed by a semiquantitative sperm cytotoxicity test. Spleen or sperm cells from a male injected into a female of the same strain evoke antisera against H-Y and only against H-Y. These antisera can then be tested against unknown sera. If an unknown serum is derived from an H-Y individual, a portion of the antiserum will be absorbed. This absorption leaves less antiserum, therefore allowing fewer sperm to be immobilized than if the unknown were not derived from an H-Y individual. The assay is tricky, and not all investigators have been able to obtain reproducible results. Assay difficulties notwithstanding, both circumstantial and direct evidence suggests that HY can direct testicular differentiation in vitro. The following circumstantial data are consistent with the hypothesis: (1) H-Y is evolutionarily conservative, being present in all tested species in

109

the sex containing the Y or W chromosome l4 ; (2) at least one locus for H-Y is near or identical with the locus for the testicular determinant(s)15, 16; (3) a 4S,XXYY male had twice the H-Y titer that 46,XY males have l7 ; (4) H-Y is present in humans and mice with testicular feminization, indicating that H-Y is not merely induced by androgens l8 ; (5) H-Y on XY cells is expressed after transfer to a female host l9 ; (6) H-Y is present in approximately 50% of mouse blastocysts,20 a stage prior to organ differentiation and, hence, prior to testicular differentiation; (7) H-Y is present in sex-reversed 46,XX true hermaphrodites 2\ (S) XY female lemmings are H-Y negative 22 ; and (9) H-Y antigen is present in the testicular but not the ovarian portion of ovo-testes. 23 In general, H-Y has thus been detected in individuals with testes, but not in those lacking testes. 15 Direct evidence also exists. If one reaggregates neonatal mouse or rat testes disassociated by Moscona type disruption, tubule-like structures form upon reaggregation. 24,25 Conversely, H-Y antiserum causes testicular cells to reaggregate into a follicle-like (female) structure. A more definite experiment-H-Y induced conversion of ovarian germ cells to tubule-like structures-has also been performed. 26 Although attractive, the H-Y theory is not without detractors. In addition to the problems in assay reproducibility, some contradictory data remain to be explained. For example, if H-Y is indeed the testicular determinant, why are XX true hermaphrodites and XX males abnormal?27 That is, why do all positive individuals not show normal testicular differentiation? In addition, some 46,XX true hermaphrodites have been reported to lack H-Y in blood and skin fibroblasts, and most patients with XY gonadal dysgenesis are H-Y positive. 28 These ostensible contradictions have led proponents of the hypothesis that H-Y is the gene product of the testicular determinant to suggest that H- Y antigen exists in multiple copies, perhaps some forms immunologically active yet biologically inactive. I find such interpretations lacking in evolutionary appeal. H-Y antigen is obviously integrally involved in testicular differentiation, although it need not necessarily be the only locus involved. If it is the only locus, it mayor may not exist in multiple copies. Receptor problems could also explain contradictory data. StaturalDeterminants. H-Y antigen and the testicular determinant are not the only loci ofinterest on the Y chromosomes. The Y may also contain statural determinants. Individuals with normal

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February 1980

testicular differentiation who apparently lack the distal portion ofYp are short. Both testicular and statural determinants appear present on Yp.9, 10 The statural determinants are apparently distal to the testicular determinants (Fig. 2), a teleologically appealing hypothesis to which we shall return after considering ovarian determinants and ovarian failure. Location of Ovarian and Statural Determinants. If two active chromosomes are necessary, it follows that specific loci are probably responsible for ovarian maintenance. If so, how may loci exist? In order to deduce the number and location of ovarian loci, one must study individuals deficient for portions of the X chromosome: 46,X,del(Xp), 46,X,i(Xq), 46,X,del(Xq), and 46,X,i(Xp). The former two configurations are characterized by deficiencies of short-arm loci, the latter two by deficiencies of long-arm loci. What are the endocrine characteristics of these and other patients with these X deficiencies? Monosomy X (45,X) is usually associated with streak gonads, short stature, and certain other anomalies comprising the Turner stigmata. Phenotypic expression varies more often than generally appreciated, as evidenced by observations that occasionally (3%) 45,X individuals menstruate or become pregnant. lO, 12, 29, 30 Deletions of most [del(Xp)] or all [i(Xq)1 of the X short arm are usually associated with short stature and certain features of the Turner stigmata. 10, 12, 29, 30 Menstruation or breast development occurs in at least 25% of 46,X,del(Xp) or 45,X/ 46,X,del(Xp) individuals, but rarely in 46,X,HXq) individuals. Deletions limited to only the distal end of Xp[46,X,deHX)(q22)] are compatible with normal ovarian function. Deletions of most [del(Xq)] or all [i(Xp)] of the X long arm are usually associated with gonadal dysgenesis; however, menstruation occurs in about

