Features associated with reproductive ageing in female rhesus monkeys

R. Dee Schramm1,3, Ann Marie Paprocki1 and Barry D. Bavister2

1 Wisconsin Regional Primate Research Center, Madison, WI and 2 Audubon Center for Research of Endangered Species, New Orleans, LA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The specific aims were to determine the effects of maternal age on the meiotic and developmental competence of oocytes and incidence of chromosomal anomalies in oocytes from a population of fertile rhesus monkeys. METHODS: Monkeys were divided into two age groups (4–15 and 16–26 years of age) and underwent ovarian stimulation for collection of oocytes. RESULTS: In the older, compared with younger, monkeys, serum basal concentrations of FSH were elevated (P < 0.05), peak concentrations of estradiol during a stimulation cycle were diminished (P < 0.05), and mean numbers of oocytes retrieved following ovarian stimulation were markedly (P < 0.05) reduced. There were no significant maternal age-related impairments in oocyte maturation, fertilization or blastocyst development. Both abnormal numbers of whole chromosomes, as well as free chromatids, were detected in a limited number of rhesus oocytes. CONCLUSIONS: Similarities between female rhesus monkeys and women in several features associated with reproductive ageing, in conjunction with our ability to perform IVF and other assisted reproductive techniques in monkeys, demonstrate the suitability of these animals for studies on human reproductive ageing and maternal age-related infertility. Although maternal age-related impairments in oocytes were not evident prior to implantation, further studies may reveal more subtle impairments, manifested during post-implantation development.

Key words: aneuploidy/macaque/maternal age/oocyte/pre-implantation development


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The introduction of assisted reproductive technology as a treatment for human infertility has emphasized the urgent need for information on the extent and causes of pre-implantation embryo mortality and spontaneous abortion. With the tendency to delay child bearing into the later reproductive years, many women between 35 and 50 years of age are still interested in having a child. However, their fertility is markedly compromised (Edwards et al., 1984Go; Gosden, 1984Go; Wood et al., 1985Go; Gindoff and Jewelewicz, 1986Go; Feldberg et al., 1990Go; Piette et al., 1990Go), either by a reduction in implantation (Edwards et al., 1984Go; Rotsztein and Asch, 1991Go) or an increase in spontaneous abortion (Edwards and Steptoe, 1983Go; Stein, 1985Go; Rotsztein and Asch, 1991Go; Hassold and Chiu, 1985Go; Warburton et al., 1986).

