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BOR - Papers in Press, published online ahead of print February 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038919
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BIOLOGY OF REPRODUCTION 72, 1336–1343 (2005)
DOI: 10.1095/biolreprod.104.038919
© 2005 by the Society for the Study of Reproduction, Inc.

Delayed Motherhood Decreases Life Expectancy of Mouse Offspring1

Juan J. Tarín 2 3, Vanessa Gómez-Piquer 3, Francisco Rausell 3, Samuel Navarro 4, Carlos Hermenegildo 6, and Antonio Cano 5

Department of Functional Biology and Physical Anthropology,3 Faculty of Biological Sciences, University of Valencia, Burjassot, 46100 Valencia, Spain Departments of Pathology,4 and Pediatrics, Obstetrics and Gynecology,5 Faculty of Medicine, University of Valencia, 46010 Valencia, Spain Research Unit,6 Hospital Clínico de Valencia and Department of Physiology, University of Valencia, 46010 Valencia, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study analyzes the long-term effects of delayed motherhood on reproductive fitness and life expectancy of offspring in the mouse. Hybrid (C57BL/6JIco x CBA/JIco) first-generation (F1) females, either at the age of 10 or 51 wk, were individually housed with a randomly selected 12- to 14-wk-old hybrid male following a breeding pen system until females reached the end of their reproductive life. Reproductive fitness of second-generation (F2) females was tested from the age of 25 wk until the end of their reproductive life. In F2 males, the testing period ranged from the age of 52 wk until their natural death. Delayed motherhood of hybrid F1 female mice was associated with a decreased percentage of male F3 offspring at birth and lower life expectancy and body weight during adulthood of F2 offspring. There was, however, no evident negative effect of delayed motherhood on several reproductive fitness variables in either male or female F2 offspring. This included between-parturition interval, litter size at birth and at weaning, body weight at weaning and preweaning mortality of F3 pups, percentage of F3 litters with at least one pup cannibalized, and time at which female and male F2 offspring ceased their reproductive life. These data clearly show that delayed motherhood in the mouse is associated with negative long-term effects on offspring survival.

aging


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present trend of women in most Western countries to pursue educational and professional goals before conceiving is increasingly forcing them to postpone motherhood until their mid-30s or even beyond [1] (i.e., the latter part of a woman's childbearing years). It is at this time that women exhibit decreased fertility and obstetrical problems, while their children exhibit increased fetal and perinatal morbidity and mortality [2–10]. Advanced maternal age at conception is also associated with a higher probability of offspring suffering from trisomy, hypospadia [11], sporadic [12] and childhood [13] leukemia, and mitochondrial DNA diseases including congenital sensorial hearing loss, cerebellar ataxia, type I (insulin-dependent) diabetes mellitus, and Alzheimer disease [2]. A recent study found that offspring from reproductively old female mice exhibit not only higher mortality, retarded sensorimotor integration, and lower body weight during preweaning development, but also decreased spontaneous motor activity and learning capacity during early adulthood [14].

Epidemiological data have shown that advanced maternal age at childbirth is associated with sons exhibiting reduced sperm quality [15] and a higher risk of being infertile [16], while daughters displaying menstrual disorders (mean cycle length ≥42 days or ≤21 days, or a within-subject variation between cycles of ≥14 days, or amenorrhea) [17] and reduced fecundity [18]. The offspring of young insects also often differ from those of old insects in morphological, physiological, and life history traits that are important to reproductive fitness. For instance, offspring from older mothers frequently develop slower and are smaller at adult emergence than offspring from young mothers [19]. Such a circumstance may limit both female capability of producing normal-sized offspring and male ability to pursue and capture mates in the field [20].

The famed inventor Alexander Graham Bell [21], after analyzing the genealogical records of 2386 descendants of William Hyde, one of the early settlers of Norwich, CT, who died in 1681, found that persons whose mothers were ≥40 yr when persons were born had {approx}25% shorter lives than persons whose mothers were <25 yr of age. More recently, Gavrilov et al. [22] analyzed the lifespan of offspring from European aristocratic families with well-known genealogy. They found no effect of maternal age at childbirth on life expectancy of sons. However, daughters born to mothers older than 40 yr of age showed a lifespan about 3.6 yr shorter when compared with daughters born to mothers younger than 40 yr. This difference was increased to 4.5 yr when data were analyzed from daughters born to long-lived mothers (those who lived 70 yr or longer) when these women were younger or older than 40 yr.