20% of adult 46,X,del(Xq) cases.lO, 12, 29, 30 None of the seven purported 46,X,i(Xp) individuals have menstruated. These data indicate that ovarian determinants exist on both Xp and Xq. Duplication of one arm (i.e., an isochromosome) fails to compensate for loss of the other arm; thus, determinants on Xp and Xq probably have different functions. In addition, each arm may contain more than a single determinant, based on differences in the frequencies of menstruation in individuals with partial versus complete deletions of a particular arm, i.e., del(Xp) versus i(Xp). If a given arm contains at least two ovarian determinants, this would be analogous to Drosophila, a species in which almost any portion of the X exerts a female-determining effect. Indeed, it might be evolutionarily advantageous if gonadal determinants were not confined to only one or two regions, for recombination involving one region of the X chromosome would not necessarily alter the mechanism of sex determination. In addition to ovarian differentiation, some interesting conclusions can be gathered with respect to statural determinants. Individuals who have deletions of the short arm, either near the centromere (Xpll) or considerably more distal (Xp22), are invariably short. However, individuals with distal breaks [46,X,deHX)(p22)] do not show gonadal dysgenesis. Thus, in the short arm the statural determinants can be separated from the gonadal determinant(s), the statural determinant(s) being more distal. Individuals with long arm deletions mayor may not contain statural determinants. The above led me to propose the map of the X chromosome illustrated in Figure 3. 11 Clinical implications of our phenotypic-karyotypic correlations can now be summarized: (1) N ormal gonadal function can be associated with X deletions, and even with a 45,X complement. This is less surprising than formerly in view of observations that oocytes require only one X to differentiate. (2) Normal stature can exist in individuals with X deletions and gonadal dysgenesis. (3) Individuals with normal gonadal function may be short yet the X deletions. Despite deduction of these important clinical observations, we still have not the slightest idea concerning mechanism of action of the ovarian determinants. StaturalDeterminants and Meiosis. A bit ofteleology does not seem inappropriate. During meiosis autosomes undergo side-to-side anastomosis at sites of homologous loci. The X and Y undergo end-to-end association, but no reason for this association has ever been clear. In recalling the pro-

110

} ? Statural Determinant (s) } Testicular Determinant (s) H-Y Antigen } Testicular Determinant (s)

FIG. 2. Diagram of postulated locations on Y chromosome for possible statural determinants, testicular determinants, and H-Y antigen. The latter two factors mayor may not be identical, and there mayor may not be more than one locus for one or both. (Reproduced with permission of the publisher from Simpson. 12)

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GENES, CHROMOSOMES, AND REPRODUCTIVE FAILURE

posed maps of the X and Y, however, we recall that the short arms of both chromosomes contain not only gonadal determinants but, more distally, statural determinants. Ifhomologous staturalloci exist, they could be the basis for end-to-end association. Existence of homologous staturalloci permit synapsis, with the inevitable recombination, without disruption of the sex determination mechanism.9, 10

A

2

I 2

B

!

x

Statural Determinant Is)

!

Ovarian Determinant Is)

Ovarian Determinant (5) ? Statural Determinant (5)

