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Morphometric Analysis of Primordial Follicle Number in Pigtailed Monkey Ovaries: Symmetry and Relationship with Age¹

^a Division of Reproductive Endocrinology, Center for Women's Medicine, Greenville Hospital System,Greenville, South Carolina 29605 ^b Department of Environmental Health, School of Public Health and Community Medicine, and ^c Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, School of Medicine, University of Washington, Seattle, Washington 98105

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We previously described a modern, three-dimensional counting method for determining primordial follicle (PF) numbers in primate ovaries using a combination of fractionator and physical dissector techniques. The purposes of our current study were 1) to apply our method to describe intraindividual differences in PF numbers between ovaries and 2) perform a linear regression analysis of age versus mean PF number per ovary. Ovaries from 16 pigtailed monkeys (Macaca nemestrina) age 0.85–12.5 yr were examined. Both ovaries were available from 11 subjects. The difference between ovaries ranged from 2% to 22% (mean ± SD, 10 ± 7%) and was not statistically significant. Regression analysis of data from all 16 subjects displayed a log-linear relationship according to the equation log N_a = 4.8542 - 0.0714(age) where N_a is the number of PF at a given chronological age. The fit for this model was highly significant (r² = 0.73, p 0.0001). Extrapolation of the model suggests that there are 71 483 PF in each ovary at the time of birth. We conclude that right and left ovaries differ little and that PF numbers follow a log-linear rate of decline during the reproductive years in this species.

	INTRODUCTION

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The determination of oocyte numbers within the primate ovary has been paramount to understanding the physiology of reproductive aging. Earlier work using human [1, 2] and monkey [3] ovaries demonstrated peak germ cell numbers antenatally, with massive follicular atresia beginning shortly thereafter. Diminution in follicle and, therefore, oocyte numbers occurs through gonadotropin-independent processes, since complete maturation of the hypothalamic-pituitary-ovarian axis does not occur until puberty, at which point less than 10% of the original gamete endowment are available for procreation [1, 4]. It is the fate of these remaining oocytes that has prompted renewed interest in primate research for extrapolation to an aging, human, "baby boom" population.

Previous investigations of follicle numbers in primate ovaries have utilized biased, model-based strategies that require one to make assumptions regarding structure. These assumptions, in turn, necessitate the use of correction factors in order to arrive at reasonable numerical estimates. We [5] have previously described a counting procedure based on the new physical dissector and fractionator stereology tools [6]. These tools combine number-weighted counting probes and systematic random sampling for unbiased estimation of the total number of objects of interest within a larger structure, without the need for correction factors. Moreover, the total number estimate is a much more biologically relevant measure than numerical density, which may be subject to anisotropic (i.e., nonuniform) distributions and differential volume changes during tissue processing [7, 8].

Histologic classification of growing follicles has proven problematic for past experiments dealing with estimation of follicle numbers. Various descriptive definitions differentiating the stages of follicle evolution from the quiescent, primordial stage to the antral, preovulatory stage have been implemented, with significant overlap and subjectivity. The lack of consistency from investigation to investigation has necessitated reclassification of existing data sets without repeat microscopic examination. The ultimate purpose in doing so was to pool data from several sources for more meaningful statistical significance in a single meta-analysis. While the focus of several of these studies was to better characterize follicle development, our purpose centers, instead, on future reproductive potential as represented by the reserve number of primordial follicles (PF). Preceding investigations demonstrated that the vast majority (approximately 80–95%) of follicles at any chronological age are nongrowing PF found in the outer cortical regions [2, 9–11]. Since they may easily be identified and do not fluctuate in number throughout the menstrual cycle [12], they are the sole consideration in this analysis of ovarian reserve.

The hypotheses to be tested in this investigation were that 1) PF numbers decrease in a log-linear fashion with time during reproductive years in the primate and 2) PF are equally distributed between left and right ovaries from the same individual.

