In humans, the number of chromosomes in each cell is 46: 22 pairs of autosomes (chromosomes 1-22) and one pair of sex chromosomes (XX or XY). The presence of a chromosome (or portions of a chromosome) in triplicate rather than duplicate is called trisomy. Most trisomies are lethal and are spontaneously aborted. Of those that occur in liveborn infants, the most common autosomal trisomies are trisomy 21 (Down syndrome) which occurs in about 1 in 700 births in North America; trisomy 18 (Edwards syndrome) which occurs in about 1 in 7,500 births; and trisomy 13 (Patau syndrome) which occurs in about 1 in 15,000 births.
Each of these trisomies results in a pattern of abnormalities including growth and development retardation. In trisomy 21 for example, affected infants have poor muscle tone, mongoloid appearance, small oral cavity and hence, a protruding tongue, low IQ, and oftentimes cardiac malformations, among other abnormalities. Early prenatal diagnosis of chromosomal disorders such as trisomy 21 is a major medical concern.
Less than one-third of human conceptions survive to birth. Of those that do, about 0.5% have a chromosome abnormality. Of those that don’t, an estimated 50% have a chromosome abnormality (Lewis, 1997). Understanding how and why these abnormalities occur is imperative to ensure human reproductive success.
The most common chromosome abnormality is trisomy (three of a particular chromosome or triplicate portions) which results preferentially from a failure of segregation (nondisjunction) homologous chromosomes at meiosis I in the female. The only etiological factor established for the occurrence of trisomy, particularly Trisomy 21, is advanced maternal age.
This study will attempt to link increased risk for trisomy in older females to sex hormone imbalance that may occur with the approach of menopause. Study participants will include in vitro fertilization patients and patients “at risk” for chromosome abnormalities. Major components of the study will be collection of reproductive histories and baseline hormone levels for all participants, cytogenetic analyses of oocytes from the in vitro group and fetal cells from both groups, and correlation of results with maternal age within study groups and the general population. If a link between hormone levels and trisomy incidence is apparent, this could lead to more accurate predictors of trisomy risk and suggest possible risk control interventions.
I. Project Description
IntroductionII. References Cited
Appendix A: Cytogenetic Technique
Chromosome abnormalities occur with astonishing frequency in humans. It is estimated they occur in 10-30% of all fertilized eggs (Hassold et al., 1996). In other mammals studied (mouse, hamster, sheep) much lower frequencies have been observed (less than 1% in preimplantation embryos – Angell et al, 1994).
The most common and clinically most important type of abnormality is trisomy which occurs in at least 4% of all recognized pregnancies and is the leading known cause of pregnancy loss and the leading genetic cause of mental retardation (Hassold et al., 1996).
Over the past 25 years, much has been learned about the incidence of trisomy in human gametes, fetuses and newborns. Molecular biological techniques in particular have uncovered some of the underlying causes. Nondisjunction has been shown to occur more often in females than in males and much more often at meiosis I than at meiosis II (95% of Trisomy 21 children receive their extra chromosome from their mother and of these, at least 80% result from an error at meiosis I – Gaulden, 1992). Some chromosomes (particularly those of D and G groups) have been shown to be more susceptible to nondisjunction than others.
The correlation between maternal age and the incidence of trisomy is well documented (risk of delivering a Trisomy 21 baby increases from 1 in 1529 at age 20 to 1 in 29 at age 45 – Clarke and Nora, 1986) but remains an enigma.
One clue, however, is that the incidence of Trisomy plotted against maternal age, beginning with age 13, forms a J-shaped curve (Fig. 1). This curve suggests that the ovarian conditions conducive to nondisjunction are primarily associated with both menarche (the establishment of the menstrual cycle at puberty) and menopause (the cessation of the menstrual cycle), which implies that hormonal imbalance may be involved.
The hormone cascade that controls the female sex cycle and oocyte maturation is complex. Luteinizing hormone-releasing hormone (LHRH) from the hypothalamus causes follicle stimulating hormone (FSH) and luteinizing hormone (LH) to be released by the anterior pituitary gland which then stimulates the ovary to release estrogen (mainly estradiol) and progesterone in an intricate feedback loop (Fig. 2). Changing levels of these hormones during the menstrual cycle cause widespread changes in the female reproduction system. Notably, they trigger resumption of meiosis I in the oocyte (Crowley and Gulati, 1979), affect the follicular environment of the oocyte and promote changes in the endometrium of the uterus in preparation for implantation of a fertilized ovum (Guyton, 1991, and Moore, 1988).
Several investigators, including Warburton (1989), Eichenlaub-Ritter and Boll (1989) and Gaulden (1989) have pursued hormone imbalance as a source of increased risk for nondisjunction. They suggest respectively that hormone imbalance affects oocyte ripeness, the rate of meiosis and spindle integrity, in each case leading to increased nondisjunction. They have not characterized the incidence of hormone imbalance in a population, however.
This study will attempt to characterize the incidence of hormone imbalance in two study groups, link hormone imbalance to maternal age and to the incidence of trisomy in both study groups, and compare these results to the incidence of trisomy in the general population correlated to maternal age.