FIG. 3. A, Schematic representation of banding pattern expected ofthe normal X chromosome. B, Diagram of postulated locations on the X chromosome for statural and ovarian determinants.
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Autosomal Control of Ovarian Differentiation. Finally, we should not leave our discussion of ovarian failure without reflecting upon a relatively unexplored area-the relationship of autosomal loci to ovarian differentiation. For several years I have been interested in one form of 46,XX gonadal dysgenesis, a disorder that appears to be caused by a mutant gene. 12 , 31 Individuals with gonadal dysgenesis who have apparently a normal female (46,XX) complement may be said to have XX gonadal dysgenesis. The external genitalia and the streak gonads of such patients are indistinguishable from those of individuals who have gonadal dysgenesis and an abnormal chromosomal complement. Likewise, the endocrine data and lack of secondary sexual development do not differ from those of other individuals with streak gonads. Most affected individuals are of normal stature (mean height 165 cm), but a few are less than 150 cm tall. Somatic anomalies are usually not present (some exceptions do exist), but both XX gonadal dysgenesis and neurosensory deafness have coexisted in multiple siblings in four typical families. 12 The coexistence could be explained by (1) varied expressivity for the allele that produces XX gonadal dysgenesis; (2) the occurrence of a mutant allele exerting a pleiotropic effect, the allele being different from that producing XX gonadal dysgenesis without deafness; or (3) the coincidental occurrence of homozygosity for more than one recessive allele, especially likely in offspring of consanguineous matings. 12 , 31 Of most immediate relevance to our discussion, however, is not the phenotype but rather the etiology of XX gonadal dysgenesis. In fifteen families more than one member had the disorder. 12 In six other kindreds, two or more X-chromatin siblings had gonadal dysgenesis. The phenomenon is thus not extremely rare. These familial aggregates contrast with 45,X gonadal dysgenesis, in which familial aggregates are very rare. In three of the fifteen familial aggregates the parents were consanguineous. These data strongly suggest autosomal recessive inheritance, a hypothesis consistent with results of our segregation analysis. Among the explanations for XX gonadal dysgenesis are (1) failure of genital ridge formation; (2) failure of germ cell migration from the yolk sac to the genital ridge; (3) increased rate of germ cell attrition, the phenomenon responsible for 45,X gonadal dysgenesis; or (4) genetically determined nondisjunction or anaphase lag limited to the germ cells (limitation to germ cells would explain failure to detect monosomic cells in lymphocytes,

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SIMPSON

skin fibroblasts, and gonadal fibroblasts of XX gonadal dysgenesis patients). No single explanation is accepted. Interestingly, however, genes cause loss of chromosomes in plants, Drosophila, and mice. lO In those species genes cause loss of chromosomes during mitosis or during both meiosis and mitosis. Similar genes in humans could cause chromosomal loss limited to the ovaries. If undetected monosomy causes XX gonadal dysgenesis, however, the phenomenon still differs from that in which individuals are known to have monosomy X on the basis of cytO" genetic studies of lymphocytes or skin fibroblasts, because in order to explain the ostensible lack of 45,X cells in XX gonadal dysgenesis on must postulate the occurrence of a cytogenetic error at a different time and in a different tissue than in 45,X gonadal dysgenesis. Consideration of the etiology and pathogenesis of XX gonadal dysgenesis has thus yielded the important biologic observation that an intact autosomal locus is necessary for normal ovarian development. In the future other evidence for autosomal ovarian control will doubtless be uncovered, and we must consider the role that these loci play in ovarian differentiation and development. CAUSES OF OVARIAN FAILURE

Having discussed control of sex determination and testicular differentiation, let us now consider ovarian differentiation and development. After Turner's report,2 Varney et a1. 32 and Albright et a1. 33 recognized in 1942 that patients with "Turner's syndrome" had increased urinary excretion

...---..,

Q

No Testosterone No Dlhydrotestosterone Wolffian Duct Regression Female External Genital ia

LoekOfY Testicular Determinants

No Miillerlan-Inhibitory Factor Mullerian Ducts Persist Fallopian Tubes Uterus Upper Vagino

FIG. 4. Diagram of hypothesis that all reproductive systems in the embryo will differentiate in female fashion-ductal, genital, and gonadal-unless directed to do otherwise, directly or indirectly, by a determinant on the Y chromosome. (Reproduced with permission of the publisher from Simpson.1l)

February 1980

j -llIq

4P

lip

t (4:,1\)

FIG. 5. Partial karyotype showing a balanced translocation between chromosomes 4 and 11, detected in a woman who experienced seven spontaneous abortions.