Although the causes of infertility in older women have not been clearly defined, several studies clearly demonstrate that infertility in older women can be reversed by oocyte donation from younger women (Levran et al., 1991Go; King and Kovacs, 1992Go; Abdalla et al., 1993Go; Sauer et al., 1993Go; Balmaceda et al., 1994Go; Navot et al., 1994Go), suggesting that uterine function in many older women is normal, but that their oocytes are defective. Chromosomal anomalies are known to impair both pre-implantation (Kornafel and Sauer, 1994Go) as well as post-implantation (Levran et al., 1991Go; Hassold et al., 1984Go) development. In human oocytes failing to fertilize, and in cleavage-arrested pre-implantation embryos, the mean incidence of aneuploidy, over all ages, ranges between 4 and 50% in women undergoing infertility treatment (Wramsby et al., 1987Go; Wramsby and Fredga, 1987Go; Plachot et al., 1988Go; Papadopoulos et al., 1989Go; Delhanty and Penketh, 1990Go; Gras et al., 1992Go). Although chromosomal anomalies in spontaneous abortions increase with maternal age, it is unclear whether higher rates of aneuploidy are present in oocytes from older women (Bowman and Saunders, 1994Go). Due to legal, ethical and experimental limitations on the use of human embryos for research, controlled in-vitro studies need to be done to determine whether maternal age-related infertility is caused by impairments in oocytes or pre-implantation embryos, are not possible. Macaques are the preferred non-human primate model for studies on human reproductive senescence (Graham et al., 1979Go; Nozaki et al., 1995Go; Gilardi et al., 1997Go). In the present study, we have determined the effects of maternal age on serum hormone concentrations, numbers of oocytes recovered following ovarian stimulation, the meiotic competence of oocytes and pre-implantation developmental potential of embryos and determined the incidence of chromosomal anomalies in oocytes from a population of fertile rhesus monkeys.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ovarian stimulation and oocyte recovery
Based upon mean age at menopause and life expectancy in captive rhesus macaques (Macaca mulatta), monkeys selected for this study were divided into two age groups (4–15 and 16–26 years of age). Many animals in the older age group were acyclic or were having abnormal and/or anovulatory menstrual cycles, as reported previously for rhesus monkeys of this age range (Gilardi et al., 1997Go; Shideler et al., 2001Go). All monkeys used for oocyte collection exhibited menstrual cycles, although not necessarily regularly, had previously produced at least one viable offspring and had not been determined to be infertile at the time of the study. Monkeys received twice daily i.m. injections of 30 IU recombinant human FSH (Organon Inc., West Orange, NJ, USA) for 7–8 days, beginning on days 1 to 3 of the menstrual cycle (day 1 = first day of menstruation). Pretreatment blood samples were taken from a subset of animals (n = 10 young; n = 10 older) for determination of basal serum FSH concentrations prior to the first injection of exogenous FSH and for determination of maximal estradiol concentrations on the final day of FSH treatment. Recombinant HCG (1000 IU; Ares Advanced Technology, NJ, USA) was administered i.m. the morning after the last day of FSH treatment for induction of oocyte maturation. Oocytes were aspirated laparoscopically 27–32 h following injection of HCG from follicles >=3 mm in diameter, using a modified Renou Device with a 20 gauge aspiration needle (Bavister et al., 1983aGo), into Tyrode's lactate (TL)-hepes medium (37°C; Bavister et al., 1983bGo) containing either 5% bovine calf serum or 0.01 mg/ml polyvinyl alcohol (PVA) and 10 IU/ml heparin.

Oocyte culture/IVF
Oocytes were retrieved from aspirates using an EM Con filter (Veterinary Concepts, Spring Valley, WI, USA). In some cases, cumulus masses were treated with 0.1% hyaluronidase to facilitate recovery of oocytes. Oocytes that did not contain an intact germinal vesicle (GV) were cultured for 1–8 h post-aspiration in 50µl drops of modified CMRL-1066 medium (mCMRL; Boatman, 1987Go) containing 20% bovine calf serum (BCS) under 3.5 ml of mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air. Oocytes were examined every 2 h for evidence of nuclear maturation and were inseminated within 4 h of first polar body extrusion.

Because sperm from older or infertile men can be genetically defective, in the current study only proven fertile males were used as sperm donors to help control for any potential paternal contribution to chromosomal anomalies and developmental impairments. Semen was collected by penile electro-ejaculation, and sperm capacitation and IVF was performed as described previously (Bavister et al., 1983aGo). Briefly, 10–20x106 washed motile sperm/ml were resuspended in 2 ml Tyrode's albumin lactate pyruvate (TALP) medium overlaid with 2 ml mineral oil, and incubated at 37°C in 5% CO2 in air for 1–6 h. Sperm were treated with 1.0 mmol/l each of caffeine and dibutyryl cAMP to induce hyperactivation (Boatman and Bavister, 1984Go) and diluted (1x105 – 2x105) into 100 µl drops of TALP medium. Sperm and oocytes were co-incubated for 12–16 h at 37°C in a humidified atmosphere of 5% CO2 in air. Sperm and remaining cumulus cells were then removed manually by drawing oocytes through a pulled glass pipette, and oocytes were examined for evidence of fertilization.

Embryo culture
All diploid (2 pronuclei) zygotes were cultured in G1 medium until the 8-cell stage followed by G2 medium beyond the 8-cell stage (Barnes et al., 1995Go; Gardner and Lane, 1997Go) in a humidified atmosphere of 5% CO2, 5%O2 and 90% N2. Embryos were placed into fresh culture medium every other day and were examined daily using differential interference contrast (DIC) optics (x200) on a Nikon inverted microscope with a heated (37°C) environmental control chamber.