The association between advanced maternal age at birth and the longevity of her offspring is not limited to human beings. Other researchers have demonstrated in a number of independent studies that older mothers have shorter-lived offspring in rotifers, duckweed, house flies, stink bugs, fruit flies, flour beetles, mealworms, nematodes, and yeast [23]. This negative effect of maternal age on offspring longevity is referred to as the Lansing effect, after Albert Lansing's widely cited work on rotifers [23]. We should note, however, that nowadays it is believed that an early demonstration by Lansing [24] that rotifer cultures propagated from older mothers had shorter lives is likely an artifact of his experimental condition [25].

The purpose of the present study is to analyze, in the mouse, the long-term effects of delayed motherhood on reproductive fitness and life expectancy of offspring. Reproductive fitness was divided into two major components, total number of offspring born (fertility) and quality of these offspring, measured as preweaning mortality and weaning weight. The variation in total number of offspring born was, in turn, attributed to other metric characters including mating success, time to pregnancy, litter size, frequency of litters, and number of litters [26].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Origin of Second-Generation (F2) Mice

All the animal experiments performed in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [27]. A detailed description of the procedures followed to produce F2 mice has been published previously [14]. Briefly, either at the age of 10 wk (young-mother group, n = 47) or 51 wk (old-mother group, n = 33), hybrid (C57Bl/6JIco female x CBA/JIco male) first-generation (F1) virgin females were individually housed with a randomly selected 12- to 14-wk-old hybrid male. When females exhibited physical evidence of pregnancy (i.e., the presence of a distended abdomen), the male was removed from the cage and females were allowed to give birth and to breastfeed her pups until weaning. When the first litter was weaned, females were left alone in the cage for another week. After this resting period, females were caged again with a new, randomly selected 12- to 14-wk-old hybrid male. This sequence of events was repeated until the 33 old females reached the end of their reproductive life. On Day 3 after birth, litter size and sex ratio of pups from young females were matched with litter size and sex ratio obtained in the old-mother group. Thus, only 33 out of the 47 fertile young mothers were initially used in the study. Thereafter, the number of fertile dams and pups from the young-mother group were reduced in successive litters so as to match the age-related decreasing number of fertile dams and pups observed in the old-mother group. The surplus females and pups from the young-mother group were killed by cervical dislocation and decapitation, respectively.

Housing of F2 Mice

At the age of 21 days (at weaning), F2 male and female offspring were separated and housed in groups of 10 in 35.5 x 23.5 x 18.5 cm plastic cages with wood shavings as bedding. Bedding was changed weekly. Mice were checked once per day, 7 days a week until natural death, to assure accurate determinations of dates of death and to preserve bodies for later necropsy. Those animals that were too autolyzed for satisfactory examination or that had been cannibalized by their cage mates were recorded as such, and data on them were used on the lifespan tables, but were excluded from the disease analysis groups. The caged groups were not rearranged after death of any member or after removal of a female or a male from each litter to test for reproductive fitness. Mice were weighed at weaning and every 10 wk starting at the age of 10 wk. Each animal was marked by ear punching/cutting after weaning. Mice were fed a standard laboratory diet and tap water ad libitum, and were maintained on a 14L:10D (0800–2200 h) photoperiod in a temperature-controlled room at 21–23°C.

Fitness Components of F2 Female Mice

At the age of 25 wk, one randomly selected F2 female (if available) from litters 1, 2, and 3 of each F1 dam (litter 1, 12 young and 12 old F1 dams; litter 2, 9 young and 9 old F1 dams; litter 3, 5 young and 5 old F1 dams) was housed in a 26.5 x 20.5 x 13.5 cm plastic cage with a randomly selected 10- to 12-wk-old hybrid male for the rest of her reproductive life, or until the male reached the age of 45 wk, at which time the male was replaced by another 10- to 12-wk-old hybrid male. Cessation of a female's reproductive life was defined as the age of the last parturition following which no more offspring were born for 3 mo. After this 3-mo period, each cohabiting male was housed with a female of 10–12 wk of age to ascertain whether he was still fertile. The F2 females were examined once daily to determine the day of parturition and to record litter size and gender of pups at birth of each consecutive litter. At weaning, third-generation (F3) offspring were weighed. After cessation of reproductive life, females were housed in groups of five in 26.5 x 20.5 x 13.5 cm plastic cages under the same light:dark cycle and temperature conditions as their virgin siblings.