of gonadotropins. Recognition of streak gonads3 and additional somatic features were later made. In 1959, Ford et a1. 7 reported that the compl~ment 45,X was associated with gonadal dysgenesis an~ the Turner stigmata. Other complements aSSOCIated with gonadal dysgenesis were subsequently described-structural rearrangements of the X and Y chromosomes, autosomal abnormalities, various forms of mosaicism, and even apparently normal male (46,XY) or female (46,XX) complements. Several fundamental questions can now be posed. Are specific determinants required for initial ovarian differentiation, or does ovarian differentiation simply represent normal embryonic homeostasis? If the latter is true, do determinants for adult ovarian differentiation exist? Where are such determinants? How many determinants exist? How do these determinants act? Pathogenesis of Ovarian Failures in 45,X. Answers to the questions posed must be gathered by deductive reasoning, utilizing abnormal individuals for clinical/scientific correlations. No biochemical markers directly assess ovarian determinants. We can illustrate the approach by considering a 45,X patient: sexual infantilism, short stature, and usually bilateral streak gonads. Because individuals with this phenotype lack one X chromosome, it is reasonable to assume that two X chromosomes are necessary for normal adult ovlirian germ cells. This does not, incidentally, imply that two X chromosomes are necessary for differentiation of the fetal ovary from the indifferent gonad. Indeed, 45,X' embryonic ovaries show oocytes and follicles in relatively normal gonadal architecture. 34 From these data one deduces that ovaries and follicles differentiate from the indifferent gonad in 45,X individuals, only to undergo attrition at a rate more rapid than that normally expected. Thi$ knowledge helps to explain not only the 3% of 45,X patients who menstruate, but the occasional patient who becomes pregnant. At any rate, I believe that the normal embryonic homeostasis is ovarian l l (Fig. 4). Such homeostasis would be consistent with ductal and genital dif-

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GENES, CHROMOSOMES, AND REPRODUCTIVE FAILURE

ferentiation, both of which occur in female direction unless specifically directed to do otherwise. Although not necessary for initial differentiation, two intact X chromosomes are usually necessary for normal adult gonadal development. At superficial inspection, this contradicts the Lyon hypothesis, a thesis which states that X chromosomes in excess of one are inactivated to form X-chromatin. However, X chromosome inactivation does not occur in oocytes. In vitro maturation studies of oocytes of individuals heterozygous at locus G6PD have been performed by Gartler and others.35 Some individuals contain a G6PD-A allele on one X chromosome and an electrophoretically different G6PD-B allele on the other X chromosome. The alleles are of no clinical significance, but they can be used as a marker of X chromosome activity. Oocyte in vitro maturation studies of heterozygous individuals reveal evidence for the active synthesis of both the A allele and the B allele, indicating that both X chromosomes are active in oocytes. Lack of X inactivation in oocytes explains the deleterious effects of 45,X and 47 ,XXY complements. CAUSES OF SPONTANEOUS ABORTION

A third area for discussion is the relationship between genetics and spontaneous abortion, both sporadic and repetitive. Preclinical Losses. Let us first discuss the extent to which embryonic losses occur prior to clinically recognized pregnancies. For years we have marveled at the meticulous work of Hertig et aI., 36 who studied women scheduled for elective hysterectomy. They recorded ovulation times, recovered preimplanted embryos and early implanted embryos, and searched for morphologic abnormalities. Four of eight preimplanted embryos were so morphologically abnormal that their continued development could not have been anticipated. Of some 26 early implanted embryos, none greater than 4% weeks after the last menstrual period, 8 were abnormal. Even in clinically unrecognized pregnancies, there is thus a tremendously high prevalence of morphologic abnormalities. No further detailed studies have been performed in humans, although in vitro fertilization studies may provide useful information. The etiology of these early losses is unknown. By analogy to animal data, however, these early morphologic abnormalities probably result from abnormal cell division-abnormal chromosomal changes. Indeed, in animals many studies have shown that abnormal

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early embryos often result from lethal chromosomal abnormalities.