Chromosome analysis
Metaphase spreads were done on mature (metaphase II) oocytes as described previously (Mikamo and Kamiguchi, 1983). Briefly, within several hours of completing meiosis (metaphase II), oocytes were placed into a hypotonic solution (0.9% Na citrate; 37°C) for 10 min and then transferred into Fix I (5:1:4 methanol:glacial acetic acid:dH2O) until the zona pellucida dissolved. Oocytes were then transferred in a very small drop of Fix I onto a glass slide and allowed to stick down. One drop of Fix II (3:1 methanol:glacial acetic acid) was immediately dropped onto the oocytes using a pulled pipette followed by two more drops to wash away any remaining cytoplasm. Slides were then transferred to a Coplin jar with Fix II for 10 min. Finally, slides were transferred to a Coplin jar with Fix III (3:3:1 methanol:glacial acetic acid:dH2O) for 60 s, and then air dried. Slides were stained with Giemsa:Sorenson's buffer (2:21) in a glass Coplin jar for 2 min, allowed to air dry, and stored in a slide box at -20°C until photographed and karyotyped.

Radioimmunoassays
Concentrations of FSH and estradiol were measured by radioimmunoassays in the Primate Center Hormone Assay Services Laboratory as previously described (Dumesic et al., 1997Go). Estradiol concentrations were determined after diethyl ether extraction of sera and solvent fraction separation by celite chromatography. The intra-assay coefficients of variation were: FSH, 4.0% and estrogen (E2), 3.4%. The interassay coefficients of variation were: FSH, 4.6% and E2, 9.8%.

Data analyses
Means or percentages for all variables were compared between age groups using t-tests. Values with P < 0.05 were considered statistically different.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pretreatment baseline serum concentrations of FSH were significantly (P < 0.05) elevated in older versus young monkeys and serum concentrations of estradiol on the final day of FSH were significantly (P < 0.05) reduced for older versus young monkeys (Figure 1Go).



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Figure 1. Serum concentrations of FSH and estradiol from young versus older rhesus monkeys undergoing ovarian stimulation. Blood samples for basal FSH were collected on day 1, 2 or 3 of menstruation, prior to the first injection of exogenous FSH. Samples for peak estradiol were collected on the last day (day 7 or 8) of FSH treatment. Young = 5–15 years of age. Older = 16–22 years of age. *P < 0.05.

 
The meiotic and developmental competence of oocytes obtained from young versus older rhesus monkeys following gonadotrophin stimulation are shown in Table IGo. Mean numbers of oocytes recovered per cycle were significantly (P < 0.05) reduced in older compared with young monkeys. There were no significant differences in the incidences of nuclear maturation (mean = 65.8%) or fertilization (mean = 65%) between the two age groups. The proportion of zygotes having more than 2 pronuclei did not differ between age groups (not shown). Development of fertilized oocytes into blastocysts was similar in young and older monkeys (mean = 61.3%). Four of the 15 monkeys in the older age group were over 20 years of age, and their rate of blastocyst development (62.5%) was not different from that of the young monkeys. The incidences of aneuploidy in mature oocytes obtained from young versus older rhesus monkeys are shown in Table IIGo. A total of 14/34 and 16/22 chromosome spreads were analysable for young and older monkeys respectively. A total of 26 of 30 analysed oocytes, over both age groups, contained the normal complement (21) of chromosomes. In the young monkeys, one of 14 oocytes was hypohaploid (20 chromosomes). In the older group, three of 16 oocytes exhibited chromosomal anomalies. One of these oocytes was highly hyperhaploid (29 chromosomes), one was hypohaploid (20 chromosomes) and one was missing a single chromatid (20 + 1/2 chromosomes). In order to avoid overestimating the incidence of aneuploidy resulting from chromosomes lost during spreading, the number of hyperhaploid oocytes can be doubled and the hypohaploid oocytes eliminated, rather than summing the total numbers of hyperhaploid and hypohaploid oocytes, to provide a more accurate estimate of aneuploidy (Koenig and Stormshak, 1993Go). Using this formulation, the adjusted incidence of aneuploidy was 0/13 (0%) for young monkeys and 2/14 (14.3%) for older monkeys. In either case, there were no statistically significant differences between age groups in this limited sample of oocytes. Representative chromosome spreads of metaphase II oocytes exhibiting normal and aneuploid karyotypes are illustrated in Figure 2Go.