Fitness Components of F2 Male Mice

Reproductive fitness of F2 males was tested when males reached the age of 1 yr (52 wk). At this time, one randomly selected F2 male (if available) from litters 1, 2, and 3 of each F1 dam (litter 1, 12 young and 12 old F1 dams; litter 2, 9 young and 9 old F1 dams; litter 3, 5 young and 5 old F1 dams) was housed in a 26.5 x 20.5 x 13.5 cm plastic cage with a randomly selected 10- to 12-wk-old hybrid female for the rest of his life. When the female was 40 wk old, she was removed from the cage and replaced by another 10- to 12-wk-old female. Females from infertile couples were housed with a 10- to 12-wk-old hybrid male to test their fertility 21 days after females reached the age of 40 wk or after the death of their respective partner. Day of parturition, litter size, gender of pups at birth, and body weight of F3 pups at weaning were recorded in each consecutive litter.

Histological Analysis

At death, necropsies were performed on all F2 mice except those that were too decomposed or eaten. Specimens of morphologically abnormal organs, tumors, or diseased areas were taken for histological examination. Samples were fixed and stored in a 10% formaldehyde solution. Histological preparations were routinely prepared by paraffin embedding and hematoxylin-eosin staining.

Statistical Analysis

Mixed-effects (some effects are random and some are fixed) nested designs of analysis of variance were used for comparisons of means. Nested designs were applied to control the potential correlation among offspring from a particular F2 female or F2 male and to avoid spurious inflation of the sample size [28]. In particular, F2 mothers and F2 fathers were nested within litter rank (i.e., litter sequence or the number of the litter within the series of litters produced by a particular mother) produced by F1 mothers, and this variable in turn was nested within F1 mothers. A Kolmogorov-Smirnov one-sample test was used to check whether variables were normally distributed. When the normality assumption was violated, logarithmic (body weight and between-labor interval) or square root (for counts) transformation of the variable was applied to induce normality. Differences in sex ratio (percentages of males), proportion of litters with at least one newborn pup cannibalized, and percentage of preweaning deaths between groups were tested using automated binomial logistic regression based on forward stepwise variable selection. A one-sample binomial test was used to test the null hypothesis that the probability of being male in each group was 0.5. Cox regression models were fitted taking into account the potential correlation among siblings (i.e., standard errors were adjusted for clustering individuals on their respective mothers) to examine the effect of several continuous and categorical independent variables on the survival of F2 mice. A two-sided Pearson chi-square test with Yates continuity correction was used for comparisons of frequencies in 2 x 2 contingency tables. Unless otherwise indicated, values given are means ± SEM. Significance was defined at P ≤ 0.05. The analyses were carried out using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL) and the Stata Statistical Software, Release 8.2, 2004 (Stata Corporation, College Station, TX).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 shows both the total number and age of fertile F1 dams at each parturition as well as the sample size of F2 male and female offspring at birth and at weaning. As litter size and sex ratio of pups from young females were matched with litter size, and sex ratio was obtained in the old-mother group on Day 3 after birth, the number of fertile F1 dams (third column) and number of pups at weaning (last two columns) from the young-mother group are exactly the same as those exhibited by the old-mother group.


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  		TABLE 1. Reproductive outcome of young and old F1 female mice paired with males for the first time at the age of 10 (young mothers) and 51 (old mothers) wk, respectively

Reproductive Fitness of F2 Females

Time to pregnancy, as estimated on the basis of between-parturition interval, was not significantly affected by age group (27.4 ± 0.6 days, n = 245 in the young-mother group vs. 27.2 ± 0.5 days, n = 257 in the old-mother group; Fig. 1A). Likewise, no significant effect was found for age group on body weight of F3 pups at weaning (11.0 ± 0.06 g, n = 1360 in the young-mother group vs. 11.0 ± 0.05 g, n = 1366 in the old-mother group; Fig. 1B), litter size at birth (6.2 ± 0.2 pups in the young-mother group vs. 5.7 ± 0.2 pups in the old-mother group; Fig. 2A), litter size at weaning (5.6 ± 0.2 pups in the young-mother group vs. 5.3 ± 0.2 pups in the old-mother group; Fig. 2B), percentage of preweaning deaths (11.1%, 169/1529 in the young-mother group and 7.2%, 107/1473 in the old-mother group), and percentage of litters with at least one pup cannibalized (22.4%, 55/245 in the young-mother group vs. 18.3%, 47/257 in the old-mother group).