Chromosomal Abnormalities in First-Trimester Abortuses. With this background it was natural that reproductive geneticists would apply the advances in chromosome methodology to a study of spontaneous abortuses. To some clinicians, the results were astonishing, for numerous studies revealed that 50% to 60% of conceptuses in firsttrimester spontaneous abortions were chromosomally abnormal. 37 The proportion of chromosomally abnormal conceptuses is thus very high. About 4% of conceptuses are trisomic, about 2% are 45,X, and about 1.5% are polyploid. Most chromosomal abnormalities in abortuses result from abnormalities during oogenesis leading to abnormal gametes. Three general classes of abnormalities are clinically relevant. First, if parental chromosomes become rearranged (e.g., translocation), meiotic segregation will inevitably produce gametes with deficiencies or duplications of genetic information (Fig. 5). Parents with translocations may have repetitive spontaneous abortions and for this reason are discussed below. Second, even if the complement of a primary oocyte is normal, homologous chromosomes may fail to pass to complementary cells during meiosis (nondisjunction). Sister chromatids may also fail to disjoin during meiosis II. Both nondisjunction and anaphase lag yield gametes without the expected number of chromosomes-aneuploidy. Third, failure of reduction of the chromosome number during meiosis I can yield gametes with two haploid complements. This phenomenon is termed polyploidy, or, more specifically, triploidy (3n = 69) or tetraploidy (4n = 92). The classes of chromosomal abnormalities described above may result from causes other than those described. Indeed, it is often not possible to reconstruct the pathogenic events that led to chromosomally abnormal offspring. For example, gametes with structural chromosomal abnormalities may result from either de novo chromosomal breakage and rearrangement or from normal meiotic segregation involving a parental translocation; parental translocations may be detected in lymphocytes, or they may be limited to gonads. Polyploid zygotes may result from double fertilization, incorporation of the second polar body into the zygote, fertilization of each of two ova contained within a single binucleated follicle, or other mechanisms. In addition, mitotic nondisjunction may lead to mosaic embryos.

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SIMPSON

These data demonstrate conclusively that chromosomal causes are the major etiologic factors responsible for spontaneous abortion, not endocrine factors or the innumerable other factors proposed over the years and upon which basis inappropriate therapy and advice were offered. In fact, chromosomal abnormalities are the only cause known to be responsible for large numbers of abortions. Nonchromosomal Causes of Spontaneous Abortion. What about the 40% of conceptuses from spontaneous abortions which show normal chromosomal complements? What deductions and relevant analogies are valid, building from observations that 60% of abortuses are chromosomally abnormal? Nongenetic possibilities include endocrine deficiencies, namely progesterone deficiencies; immunologic factors (lack of blocking antibodies); infectious agents; psychologic factors; and trauma. Available data, reviewed recently by Glass and Golbus,38 indicate that none of these factors are frequent causes. Two other genetic etiologies could, however, be responsible-Mendelian mutations and polygenic factors. Indeed, on the basis of observations that 60% . of abortuses are chromosomally abnormal, whereas no nongenetic causes are nearly as frequent, I believe that Mendelian and polygenic factors will be shown to be the major causes for the remaining 40%. That is, at birth, 0.5% of liveborn infants have a chromosomal abnormality, 1% will show a disorder that results from a single mutant gene, and approximately 1% will show a disorder inherited in polygenic fashion. It seems naive to assume that the 40% of first-trimester abortions with n~rmal complements result predominantly from nongenetic causes, for this would ignore an obvious analogy gathered from liveborn infants and ignore the lesson that nature preferentially selects against abnormal fetuses. Mutant genes might produce metabolic errors that interfere with synthesis of a crucial protein or steroid. Polygenic factors might cause failure of differentiation of certain important embryonic structures. In forthcoming years, I predict that virtually all first-trimester spontaneous abortions will prove genetic in etiology. However, the deficient gene product could in some cases yield hormonal inbalance, allowing a wedding of genetic and endocrine proponents of abortion causation.

Repetitive Abortions. Not only have chromosomal studies helped to elucidate causes for sporadic cases of spontaneous abortions, but they have provided important data with respect to repetitive abortions. These observations especial-