	MATERIALS AND METHODS

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Ovariectomy was performed on 16 feral- or colony-born, female pigtailed monkeys (Macaca nemestrina), ages 0.85–12.50 yr, scheduled to undergo abdominal surgery as part of other study protocols (NIH Grant AI-22082 and University of Washington, Division of Research Resources Grant RR-00166). All animals were housed in the University of Washington Regional Primate Research Center and were free of any chronic disease or reproductive abnormality. All procedures met the prior approval of the Institutional Animal Care Committee. Bilateral ovariectomy was performed in 11 cases, while unilateral ovariectomy was performed in 5 cases. In all subjects, both ovaries were intact at the time of surgery.

A PF was defined as a single oocyte surrounded by a layer of granulosa cells, at least half of which were flattened, corresponding to Block's nongrowing follicles [4] and Lintern-Moore's category B and B/C follicles [13]. Processing of ovaries and counting of PF were performed as previously described [5]. In short, after fixation for a minimum of 7 days in 10% formalin, ovaries were embedded in agar and cut into 1.13-mm slabs. Using the fractionator method of Gundersen and colleagues [6, 14], every other slab was dehydrated in an ethanol series and embedded in glycolmethacrylate. The resulting blocks were serially sectioned at 20- or 30-µm intervals, with sections sequentially placed on acid-washed glass slides. After drying overnight in a 70°C oven, sections were stained with methylene blue-Azure II and mounted with coverslips. Systematic placement of physical dissectors [15] was performed microscopically by a single investigator (JC) who was blinded to the age of the monkeys. Unbiased estimation of numerical density (N_v), volume (V), and total number (N) of PF within each ovary was performed as previously described [5].

The data are expressed as the total number of PF per ovary. The sampling scheme was analyzed according to our previously described methods to determine that the greatest contributor to the observed variance was the true biological variation [5]. The difference between the two sides within the same individual was analyzed using a chi-square test, with mean ± SD percentage difference calculated for the entire group.

For the relationship of age and PF number, the basic statistical unit was considered to be the mean number of PF per ovary per individual. Mean PF numbers were transformed logarithmically prior to regression analysis to avoid heteroscedasticity. A single investigational model was tested:

where N_a = the mean PF count per ovary per individual at a given chronological age (a); N_o = the mean PF count per ovary per individual at birth; R₁ = a constant rate of decay.

This model was chosen after an earlier analysis of human follicle populations by a prior investigator showed excellent fit using this equation [17]. This same analysis demonstrated improved fit for a bi-exponential model, but because of our limited sample size and the age of our subjects, we chose the simpler formula (see Discussion). Overall quality of the model was judged based on the residual sum of squares, the plot of the residuals, and calculation of the coefficient of determination (r²) using SAS 6.07 software (SAS Institute, Inc., Cary, NC).

	RESULTS

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Intraindividual Symmetry

Age and PF numbers for subjects studied are displayed in Table 1. The difference between sides ranged from 2% to 22% (mean ± SD, 10 ± 7%), with no statistical difference appreciated (p 0.05). Given these values, we had an overall power of 98% to detect a difference (alpha = 0.05).

Regression Model

A scatterplot of log PF versus age is shown in Figure 1. The superimposed regression equation of log N_a = 4.855 - 0.0715(age) shows an excellent fit, with r² = 0.73 (p 0.0001). According to this equation, the mean number of PF per ovary for pigtailed monkeys at the time of birth (N_o) is 71 614. The time for complete exhaustion of the stock of PF is 68 yr.

View larger version (14K): [in this window] [in a new window]   FIG. 1. A scatterplot of log PF versus age (years). The solid line represents the regression line log PF = 4.8542 - 0.0714(age). The dashed lines delineate the 95% confidence interval for the regression line. The coefficient of determination (r2) is 0.73 (p 0.0001).

	DISCUSSION

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This investigation successfully integrated modern stereologic techniques to accurately determine a rate of PF decline in pigtailed monkeys during their premenopausal years. Similar earlier reports in monkeys [3, 10, 18] and humans [1, 9, 12, 13, 17, 19–23] relied on two-dimensional counting of histologic profiles, a technique that has proven problematic in a variety of organ systems. Single histology sections represent volume-weighted, not number-weighted probes; that is, larger objects produce more section profiles, which in turn tend to be encountered and counted more frequently using the former technique. Therefore, estimates based on histologic profiles require model-based correction factors of unknown and unproven validity to convert profile counts into "meaningful" number estimates. Several investigators have endeavored to calculate total PF numbers by using more modern stereologic methods, but they have failed to account for basic technical aspects such as tissue shrinkage [2, 24], which may be as great as 33.4% using common methods of preparation [4].