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  		FIG. 1. Effect of maternal age at birth and rank of litters produced by F2 females on between-parturition interval (A) and body weight of F3 pups at weaning (B). Error bars are ± SEM. Some points are devoid of error bars because the SEMs were too small to be drawn



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  		FIG. 2. Effect of maternal age at birth and rank of litters produced F2 females on litter size at birth (A) and at weaning (B). Error bars are ± SEM

In contrast, litter rank of litters produced by F2 females had a significant effect on between-parturition interval (P ≤ 0.0005; Fig. 1A), body weight of F3 pups (P ≤ 0.0005; Fig. 1B), litter size at birth (P ≤ 0.0005; Fig. 2A), litter size at weaning (P ≤ 0.0005; Fig. 2B), percentage of litters with at least one pup cannibalized (P ≤ 0.0005; Fig. 3A), and percentage of preweaning deaths (P ≤ 0.0005; Fig. 3B). Body weight of F3 pups at weaning was also significantly affected by gender of pups (11.3 ± 0.06 g in males vs. 10.8 ± 0.05 in females; P ≤ 0.0005) and the covariate litter size at weaning (P ≤ 0.0005; regression coefficient ± standard error = –0.304 ± 0.018).



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  		FIG. 3. Effect of rank of litters produced by F2 females on percentage of litters with at least one pup cannibalized (A) and percentage of preweaning deaths (B)

Sex ratio (percentage of males) of offspring at birth (49.8%, 762/1529 in the young-mother group vs. 43.7%, 643/1473 in the old-mother group; P ≤ 0.001) and at weaning (49.9%, 679/1360 in the young-mother group vs. 43.8%, 598/1366 in the old-mother group; P ≤ 0.001) was significantly lower in the old-mother group. In this group, the percentage of males was significantly (P ≤ 0.0005) lower than 50% both at birth and at weaning. In contrast, in the young-mother group, the percentage of males at birth and at weaning was not significantly different from 50%. Rank of litters produced by F1 and F2 females and litter size at birth did not affect the sex ratio of offspring at birth and at weaning. Likewise, preweaning mortality was not dependent on gender of pups (male mortality, 49.1%, 83 males out of 169 dead pups in the young-mother group; and 42.1%, 45 males out of 107 dead pups in the old-mother group). No pups with morphological abnormalities were found in the old-mother group. The only abnormality observed was a stillborn, abrachian, young-mother female pup lacking her right foreleg.

Whereas the period of time between the end of female reproductive life and death was significantly (P ≤ 0.01) lower in the old-mother group (21.5 ± 4.4 wk vs. 38.3 ± 4.7 wk in the young-mother group), no significant effect was observed for delayed motherhood on time at which female offspring ceased their reproductive life (at the age of 63.8 ± 4.4 wk in the young-mother group vs. 60.8 ± 4.2 wk in the old-mother group), total number of litters produced (247 litters, 9.9 ± 0.6 litters/dam in the young-mother group vs. 257 litters, 10.3 ± 0.7 litters/dam in the old-mother group), frequency of litters (one litter every 3.9 ± 0.1 wk in the young-mother group vs. 3.8 ± 0.1 wk in the old-mother group), and total number of offspring born (1529 pups, 61.2 ± 4.5 pups/dam in the young-mother group vs. 1473 pups, 58.9 ± 4.0 pups/dam in the old-mother group).

All the siring males were still fertile, as estimated on the basis of their reproductive performance when housed with a 10- to 12-wk-old female, after their respective females either died or had ceased their reproductive life.