ly contrast with the paucity of knowledge under which obstetrician-gynecologists and endocrinologists operated in 1944. In 1944, physicians were using the theoretical calculations by Malpas,39 who reasoned in 1938 that the frequency of spontaneous abortion was a direct function of the number of previous abortions. After three abortions, Malpas calculated that the likelihood of recurrence was about 80%, a frequency that led such women to be labeled "habitual aborters." Acceptance of this notion made it inevitable that a generation of patients and physicians accepted the usefulness of many external remedies, including hormones. If indeed one believes that an abortion rate of 80% was to be expected, practically any remedy would have decreased this rate. In 1964, Warburton and Fraser40 showed that the likelihood of abortion was not nearly so high as what Malpas had concluded. After one abortion the likelihood of recurrence increased slightly, to 25%; however, the risk did not increase appreciably thereafter (Table 1). In couples with no liveborn offspring the risk may be slightly higher, but not above 40% to 45%.41 Chromosomal abnormalities were subsequently shown to be related to repetitive abortion in two different ways. First, in approximately 5% to 10% of couples with repetitive abortions, one parent will have a translocation or inversion42-49 (Table 2). I suspect that the frequency may be higher as chromosomal inversions are detected more often. Like those with balanced translocations, individuals with inversions are normal; however, as the result of recombination occurring within the inversion loop, recombinant unbalanced gametes can be produced. Elegant cytogenetics notwithstanding, parental chromosomal rearrangements will be detected in only a few families with repetitive abortions. Instead, there is apparently a second mechanism by which chromosomal abnormalities cause repetitive abortions. Successive abortuses are either TABLE 1. Empiric Data Concerning the Risks of Spontaneous Abortion following a Given Number of Abortions" No. of previous

(~

of abortions

abortions

o 1 2 3

4

12.3 23.7 26.2 32.2 25.9

aTabulated from data of Warburton and Fraser. 4o Sample includes couples with at least one live-born offspring.

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GENES, CHROMOSOMES, AND REPRODUCTIVE F AlLURE

TABLE 2. Frequency of Parental Chromosomal Rearrangements (e.g., Balanced Translocation) among Selected Series of Couples with Two or More Spontaneous Abortions" Investigator

Couples with rearrangement

Kim et al. 42 Schmidt et al. 43b Stenchever et al. 44 Byrd et al. 45 Mennuti et al. 46 Kaji and Ferrier47 Luthy and Karp48 Simpson et al. 49

4/16 4/39 7/28 6/55 5/34 2173 4/36 3/65

Total

35/316 !9.0'k)

UBanding techniques were used in all cases. Samples included couples who also had live-born progeny, normal and abnormal. Couples who had experienced both repetitive abortions and live-born abnormal progeny were in general more likely to have a rearrangement than those who had had abortions alone; however, a difference was not observed in all samples. bOne 45,Xl46,XX mother.

usually all chromosomally abnormal or all chromosomally normal. 47 ,50-52 If the first abortus is trisomic, the likelihood is about 80% that the second will also be trisomic (Table 3). Conversely, if the first is normal the likelihood is 60% that the second will be normal. The clinical significance of these observations is enormous. Some couples have a tendency to have repetitive spontaneous abortions because they produce chromosomally abnormal gametes, not as result of translocation but presumbly because of a tendency toward nondisjunction. Identification of these individuals would permit antenatal diagnosis in subsequent pregnancies and facilitate our understanding of the causes of nondisjunction. Conversely, among those couples whose abortuses are chromosomally normal, one would search for either nongenetic, Mendelian, or polygenic causes. CONCLUSION

In this presentation a few of the ways in which genes and chromosomes influence reproductive failure have been discussed. Since the founding of TABLE 3. Relationship between Karyotypes of Successive Abortuses" Complement of first abortus

Normal Trisomy 45,X Polyploidy Translocation

Complement of second abortus Normal

@ 4 4 1 0

Trisomy

119 2 3 0

45.X

Polyploidy

Translocation

2 1 1 1 0

5 1 1 1 0

0 0 0 0

®

"Tabulation from Kaji and Ferrier47 from some reported series.47.50.52 Circled numbers suggest nonrandom losses.