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Table I. Meiotic and developmental competence of oocytes from young versus older rhesus macaques following gonadotrophin stimulationa
 

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Table II. Incidence of maternal age-related aneuploidy in metaphase II oocytes from gonadotrophin stimulated rhesus macaques
 


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Figure 2. Representative chromosome spreads of metaphase II macaque oocytes exhibiting (a) normal, (21 chromosomes) and (b) aneuploid, (29 chromosomes) karyotypes.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pretreatment baseline serum concentrations of FSH were significantly elevated in older compared with young monkeys, consistent with previous studies in rhesus monkeys (Hodgen et al., 1977Go; Graham et al., 1979Go) and women (Sherman et al., 1976Go; Mussey et al., 1987Go; Klein et al., 1996Go). Additionally, serum concentrations of estradiol during gonadotrophin stimulation and numbers of oocytes retrieved per cycle were markedly reduced in older versus young monkeys, similar to that previously reported for women (Janny and Menezo, 1996Go), and in some older monkeys, no oocytes were recovered. Taken together these results are consistent with depletion of the follicular reserve with the approach of menopause (Richardson et al., 1987Go; Faddy et al., 1992Go; Hull et al., 1996Go). In a recent study, Shideler et al. failed to detect differences in urinary FSH or estradiol levels within aged monkeys (18–26 years of age) prior to the onset of abnormal menstrual cycles (Shideler et al., 2001Go). However, FSH levels were not compared with those of young monkeys, and estradiol was measured in naturally cycling, rather than gonadotrophin stimulated, animals. Thus, while estradiol levels arising from a single dominant follicle during a natural cycle may be similar among young and older monkeys, the reduced levels observed following gonadotrophin stimulation in the present study likely reflect a reduction in the numbers of follicles recruited. Therefore, results of the present study reveal similarities between rhesus macaques and women in age-related alterations in ovarian and pituitary function and further support the suitability of the rhesus macaque as a model for study of the menopausal transition (Gilardi et al., 1997Go; Shideler et al., 2001Go). Since abnormal and/or anovulatory menstrual cycles are characteristic of rhesus monkeys >18 years of age (Gilardi et al., 1997Go; Shideler et al., 2001Go; present study), and age-related changes in endocrine function and follicular populations were evident in the present study, results were unlikely to have been confounded by homogeneity in the ages of the groups of monkeys selected, as is often the case in most, if not all clinical studies.

Two probable causes of infertility in older women are an unreceptive uterine environment (Edwards et al., 1984Go; Feldberg et al., 1990Go; Piette et al., 1990Go; Dicker et al., 1991Go; Rotsztein and Asch, 1991Go) or oocytes that are not capable of normal fertilization and/or embryonic development (Feldberg et al., 1990Go; Rotsztein and Asch, 1991Go). Studies involving oocyte donation suggest that both poor oocyte quality as well as an inadequate uterine environment may contribute to infertility in older women. Circumstantial evidence exists for an age-related decline in uterine receptivity (Asch et al., 1987Go; Rotsztein and Asch, 1991Go; Flamigni et al., 1993Go), although controlled, scientifically sound experiments have not been conducted in women. It was suggested, however (Stein, 1985Go), that the effects of ageing on uterine function appear at later ages than do the effects of ageing on the ovum. Studies in women involving oocyte donation clearly demonstrate that infertility in older women can be reversed by oocyte donation from younger women (Levran et al., 1991Go; Navot et al., 1991Go; Rotsztein et al., 1991; King and Kovacs, 1992Go; Sauer et al., 1992Go; Balmaceda et al., 1994Go), suggesting that uterine function in most older women is normal, but that their oocytes are defective. However, oocyte donation studies are often confounded and difficult to interpret because when older women are recipients of their own embryos, they are often replaced during the same stimulation cycle, but when they receive donated oocytes from younger women, embryos are replaced during a normal non-stimulated cycle, which likely leads to an increased implantation and delivery rate. Although it is reasonable to infer that oocytes from older women are developmentally impaired, neither the specific age-related defects in oocytes nor the time during development (pre- or post-implantation) when they are manifested have been clearly defined.