Reproductive Fitness of F2 Males

Between-parturition interval (27.5 ± 0.6 days, n = 225 in the young-mother group vs. 26.2 ± 0.5 days, n = 206 in the old-mother group; Fig. 4A), body weight of F3 pups at weaning (10.5 ± 0.04 g in the young-mother group vs. 10.7 ± 0.04 g in the old-mother group; Fig. 4B), litter size at birth (9.1 ± 0.2 pups in the young-mother group vs. 9.6 ± 0.2 pups in the old-mother group; Fig. 5A), litter size at weaning (8.7 ± 0.2 pups in the young-mother group vs. 8.9 ± 0.2 pups in the old-mother group; Fig. 5B), preweaning mortality (4.0%, 81/2030 in the young-mother group vs. 6.3%, 124/1956 in the old-mother group), and percentage of litters with at least one pup cannibalized (1.8%, 4/225 in the young-mother group vs. 1.9%, 4/206 in the old-mother group) were not significantly affected by age group. Likewise, no significant differences between age groups in percentage of male offspring were observed at birth (49.1%, 996/2030 in the young-mother group vs. 48.4%, 947/1956 in the old-mother group) or at weaning (48.7%, 950/1949 in the young-mother group vs. 48.9%, 896/1832 in the old-mother group). In both age groups, the percentage of male offspring was not significantly different from 50%.



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  		FIG. 4. Effect of maternal age at birth and rank of litters produced by F2 males on between-parturition interval of female mice paired with these males (A) and body weight of F3 pups at weaning (B). Error bars are ± SEM. Some points are devoid of error bars because the SEMs were too small to be drawn



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  		FIG. 5. Effect of maternal age at birth and rank of litters produced by F2 males on litter size at birth (A) and at weaning (B). Error bars are ± SEM. Some points are devoid of error bars because the SEMs were too small to be drawn

Rank of litters produced by F2 males had a significant effect on between-parturition interval (P ≤ 0.005; Fig. 4A), body weight of F3 pups at weaning (P ≤ 0.0005; Fig. 4B), litter size at birth (P ≤ 0.0005; Fig. 5A), and at weaning (P ≤ 0.005; Fig. 5B). Body weight of F3 pups at weaning was also significantly affected by gender of pups (10.9 ± 0.04 g in males vs. 10.3 ± 0.03 in females; P ≤ 0.0005), the covariate's litter size at weaning (P ≤ 0.0005; regression coefficient ± standard error = –0.296 ± 0.012), and rank of litters produced by females paired with F2 males (P ≤ 0.0005; regression coefficient ± standard error = –0.068 ± 0.011).

Percentage of litters with at least one pup cannibalized was negatively correlated with litter size at birth (logistic regression coefficient ± standard error –0.586 ± 0.141; P ≤ 0.0005; Fig. 6A); and preweaning mortality positively correlated with rank of litters produced by females paired with F2 males (logistic regression coefficient ± standard error, 0.089 ± 0.029; P ≤ 0.005; Fig. 6B). Preweaning mortality was not dependent on gender of pups (male mortality: 56.8%, 46 males out of 81 dead pups in the young-mother group and 41.1%, 51 males out of 124 dead pups in the old-mother group). Only two female pups exhibited morphological abnormalities: an abrachian pup from the young-mother group lacking her left hind leg, and a pup from the old-mother group with her left hind leg smaller than the right hind leg and with only three toes. Both female pups were alive at weaning.



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  		FIG. 6. A) Effect of litter size at birth on percentage of litters produced by F2 males with at least one pup cannibalized. Note that litter size at birth = 0 represents those litters in which all the pups were cannibalized at birth. B) Effect of rank of litters produced by females paired by F2 males on percentage of preweaning deaths

There was no significant effect of delayed motherhood on time at which F2 males ceased their reproductive life (at the age of 90.1 ± 4.2 wk in the young-mother group vs. 87.7 ± 3.2 wk in the old-mother group), period of time between the end of reproductive life and death (9.2 ± 2.6 wk in the young-mother group vs. 10.4± 2.8 wk in the old-mother group), total number of litters produced (225 litters, 9.8 ± 1.1 litters/dam in the young-mother group vs. 206 litters, 9.4 ± 0.9 litters/dam in the old-mother group), frequency of litters (one litter every 4.1 ± 0.2 wk in the young-mother group vs. 4.0 ± 0.3 wk in the old-mother group), and total number of offspring born (2030 pups, 88.3 ± 10.4 pups/dam in the young-mother group vs. 1956 pups, 88.9 ± 9.3 pups/dam in the old-mother group).

All the females from infertile couples were fertile, as estimated on the basis of their reproductive performance when housed with a 10- to 12-wk-old male, either when they reached the age of 40 wk and were replaced by another 10- to 12-wk-old hybrid female, or after the death of their respective F2 male.