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The American Fertility Society 35 years ago, genes and chromosomes have been shown to direct the control of sex determination and to play key roles in the etiology of ovarian failure and spontaneous abortions. In the future, genetic advances may be expected in other areas. The molecular basis of testicular and ovarian differentiation should become elucidated, and the knowledge clinically applicable. Increasing emphasis upon the genetic basis for preclinical embryonic losses seems likely, with unrecognized clinical losses possibly shown as an explanation for previously idiopathic infertility. Mendelian mutations will be shown responsible for some repetitive first-trimester abortions. The years 1944-1979 have seen remarkable genetic advances. By contrast, nongenetic causes have diminished in importance as factors responsible for sex determination, for ovarian failure, and for spontaneous abortion. In the next 25 years there is every expectation that the role of genetics will further be elucidated and expanded. REFERENCES 1. Bridges CB: Triploid intersexes in Drosophila melanogaster. Science 54:252, 1921 2. Turner HH: A syndrome of infantilism, congenital webbed neck and cubitus valgus. Endocrinology 23:566, 1938 3. Wilkins L, Fleischmann W: Ovarian agenesis. Pathology, associated clinical symptoms and the bearing on the theories of sex differentiation. J Clin Endocrinol Metab 4:357,1944 4. Avery OT, MacLeod CM, McCarty M: Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med 79:137, 1944 5. Watson JD, Crick FC: Molecular structure of nucleic acids. Nature 71:737, 1953 6. Tjio JH, Levan A: The chromosome number of man. Hereditas 42:1, 1956 7. Ford CE, Jones KW, Polani PE, de Almeida JC, Briggs JH: A sex-chromosome anomaly in a case of gonadal dysgenesis (Turner's syndrome). Lancet 2:403, 1959 8. Jacobs PA, Strong JA: A case of human intersexuality having a possible XXY sex-determining mechanism. Nature 183:302, 1959 9. German J, Simpson JL, McLemore G: Abnormalities of human sex chromosomes. I. A ring Y without mosaicism. Ann Genet (Paris) 16:225, 1973 10. Simpson JL: Disorders of Sexual Differentiation. Etiology and Clinical Delineation. New York, Academic Press. 1976 11. Simpson JL: Genetics of human reproduction. In Human Reproduction, Edtied by ESE Hafez. New York. Harper and Row. In press 12. Simpson JL: Gonadal dysgenesis and sex chromosome abnormalities. Phenotypic/karyotypic correlations. In Genetic Mechanisms of Sexual Development, Edited by HL Vallet, IH Porter. New York, Academic Press, 1979. p 365

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13. German J, Simpson JL, Chaganti RSK, Summitt RL, Reid LB, Merkatz IR: Genetically determined sex-reversal in 46,XY humans. Science 202:53, 1978 14. Wachtel SS, Ohno S, Koo GC, Boyse EA: Possible role for H-Y antigen in the primary determination of sex. Nature 257:235, 1975 15. Wachtel SS: H-Y antigen and the genetics of sex determination. Science 198:797, 1977 16. Silvers WK, Wachtel SS: H-Y antigen: behavior and function. Science 195:956, 1977 17. Wachtel SS, Koo GC, Breg WR, Elias S, Boyse EA, Miller OJ: Expression of H -Y antigen in human males with two Y chromosomes. N Engl J Med 293:1070, 1975 18. Ohno S: Major regulator genes for mammalian sexual development. Cell 7:315, 1976 19. Wachtel SS, Goldberg EH, Zuckerman E, Boyse EA: Continued expression of H-Y antigen on male lymphoid cells resident in female (chimaeric) mice. Nature 244:102,1973 20. Krco CJ, Goldberg EH: H-Y male antigen: detection in eight-cell mouse embryos. Science 193:1134, 1976 21. Wachtel SS, Koo GC, Breg WR, Thaler HT, Dillard CM, Rosenthal 1M, Dosik H, Gerald PS, Saenger P, New M, Lieber E, Miller OJ: Serologic detection of a Y-linked gene in XX males and true hermaphrodites. N Engl J Med 295:750, 1976 22. Wachtel SS, Koo GC, Ohno S, Gropp A, Dev VG, Tantravahi R, Miller DA, MIller OJ: H-Y antigen and the origin of XY female wood lemmings (Myopus schistocolor). Nature 264:638, 1976 23. Winters JJ, Wachtel SS, White BJ, Koo GC, J avadpour N, Loriaux DL, Sherins RJ: H-Y antigen mosaicism in the gonad of a 46,XX true hermaphrodite. N Engl J Med 300:745, 1979 24. Ohno S, Nagai Y, Ciccarese S: Testicular cells lysostripped of H-Y antigen organize ovarian follicle like aggregates. Cytogenet Cell Genet 20:351, 1978 25. Zenzes MT, WolfU, Gunther E: Studies on the function of H-Y antigen: dissociation and reorganization experiments on rat gonadal cells. Cytogenet Cell Genet 20:365, 1978 26. Zenzes MT, Wolf U, Engel W: Organization in vitro of ovarian cells into testicular structures. Hum Genet 44:333, 1978 27. Simpson JL: True hermaphroditism: etiology and phenotypic considerations. Birth Defects 14(6C):9, 1978 28. Jones HW, Rary JM, Cummings D: The role ofH-Y antigen in human sexual development. Johns Hopkins Med J 145:33, 1979 29. Simpson JL: Genetic aspects of reproductive failure: deficiencies of germ cell number (gonadal dysgenesis) and abnormalities of oogenesis (spontaneous abortion). In The Infertile Female, Edited by JR Givens. Chicago, Year Book Publishers. In press 30. Simpson JL: Gonadal dysgenesis and abnormalities of the human sex chromosomes: current status of phenotypickaryotypic correlations. Birth Defects 11(4):23, 1975 31. Simpson JL, Christakos AC, Horwith M, Silverman F: Gonadal dysgenesis associated with apparently normal chromosomal complem!lnts. Birth Defects 7(6):215, 1971

