Brody School of Medicine, East Carolina University, Greenville, NC 27858-4354, USA
1 Email:boklagec{at}mail.ecu.edu
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Abstract |
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Key words: fertilization/gamete/imprinting/sex ratio
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Introduction |
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From the perspective of developmental biology, the most critical work of embryogenesis is done before the existence of pregnancy is recognized. At or about 8 weeks after fertilization, when the second consecutive menses has been missed and a clinical appointment has been made and met, a normal embryo for which all has gone well is about the size of an adult fingernail, but has already well begun building its own fingernails. Body symmetry is long-since clearly framed and well elaborated. All organs and organ systems are in place and growing. Nearly all cell types required to perform the body's many functions have differentiated. Very nearly all of what might go wrong already has.
It is against the background of that understanding that we should consider questions concerning sex ratio at birth. There is a large literature on variations in the secondary sex ratio. Variations from the common values are generally considered to represent the effects of anomalies in or of the mother's world around the time of conception, on the order of war, famine, plague and pollution (Whorton et al., 1989; Graffelman and Hoekstra, 2000
; Grech et al., 2003
; Jongbloet, 2004
). In general, a reduction from the customary excess of males at birth is taken as a sign of some negative influence. William H.James alone has published some 200 papers about the effects on secondary sex ratio of various substances, events, circumstances or behaviours. Some of his most recent contributions (James, 2004a
e
) should lead to the others.
Such observations are seldom lately allowed into print without demonstrations of statistical significance, but their publications are often followed closely in the literature by reports that a particular sex-ratio result could not be repeated in an apparently equivalent sample. Many of the opinions and interpretations in that literature are contradictions of others (e.g. Boklage, 1997; Cagnacci, 2004
; James, 2004d
). However consistently frustrating, the subject remains somehow deeply intriguing, as if something close to 106 males per 100 females among newborns of European ancestry is to be considered normal and any smaller fractions of males is an anomaly the meaning of which must be sought. If the ratio of male/female newborns in a white European population falls toward the lower fraction of males typical of a population of newborns of African ancestry, something must be wrong, and such problems are generally imagined to have occurred somewhere near conception.
One major theme throughout all of those considerations is the enduring supposition that the primary sex ratio at conception must be quite high to explain the consistent observation of male excess at birth (secondary sex ratio, e.g. Chahnazarian, 1988; Charnov and Bull, 1989
), in spite of the not-always-but-more-often-than-not observed male excess in losses throughout recognized pregnancy (Hassold et al., 1983
; Cooperstock and Campbell, 1996
; Mizuno, 2000
; Ingemarsson, 2003
). Creasy, as long ago as 1977, felt the need to publish an argument against the prospect of high primary sex ratio, but did not settle the issue (Creasy, 1997
). The question has clearly survived to very recent times, and the prevailing prejudice is apparently still to the effect that secondary sex ratio depends on primary sex ratio, which must somehow depend on circumstances of conception. (You might notice that there are in play several understandings of the meaning of conception at a cellular level. It would make a great deal of sense to me to consider successful fertilization, implantation and embryogenesis as events and processes leading to conception, which would then correspond nicely to maternal and clinical recognition.)
There has been a sizeable number of sound research efforts that would have discovered any consistent bias in spermatogenesis or fertilization, but did not. Such studies have consistently found no significant departure from X=Y among sperm cells or among sperm-derived chromosome sets after fertilization. The accumulated evidence shows that there is in fact a significantly consistent small excess of fertilizations by X-bearing sperm. This X-excess is generally not significant in the numbers from any one study (Martin et al. 1982, 1983
, 1987
; Brandiff et al., 1986a
; Kamiguchi and Mikamo 1986
), but, when the sample numbers and results of these studies are combined, the excess of fertilization by X-bearing sperm does reach statistical significance: among a total of 7100 sperm from the five studies just mentioned, 3400 (47.9%) showed a Y chromosome (
21df=12.4, P<0.001 versus the null hypothesis of 50%). In other studies: 9225 sperm showed no significant variation from X:Y::1:1 with varying age of the donor (Martin and Rademaker, 1992
); 1960 sperm showed no effect of cryopreservation on X:Y ratio, which did not depart significantly from 50% (Martin et al., 1991
); 630 sperm showed no significant variation of X:Y ratio from 1:1 before or after selection for high motility (Brandiff et al., 1986b
); Lobel et al. (1993)
found no effect of manipulation by swim-up or Sephadex filtration, and 10 664 sperm assessed directly by multicolour fluorescent in situ hybridization for X versus Y chromosome content did not depart significantly from a 1:1 ratio before or after chemotherapy (Martin et al., 1995
).
The evidence provided by these results is indirect. That is unlikely to change. Ethical, technical and financial considerations argue against the destruction for karyotyping of statistically sufficient numbers of products of natural human fertilizations. Granted, any bias of the sort we seek to discover and understand might, after all, operate differently, or not at all, in sham fertilizations of hamster oocytes, versus oocytes from artificially induced human ovulations, versus naturally fertilized human oocytes. The best evidence we have, or are likely to have in the foreseeable future, indicates that the consistent excess of males observed in human births does not originate in a consistent bias in spermatogenesis or in fertilization.
The usual excess of males is present through pregnancy beyond clinical recognition, but not present at fertilization. The difference, therefore, must arise between fertilization and clinical recognition, through a preferential loss of females during embryogenesis.