If such a hormone imbalance link can be demonstrated, it could account for the preferential meiosis I origin of nondisjunction (while not precluding a meiosis II or mitosis origin); could account for the increased incidence of trisomy in both very young women and older women approaching menopause (see Figure X); and could explain why women of all reproductive ages may have a Trisomy 21 child. Further studies would be required to determine the precise mechanisms by which hormone imbalance might disrupt chromosome/chromatid segreation and to determine whether paternally derived trisomies might also result from hormone imbalance.
1. Women undergoing fertility treatment willing to donate oocytes that fail to fertilize, preimplantation embryos that fail to thrive, and fetal cells from any pregnancy outcome.
An extensive questionnaire will be administered to all participants to ascertain menstrual, reproductive and medical history. Menstrual cycle length and plasma concentrations of FSH, LH, estradiol and progesterone at several cycle checkpoints will be used to determine hormone imbalance. Hormone levels will be ascertained in more than on cycle including the cycle in which conception occurs where possible.
The hormone stimulation protocol for superovulated oocytes will be kept constant where possible. Only these oocytes will be analyzed to determine the incidence and meiotic origin of trisomy.
Oocytes and fetal cells will be prepared
for cytogenetic anlaysis using the three-stage fixation technique of Mikamo
and Kamiguchi (1983 – See Appendix A) and will be examined for evidence
of cell cycle disturbance. Particular chromosomes will be identified
where possible. Correlations between maternal age, hormone levels
and trisomy will be identified.
If the results of this study demonstrate a correlation between levels of particular hormones, maternal age, and the incidence of trisomy, then this could suggest a causative relationship between hormone imbalance and trisomy.
If the results do not demonstrate such a correlation, then this could mean there is no causative relationship between hormone levels and trisomy. It could also mean, however, that the hormone stimulation protocol used with infertility patients obscures typical hormone patterns or that method of measuring hormone levels are not sufficiently sensitive to identify imbalances.
Regardless of the results of this study,
it seems reasonable to suggest that more extensive hormone analysis could
be helpful in understanding the causes of human infertility and trisomy.
A fully equipped lab for cytogenetic analysis
with modern molecular biological analysis tools, trained technicians and
statistical support will be required. Students (with sensitivity
training) could be used to administer questionnaires and compile results.
Lewis, R. (1997) Human Genetics Concepts
& Applications, 2nd ed.,
Hassold T, Abruzzo M, Adkins K, et al. (1996) Human Aneuploidy; Incidence, Origin & Etiology, Environmental and Molecular Mutagenesis, 28:167-175.
Angell R, Xian J, Keith J, et al, (1994) First meiotic division abnormalities in human oocytes: mechanisms of trisomy formation, Cytogenetic Cell Genetics 65:194-202.
Eichenlaub-Ritter U, and Boll I, (1989) Nocodazole sensitivity, age-related aneuploidy and alterations in the cell cycle during maturation of mouse oocytes, Cytogenetic Cell Genetics, 52:170-176.
Gaulden M, (1992) Maternal age effect: the enigma of Down syndrome and other trisomic conditions, Mutation Research, 296:69-88.
Clarke F, Nora J, (1986) Genetics of man, 2nd ed., Philadelphia, Lea & Febiger, p. 98.
Moore K, (1988) The Developing Human, 4th ed., New York, Harcourt Brace Jovanovich, p. 167.
Eichenlaub-Ritter U, (1996) Perental Age-Related Aneuploidy in Human Germ Cells and Offspring, Environmental and Molecular Mutagenesis, 28:211-236.
Crowley P, Gulati D, et al. (1979) A chiasma-hormonal hypothesis relating Down Syndrome and maternal age, Nature, 280:417-418.
Pellestor F, (1991) Frequency and distribution of aneuploidy in human female gametes, Human Genetics 86:283-288.
Guyton A, (1991) Textbook of Medical Physiology, 8th ed. New York, Harcourt Brace Jovanovich, pp. 826-899.
Hassold T, Hunt P, and Sherman S, (1993)
Trisomy in Humans: Incidence, Origin and Etiology, Current Opinions in
Genetics and Development, 3:398-403.
The cytogenetic technique to be used throughout for fixing the oocytes will be based on the gradual, three-stage fixation technique of Mikamo and Kamiguchi (1983). After hypotonic treatment (0.9% sodium citrate for 10 mins) the oocyte will be placed in fix I (methanol:acetatic acid: H2O, 5:1:4) for about 3 mins. To dissolve the zone pellucida. It will then be transferred to a slide and fix II (methanol:acetic acid 3:1) added gently.
The slide will be put in a Coplin jar with fix II for about 10 mins. Then transferred to fix III (methanol:acetic acid:H2O, 3:3:1) for exactly 60 secs., slowly removed and control dried in a stream of moist warm air.
This technique provides excellent chromosome morphology and virtually no chromosome scatter. The slides will be stained with Giemsa initially for analysis, but those requiring further chromosome identification will be subsequently destained and restained with methyl green/DAPI for examination by fluorescence microscopy.