Longevity of F2 Offspring

Figure 7 shows the cumulative survival functions of young- and old-mother offspring. The curve for mice from the old-mother group was significantly (P ≤ 0.01) lower than that exhibited by mice from the young-mother group (hazard ratio = 1.549 [1.260–2.130, 95% CI]; median life span, 104.0 wk [100.79–107.21 wk, 95% CI] in the young-mother group vs. 92.0 wk [89.48–94.52 wk, 95% CI] in the old-mother group). The mean ages at death of the 10th deciles of survivorship, which affords a reasonably accurate estimate of physiological aging [29], were 128.8 ± 1.0 wk, n = 18, in the old-mother group and 133.4 ± 1.3 wk, n = 18, in the young-mother group; P ≤ 0.01. Gender, litter size at birth, virginity status, rank of litters produced by F1 females, and all the two-way interactions between variables had no significant effect on life expectation survival times of offspring.



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  		FIG. 7. Effect of maternal age at birth on expectation of survival times of F2 offspring

The lower expectation of survival times exhibited by old-mother offspring was associated with lower body weights in virgin and nonvirgin offspring of both sexes, although differences were significant only in the virgin subgroup (Fig. 8). This association, however, was not causative. On the contrary, Cox regression analysis showed that as the value of body weight of virgin offspring increased at the age of 40 wk (the point at which the body weight of young- and old-mother offspring started to display a distinct pattern [see Fig. 8]), so did the risk of death (estimated regression coefficient ± standard error = 0.03 ± 0.01; P ≤ 0.0005). As expected, males were significantly (P ≤ 0.0005) heavier than females (39.3 ± 0.2 g vs. 36.2 ± 0.2 g, respectively). The covariate litter size at birth did not affect body weight of offspring.



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  		FIG. 8. Effect of maternal age at birth on body weight of F2 virgin (A and B) and nonvirgin (C and D) female (A and C) and male (B and D) offspring. Error bars are ± SEM. Some points are devoid of error bars because the SEMs were too small to be drawn. *P ≤ 0.05; **P ≤ 0.01 after Bonferroni correction

Histological Analysis

The histological examination of morphologically abnormal organs, tumors, or diseased areas of necropsied F2 mice showed a significantly (P ≤ 0.005) lower incidence of malignant tumors in the old-mother group compared with their young-mother counterparts (Table 2). No significant differences between groups in other tissue conditions were found.


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  		TABLE 2. Effect of maternal age at birth on incidence of tumors and diseases of F2 offspring


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that delayed motherhood of hybrid F1 female mice is associated with a decreased percentage of male F3 offspring at birth and lower expectation of survival times and body weight during adulthood of F2 offspring. Delayed motherhood, however, did not have negative effects on several reproductive fitness variables in either male and female F2 offspring, including the following: between-parturition interval, litter size at birth and at weaning, body weight at weaning and preweaning mortality of F3 pups, percentage of F3 litters with at least one pup cannibalized, and time at which male and female F2 offspring ceased their reproductive life. Although these data agree with the study by Wang and vom Saal [30] showing that litter size produced by CF-1 female mice is not affected by the age of their mothers at pregnancy, the lack of effect of delayed motherhood on offspring's reproductive fitness found in the present study suggests that the differences reported by Wang and vom Saal [30] (delayed puberty in females and diminished reproductive organ weights in males from middle-aged mothers) are irrelevant to overall reproductive fitness, at least in the laboratory. Moreover, our data are at variance with previous epidemiological studies in human beings, suggesting that advanced maternal age at childbirth is associated with sons exhibiting reduced sperm quality [15] and higher risk to be infertile [16], as well as daughters displaying menstrual disorders [17] and reduced ability to achieve pregnancies and carry these to the birth of healthy singletons [18]. Although discrepancies between studies may be explained by intrinsic strain and species differences in reproductive traits, we should bear in mind that whereas we used hybrid mice, Wang and vom Saal [30] used the inbred CF-1 strain, which may concentrate deleterious recessive genes with strong effects on both reproduction and aging. Furthermore, the present study is based on a prospective and controlled design, whereas the epidemiological studies in human beings followed a retrospective, noncontrolled methodology.