Fewer than 25% of natural human fertilizations survive to term, even with healthy parents of proven fertility and of healthy reproductive age, under good medical attention. It seems reasonable to suppose that the reproductive efficiency of the population as a whole is less than that and quite plausibly substantially less. The majority of the losses between fertilization and term occur in embryogenesis, with two-thirds failing before clinical pregnancy recognition and only 10% of the remainder failing in the remaining
30 weeks (Boklage, 1990
).
Among mammalian embryos, those earliest stages of development progress more rapidly for male embryos than for females (Mittwoch and Mahadevaiah, 1980; Bourgoyne, 1993
; Mittwoch, 1993
, 1997
; Pergament et al., 1994
; Krackow et al., 2003
). Since some of the products of these earliest stages are signals from one part of the embryo to another, or from the embryo to the maternal physiology, signals which are required for continuation and maintenance of the pregnancy, the establishment of viable, clinically recognizable pregnancy is correspondingly more efficient in general for male embryos (Krackow, 1995
; Kochhar et al., 2003
).
Sex differences such as these in rates and efficiencies of early embryonic development are widely thought to depend at least in part on genomic imprinting. In the extreme, absence of the maternal imprint normally imposed in oogenesis causes lethally poor growth of the embryo proper; absence of the normal paternal imprint from spermatogenesis causes lethally poor growth of extra-embryonic support tissues (Hurst, 1994; Moore et al., 1995
; Werren and Hatcher, 2000
). Thornhill and Burgoyne (1993), in particular, showed that a paternally imprinted X chromosome (possessed by all normal mammalian females and no normal males) retards early development in the mouse. Khoury et al. (1984)
and Ruder (1986)
showed that the most consistent of all reported variations in human secondary sex ratio, the race difference, is determined by paternal factors in births to interracial couples. Tesarik et al. (2002)
have shown that some such paternal developmental effects operate in the first cell cycle of embryogenesis.
Another source of excess loss of female embryos which may help to illustrate the importance in human embryogenesis of paternal epigenetic factors concerns X monosomy, the most common cause of Turner syndrome. The frequency of the 45,X karyotype at birth is in the order of 1/3000 newborn females, which represents a very small fraction of their frequency among first-trimester spontaneous abortions. At least 95% of the known lost 45,X conceptuses fail as embryos, with the mean developmental age of those found in first trimester abortions being 6 weeks (cf. Epstein, 1986
; at least because we only know about those that lasted long enough to become observable spontaneous abortions). Most liveborn 45,X Turner females have a maternal X chromosome (Hassold et al., 1990
, 1992
; Kelly et al., 1992
). With no thought to parental genome imprinting at the time of that work, those observations have been interpreted to mean that loss of the X-chromosome from XY segregation in spermatogenesis is the primary source of the X-monosomy anomaly (although the majority of anomalies of chromosome number generally are attributed to maternal meiotic errors). Given present understanding of the potential differences due to imprinting, and observations of the developmental effects of a paternally imprinted X, it is more than reasonable to suppose that the frequency of maternal X chromosomes in liveborn 45,X girls is due to preferential loss of embryos bearing paternally imprinted monosomic X chromosomes.
To establish during embryogenesis an excess of males to be present at the onset of the fetal period, sufficient to retain a male excess throughout gestation to term birth in spite of consistent male excess in pregnancy wastage at all stages, can easily be arranged. There need only be an excess of female losses during embryogenesis, which may in fact be readily demonstrated. To generate numbers consistent with the long history of observations requires a plausibly small excess of female losses in embryogenesis (Figure 1).
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This understanding leaves it reasonable to suppose that populational conditions which might affect the progress of embryogenesis, particularly by way of epigenetic processes (cf. Gosden, 2002; McEvoy, 2003
; Fleming et al., 2004
; Lucifero et al., 2004
), might change sex ratios at birth. It also sets some conceptual limits on what conditions or changes might reasonably be considered more or less likely to affect sex ratios.
Evdokimova et al. (2000) offer results consistent with this proposal. They conducted cytogenetic examination of 342 embryonic spontaneous abortions divided into three clinical groups on the basis of the timing of the failure of the embryo: spontaneous abortions strictly so called, including a developed embryo without clearly delayed development (n=100), non-developing pregnancies (n=176), and anembryonic conceptuses (n=66). The frequency of chromosomal mutations in these groups was 22.0, 48.3 and 48.5% respectively. Among the members of each group with normal karyotypes, sex ratios were: 0.77 for the most advanced embryos; 0.60 for the non-developing pregnancies; and 0.31 for the anembryonic conceptuses. The earlier the failure most likely occurred, the greater fraction female. Within this sample, the fraction male was higher among better-formed conceptuses with or without aneuploidy. These authors proposed that the expression of genes of the single, maternal (maternally imprinted) X chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos (with one paternally imprinted X chromosome).
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Discussion |
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The prospect of gathering evidence more direct than provided here is problematical. Remains from spontaneous failures of biochemically recognized pregnancies, or products of early pharmaceutical abortions (cf. Joffe and Weitz, 2003; Weismiller, 2004
), might be ranked by developmental versus gestational age, and analysed for sex ratios accordingly. Because sex is unlikely to be anatomically visible in embryos of the ages in question, analyses must be cytogenetic and/or molecular (Daniely et al., 1998
; Lomax et al., 2000
), testing for Y-chromosome sequences and X-linked alleles specific to the father which should represent products of conception. (Parental samples will be required as well.) It would seem that this hypothesis should predict that fraction male among these remains should be lowest among the earliest failures (lowest developmental ages or developmental/gestation age ratios) and rise through embryogenesis.
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References |
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Submitted on August 11, 2004; accepted on November 19, 2004.