We further chose to avoid ambiguities of past reports by focusing on the quiescent PF, rather than attempting to classify all follicles observed in the ovarian specimens. Alternatively, others have sought to count all follicles and categorize them based on their overall diameter [11], or according to the absolute number of granulosa cell layers on the largest sections [10, 13]. Both of these scenarios require observers to subjectively make decisions about three-dimensional structure based on two-dimensional profiles that fail to accurately reflect the uneven distribution and asymmetry of cells in vivo. Furthermore, the percentage of growing follicles present at various stages of development appears to fluctuate throughout the menstrual cycle, making counting and classification cycle dependent [10, 11, 18]. Our scientific question of interest concerned the reproductive reserve of the animal at a given chronological age, rather than characterization of follicle development as it relates to age and total follicle number. Since PF are the storage form of female gametes, are the most abundant follicle type, do not fluctuate with menstrual cycle stage, and may be unambiguously identified, they allow the most accurate and unbiased assessment of reproductive potential.

The similarity in PF estimates between ovaries from the same individual is not surprising. On the basis of an alternating pattern of ovulation from one side to the other in consecutive cycles [25], one would expect to find nearly identical factors affecting overall numbers: namely, similar endowments at birth and similar rates of follicle loss through growth and atresia. The statistical power of our own data set adds support to the findings of past investigators in humans [9, 19] and rhesus monkeys [10, 11, 18]. Because we found no statistically significant difference between sides, and found the mean percentage difference to be acceptably small, we included in the table of mean values the follicle data from the five subjects for which only one ovary was available, understanding that we were introducing greater variance for those figures. Despite our findings, we feel that greater sample sizes must be examined using uniform methods before one draws definitive conclusions regarding left-right symmetry.

The regression analysis revealed a strong log-linear correlation between age and PF number. This finding is similar to those of Faddy and colleagues [17, 20], who formulated equations based on larger, combined data sets from several different studies. What differs is the failure of our data to show any point of accelerated PF loss as our subjects aged, as the others discovered in their human studies. This may, in part, be due to the limited sample size used in our analysis. Rather than this statistical explanation, a more physiologic reconciliation of these differences emerges if one considers the comparative biology among primate species. Using the most replete data set from humans [20], one sees accelerated PF loss at approximately 38 yr of age, or after roughly three fourths of a woman's reproductive life span. Although the data are scarce, it appears from the work of Graham et al. [26] that the ovaries of pigtailed monkeys contain few or no follicles at age 20 yr, roughly equating this time with human menopause. If the two species follow similar patterns of PF decline, we may expect to see an inflection in the log-linear rate of loss at approximately 15–16 yr of age, i.e., beyond the chronological ages of the animals available for this study. Failure to study subjects beyond this age resulted in an unnatural projected age of complete follicle exhaustion (68 yr), not unlike the 70-yr mark that Faddy et al. [20] calculated when applying a first-order model to a human data set that included ovaries from women up to age 50. Although we chose not to test any more sophisticated models, and it is unlikely that we would have achieved a better fit had we done so, the future inclusion of subjects more than 15 yr of age may warrant such an analysis.

In summary, unbiased stereologic estimates of PF number in pigtailed monkeys demonstrate relative symmetry between left and right ovaries from the same individual. Furthermore, a continuous log-linear rate of PF loss occurs throughout peak reproductive years, similar to that seen in humans. Future application of our technique in both human and nonhuman primates offers a clear advantage in accuracy over previous methodologies.

	ACKNOWLEDGMENTS

 
The authors wish to thank Ms. Dawn Blackhurst for her assistance with the statistical analysis in this report.

	FOOTNOTES

 
¹ Supported by a grant from Serono Laboratories, Inc., Norwell, MA.

² Correspondence: Paul B. Miller, 890 W. Faris Road, Suite 470, Greenville, SC 29605. FAX: 864 455 8489; pmiller{at}ghsms.ghs.org

Accepted: March 30, 1999.

Received: October 19, 1998.

	REFERENCES

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