In the present study, we have compared the meiotic and pre-implantation developmental competence of oocytes from young versus older monkeys. Similar studies would be difficult to carry out in women because embryos are typically either transferred at the early cleavage stages or cryopreserved for subsequent transfers. Results demonstrate that both the meiotic and developmental competence of oocytes, including their competence to develop into blastocysts, were equivalent between young and older monkeys, indicating that pre-implantation development is not compromised by age of the oocyte donor. In fact, four of the 15 monkeys in the older age group were >20 years of age, and their rate of blastocyst development (62.5%) was not different from that of the young monkeys. This is consistent with studies of pregnancy failure in women demonstrating that miscarriage rate, but not implantation rate, was adversely affected by age of oocyte donor (Abdalla et al., 1993Go), suggesting that maternal age-related embryo defects typically may not be manifested until after implantation. Similarly, other studies indicate that the majority of chromosomal anomalies in aneuploid embryos are not lethal until after implantation (Hassold and Jacobs, 1984Go). In contrast, however, Janny and Ménézo reported that human blastocyst development and expansion in vitro declined in women over 30 years of age, but neither post implantation pregnancy loss nor delivery rates were influenced by donor age following blastocyst transfer (Janny and Ménézo, 1996Go). The apparent discrepancy between the present study in monkeys and the former study in women with regard to pre-implantation development may be attributed to differences in the fertility status of the study population. None of the monkeys in the present study was considered to be infertile, whereas many older women presenting for infertility treatment in the former study were obviously infertile, or at least subfertile, which may have biased the results. Alternatively, the reduced blastocyst development observed in older women may have resulted from the known high incidence of age-related aneuploidy in human embryos, which may impair pre-implantation embryo development (Kornafel and Sauer, 1994Go). The present study did not address aneuploidy in pre-implantation embryos, and an age-related increase in the incidence of aneuploidy was not detected in the limited number of oocytes evaluated. Although a larger sample size may have revealed an increased incidence in maternal age-related aneuploidy, the developmental data clearly indicate that age-related chromosomal anomalies, if present, are not lethal prior to implantation. Thus, while pre-implantation development may not be compromised with increasing maternal age, more subtle impairments in embryos, including blastocysts, such as abnormal or inappropriate expression of developmentally important genes, may be manifested during or after implantation, leading to developmental failure and pregnancy loss. It has been speculated that mitochondrial DNA deletions may contribute to maternal age-related pregnancy loss. In women, mitochondrial DNA deletions have been detected in ova (Chen et al., 1994Go) and appear to occur more frequently with increasing maternal age (Keefe et al., 1995Go). However, a large-scale study on macaque oocytes failed to reveal a maternal age-related increase in mitochondrial DNA deletions (R.D.Schramm, unpublished data).

While a limited number of studies in women indicate that the incidence of aneuploidy in oocytes increases with maternal age (Plachot et al., 1988Go), the majority of studies do not support this conception (Delhanty and Penketh, 1990Go; Gras et al., 1992Go; Angell et al., 1993Go; Almeida and Bolton, 1993Go; Kamiguchi et al., 1993Go; Angell et al., 1994Go). Although the true incidence of maternal age-related chromosomal anomalies in human oocytes is unclear, the incidence, over all ages, of aneuploidy in human oocytes failing to fertilize ranges from 4–50% (Wramsby et al., 1987Go; Plachot et al., 1988Go; Papadopoulos et al., 1989Go). Results from human studies are generally difficult to interpret for a variety of reasons. Oocytes are obtained from patients who are infertile, and their average ages are fairly homogeneous. Additionally, data are typically obtained from rejected oocytes that had failed to fertilize and were analysed several days after aspiration, which may lead to increased predivision of chromatids (Dailey et al., 1996Go), or from embryos that had undergone cleavage arrest, which may also bias the results.