We should note that in the present study, F1 females ceased their reproductive life later than F2 females. This difference could be due to the fact F1 females displayed higher hybrid vigor or heterosis (from the mating of C57Bl/ 6JIco females with CBA/JIco males) than F2 females (from the mating of hybrid F1 females with hybrid F1 males). Furthermore, it is important to mention that the age at which F2 females ceased their reproductive life would be analogous to the age at which women from natural fertility populations become sterile (at a mean age of 40–41 yr), which takes place approximately 10 yr before menopause (at a mean age of 50–51 yr) [31].

As mentioned above, the present data show that F2 female mice from the old-mother group produced a lower percentage of male offspring at birth compared with F2 female mice from the young-mother group. Nevertheless, this finding should be interpreted with caution. Literature shows that advanced maternal age at birth is associated with a small but significant decrease in sex ratio in human beings [32–34]. In contrast, in other mammalian species, the effects of maternal age on sex ratio of offspring are variable. Whereas some species exhibit a trend for the sex ratio to decline with maternal age, others show a trend for it to increase or even to exhibit no clear tendency at all [35]. In the mouse, examination of breeding records of 30 [36] and 10 [37] inbred strains has revealed that no generalizations for all strains can be made about the relationship of sex ratio with age of parents. In fact, no consistency in the sign and significance of the correlations between these variables were found. Within this context, it should be noted that in the present study, the proportion of male offspring at birth produced by F1 mothers was not dependent on maternal age (see Table 1). It was only associated with litter rank in such a way that the higher the rank of litter, the lower the percentage of male offspring produced.

The shorter longevity exhibited by F2 offspring from the old-mother group observed in the present study is largely consistent with the results obtained in previous studies in human beings [21, 22] and many invertebrate species [23] showing that offspring from older mothers have shorter lives than offspring from younger mothers. However, in contrast to several studies in human beings [22, 38] and Drosophila melanogaster [23] reporting that maternal age has a much larger influence on daughters than sons, we did not detect gender differences in the effect of maternal age on offspring longevity, nor was there any significant effect of virginity status on lifespan of offspring observed.

It is important to note that the lower life expectancy exhibited by old-mother offspring was associated with decreased body weights during adulthood. Although this association was not causative, this finding endorses the study by Albert et al. [39] using the high mammary tumor C3H/ Sp mouse strain, in which increasing maternal age was accompanied by decreases not only in survival but also in body weight and in the absolute weight and mitotic activity of all lymphoreticular organs (lymph nodes, spleen, and thymus) of offspring at the age of 61/2–7 wk. Likewise, our data are consistent with those of the study by Wang and vom Saal [30], in which grown offspring from middle-aged CF-1 female mice had lower body weight than those produced by young-adult mothers. These changes were associated with lower serum estradiol and different pattern of serum testosterone in middle-aged pregnant mothers between Gestation Day 16 and the last day of pregnancy compared with that of young-adult pregnant females. Such a circumstance let Wang and vom Saal [30] to suggest that these hormones in utero may permanently imprint the function of cells in reproductive organs, the brain, and many other tissues. Furthermore, Wang and vom Saal [30] observed that pups with middle-aged grandmothers were significantly lighter at birth than pups with grandmothers that had become pregnant at early ages (i.e., the negative effect of maternal age on offspring weight passed on to a subsequent generation). Although in the present study the body weights of F3 pups from the old-mother group may have caught up to those exhibited by pups from the young-mother group during preweaning development, we did not detect any transgenerational transmission of body weight traits.

Finally, the lower incidence of malignant tumors exhibited in the present study by offspring from reproductively old mothers may be a direct consequence of the lower survival times observed in this group of mice. Indeed, basic research in both gerontology and oncology has led to an understanding that normal aging and the development of cancer are closely related [40].


   ACKNOWLEDGMENTS
 
We are grateful to Prof. Juan Brines, Department of Pediatrics, Obstetrics, and Gynecology, University of Valencia, for his constant support and helpful discussions on reproductive and developmental issues. We also acknowledge Prof. Santiago Pérez-Hoyos, Epidemiology and Statistics Unit, Valencian School for Health Studies, for performing the survival statistical analyses.


   FOOTNOTES
 
1 Supported by FIS 01/0138 from Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER), and grant BFI2003-04761 from Ministerio de Ciencia y Tecnología, cofinanced by the FEDER. Back

2 Correspondence: Juan J. Tarín, Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibañez 17, 46010 Valencia, Spain. FAX: 34 96 386 4815; tarinjj{at}uv.es Back

Received: 9 December 2004.

First decision: 5 January 2005.

Accepted: 31 January 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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