February 1980 32. Varney RF, Kenyon AT, Koch FC: An association of short stature, retarded sexual development and high urinary gonadotropin titers in women: ovarian dwarfism. J Clin Endocrinol Metab 2:137, 1942 33. Albright F, Smith PH, Fraser R: A syndrome characterized by primary ovarian insufficiency and decreased stature. Report of 11 cases with a digression on hormonal control of axillary and pubic hair. Am J Med Sci 204:625, 1942 34. Singh RP, Carr DH: Anatomic findings in human abortions of embryos and fetuses. Anat Rec 155:369, 1966 35. Gartler SM, Liskay RM, Campbell BK, Sparkes R, Gant N: Evidence of two functional X chromosomes in human oocytes. Cell Diff 1:215, 1972 36. Hertig AT, Rock J, Adams EC: A description of34 human ova within 17 days of development. Am J Anat, 98:435, 1956 37. Boue J, Boue A, Lazar P: Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous abortions. Teratology 12:11. 1975 38. Glass RH, Golbus MS: Habitual abortion. Fertil Steril 29:257, 1978 39. Malpas P: A study of abortion sequences. J Obstet Gynaecol Br Commonw 45:931, 1938 40. Warburton P, Fraser FC: Spontaneous abortion risks in man: data from reproductive histories collected in a medical genetics unit. Am J Hum Genet 16:1, 1964 41. Poland BJ, Miller JR, Jones DC, Trimble BK: Reproductive counseling in patients who have had a spotaneous abortion. Am J Obstet Gynecol 127:685, 1977 42. Kim HJ, Hsu LYF, Paciuc S, Christian S, Quintana A, Hirschhorn K: Cytogenetics offetal wastage. N Engl J Med 293:844, 1975 43. Schmidt R, Nitowsky HM, Dar H: Cytogenetic studies in reproductive loss. JAMA 236:369, 1976 44. Stenchever MA, Parks KJ, Daines TL, Allen MA, Stenchever MR: Cytogenetics of habitual abortion and other reproductive wastage. Am J Obstet Gyneco1127:143, 1977 45. Byrd JR, Askew DE, McDonough PG: Cytogenetic findings in fifty-five couples with recurrent fetal wastage. Fertil Steril 28:246, 1977 46. Mennuti MT, Jingeleski S, Schwartz RH, Mellman WJ: An evaluation of cytogenetic analysis as a primary tool in the assessment of recurrent pregnancy wastage. Obstet Gynecol 52:308, 1978 47. Kaji T, Ferrier A: Cytogenetics ofaborters and abortuses. Am J Obstet Gynecol 131:33, 1978 48. Luthy DA, Karp LE: What part do chromosomes play in repeated reproductive failure? Contemp Ob/Gyn 13:98, 1979 49. Simpson JL, Martin AO, Elias S: Unpublished data 50. Boue JG, Boue A: Chromosomal analyses of two consecutive abortuses in each of 43 women. Humangenetik 19:275, 1973 51. Alberman E, Elliott M, Creasy M: Previous reproductive history in mothers presenting with spontaneous abortions. Br J Obstet Gynaecol 83:366, 1975 52. Lauritsen JG: Aetiology of spontaneous abortion. A cytogenetic study of 288 abortuses and their parents. Acta Obstet Gynecol Scand [Suppl] 52:1, 1976

Received August 28, 1979. Reprint request: Joe Leigh Simpson, M,D., Section of Human Genetics, Prentice Women's Hospital, 333 East Superior Street, Chicago, Ill. 60611.