To date, results of the present study are the only data that exist for aneuploidy in non-human primate oocytes. Although limited in nature, these results are of considerable significance because unlike many women undergoing infertility treatment, monkeys selected for the present study were not considered infertile, and data obtained were not from oocytes that had failed to fertilize or from embryos that had undergone cleavage arrest. Furthermore, results were unlikely to have been confounded by homogeneity in the ages of the groups of monkeys studied, unlike the situation in clinical studies. Moreover, only proven fertile males were used as sperm donors to help control for any potential paternal contribution to chromosomal anomalies and developmental impairments. Thus, most, if not all, of the factors that confound interpretations in human studies were avoided. In the present study, only half of the oocytes were clearly analysable due to lost spreads, overspreading and underspreading, similar to results for human oocytes (Angell et al., 1994Go). Although the sample size was limited, assessment of all chromosomes, rather than just a few select chromosomes, as is often the case in clinical studies using fluorescent in-situ hybridization (FISH), was a more powerful means for detecting any chromosomal anomalies present in a given oocyte. Chromosomal anomalies were manifested as both missing and extra whole chromosomes, as well as free chromatids, as reported previously for human oocytes (Dailey et al., 1996Go; Dyban et al., 1996Go; Angell, 1997Go; Marquez et al., 1998Go). Although there is still much debate on the origins and causes of age-related aneuploidy (Angell, 1997Go; Lamb et al., 1997Go; Wolstenholme and Angell, 2000Go), our findings support the theory that loss of cohesion between either homologous chromosomes or chromatids is the predominant mechanism of aneuploidy in women (Wolstenholme and Angell, 2000Go), likely resulting from the prolonged retention of the oocyte in the ovary before ovulation (Eichenlaub-Ritter et al., 1986Go, 1988aGo,bGo). Unfortunately, it was not possible to draw any firm conclusions with respect to maternal age-related differences in the incidence of aneuploidy due to the statistical constraints associated with the limited numbers of oocytes analysed in the present study.

In conclusion, we have demonstrated similarities between female rhesus monkeys and women in several physiological features associated with reproductive ageing. These features, in conjunction with our ability to perform IVF and other assisted reproductive techniques, including ICSI and embryo transfer in monkeys, encourage the use of rhesus monkeys as non-human primate models for studies on human reproductive ageing. Although animals and oocytes selected for the present study may not have been representative of the human population in clinical infertility studies, they may provide a more accurate interpretation of the effects of maternal age on infertility in the general human population. In rhesus monkeys, maternal age-related developmental impairments in oocytes and embryos were not evident prior to implantation. Further studies may reveal more subtle developmental impairments, manifested during post-implantation development. Detection of chromosomal anomalies in rhesus monkey oocytes warrants further studies on maternal age-related aneuploidy in a non-human primate model.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This research was supported by research grants NIH RR00167 and NIA AG12179. This is publication number 41–015 of the WRPRC. The authors gratefully acknowledge Chuck Harris and Dr Lorraine Meisner of the Cytogenetics Department for analysing the chromosome spreads. We are grateful to Steve Eisele, Lisa Knowles, Melissa Brown and Michele Shotzko of Reproductive Services for menstrual cycle monitoring, hormone injections, blood sampling, and sperm collection, and to Denny Mohr and Michele Shotzko for surgical assistance with oocyte collection. We further thank Fritz Wegner and Dan Wittwer for hormone assay services. We are grateful to Organon Inc., West Orange, NJ, USA for the generous supply of recombinant human FSH and Serono Laboratories (Ares Advanced Technology), Randolph, MA, USA for the gift of recombinant HCG.


    Notes
 
3 To whom correspondence should be addressed at: Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, WI 53715, USA. E-mail: schramm{at}primate.wisc.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 11, 2001; resubmitted on January 21, 2002; accepted on February 25, 2002.