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Departments of Pediatrics (M.M., V.P.) and the Reproductive Sciences Program (M.M., T.L.S., V.P.), and the Center for Statistical Consultation and Research (K.W.), University of Michigan, Ann Arbor, Michigan 48109; and Division of Animal and Veterinary Sciences, West Virginia University (E.K.I.), Morgantown, West Virginia 26506
Address all correspondence and requests for reprints to: Dr. Vasantha Padmanabhan, Department of Pediatrics and Reproductive Sciences Program, University of Michigan, 300 North Ingalls Building, Room 1109 Southwest, Ann Arbor, Michigan 48109-0404. E-mail: vasantha{at}umich.edu.
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Introduction |
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Some believe that androgen excess early in life may provide a hormonal insult that results in manifestation of PCOS in adulthood (7, 17). For instance, polycystic ovarian morphology is highly associated with conditions in which the fetus has been exposed to high amounts of sex steroids before birth. Women with classical 21-hydroxylase deficiency mimic PCOS, exhibiting anovulation, ovarian hyperandrogenism, and LH hypersecretion (18). Animal models serve as a good resource for understanding the developmental origin of PCOS. Prenatal testosterone (T)-treated rhesus monkeys have evolved into an excellent model for understanding the etiology of PCOS (19), although the prohibitive cost of subhuman primates poses some constraints. In recent years the sheep has evolved as an alternative model for understanding the etiology of PCOS. Prenatal T-treated sheep, like women with PCOS and prenatal T-treated monkeys, manifest anovulatory infertility (20, 21), hyperinsulinemia (22), hypergonadotropism (23, 24, 25), neuroendocrine feedback defects (24, 25, 26, 27, 28), functional hyperandrogenism (29, 30, 31), and polycystic ovaries (29). Therefore, sheep provide an excellent resource for probing the mechanisms and understanding the developmental origin of the PCOS phenotype.
From an ovarian perspective, the polycystic ovarian morphology seen in women with PCOS and the prenatal T-treated sheep may be the result of increased follicular recruitment and/or, alternatively, the result of follicular persistence from failure to regress. Recently, we found decreased numbers of primordial and increased numbers of primary, preantral, and antral follicles in 140-d-old prenatal T-treated ovine fetuses (32). Studies that addressed follicular recruitment in women with PCOS, however, have yielded conflicting results; only one (33) of three studies (33, 34, 35) demonstrated increased recruitment. Such differences may relate to the heterogeneity of PCOS in the women studied or, alternatively, to the site and extent of ovarian tissue biopsy. Follicular arrest is believed to be a contributing factor in the development of polycystic morphology in women with PCOS (36, 37).
The primary goal of this study was to determine whether follicular persistence contributes to the development of polycystic ovarian morphology in prenatally T-treated sheep. As discussed in a recent review (30), prenatally T-treated sheep are particularly useful for understanding the mechanisms underlying follicular persistence through sequential ultrasonography. The second goal of the study was to determine whether cyclic progesterone (P) treatment will help overcome the ovarian deficits. The rationale for choosing cyclic P treatment stems from clinical use of gonadal steroids in women with PCOS (14, 16), whose phenotype the prenatal T females resemble. Treatment with P and estrogen has been found to be beneficial in some anovulatory women with PCOS, leading to initiation of normal follicular development (15, 16). Successful use of P to overcome cystic follicles and anovulatory infertility in cattle (38, 39) provides additional rationale, although the cystic follicular condition in cattle differs from ovarian follicular morphology in PCOS women in the increased size of follicles (40).
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Materials and Methods |
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Experimental design
The study design is summarized in Fig. 1
and comprised three study periods: the first breeding season [period 1 (P1)], the anestrous season after the first breeding season (P2), and the second breeding season (P3). In P1, twice-weekly blood samples were obtained from 16 C and 14 T60 (prenatally T-treated from d 30–90 of pregnancy) sheep beginning at 6–8 wk of age to determine the timing of puberty and maintenance of cyclicity during the first breeding season. To obtain a preliminary assessment of changes in follicular dynamics, eight C and 14 T60 females underwent transrectal ovarian ultrasonography from the day of estrus (based on ram markings) for 8 d in late October-November 2002 during the first breeding season. During P2 (anestrus; April-August, 2003), seven C and six T60 sheep were implanted sc either in the axillary or inner thigh region with a controlled internal drug-releasing device (CIDR; 0.3 g P) that had the wings cut off (InterAg, Hamilton, New Zealand) for 13–14 d every 17 d to simulate plasma P patterns of normal estrous cycles observed during the breeding season. These animals are referred to from now as CP and TP, respectively. Nine C and eight T60 ewes did not receive cyclic P implants. Empty implants or sham procedures were not used, because previous studies failed to show effects of these manipulations on LH or P secretion (42). Twice-weekly blood samples were collected from all CIDR-implanted sheep during anestrus and a subset of unimplanted sheep (six C and eight T60) to confirm patterns and plasma P increases achieved in CIDR-implanted sheep.
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(PGF2; 5 mg/ml; Lutalyse, Pfizer Animal Health, Kalamazoo, MI) given 11 d apart. Beginning from the day of the second injection, ovarian ultrasonography was carried out daily on these sheep. After the initial 7 d, because of the lack of differences between C and CP in follicular dynamics and twice-weekly progestogenic cycles, only a subset of C (five of nine) and CP (three of seven) ewes and all T60 (eight of eight) and TP (five of six; one died at the beginning of second breeding season) ewes were scanned for a minimum of 18 [if ovulation and corpora lutea (CL) were detected] to a maximum of 21 d. Data from CP and C groups were combined to form a single C group for yr 2.
Ultrasonography
Transrectal ovarian ultrasonography was performed using an Aloka 500 scanner (Aloka Co. Ltd., Wallingford, CT) fitted with a 7.5-MHz transducer as reported previously (43). Sheep were restrained in a chute during ultrasonography. A coating of carboxymethyl cellulose (Sigma-Aldrich Corp., St. Louis, MO) as a 3.5% gel was applied to the probe as a coupling medium and to provide lubrication during probe insertion. After careful insertion of the probe into the rectum, an image of uterine horns and bladder was observed. Then the probe was rotated to scan the ovaries. Follicles with antral diameter of 2 mm or more and CL were identified and measured. The sizes and relative positions of the follicles and CL were sketched on ovarian charts and were used to assess daily changes in follicular dynamics. Thermal prints and digital video images of the ovarian scans were obtained to document follicular and luteal changes. Two investigators jointly performed ultrasonography throughout the study to avoid subjectivity of measures. Follicles were tracked across successive days using the CL in cycling females and/or the largest follicles in anovulatory T60 females as landmarks. Daily blood samples were collected from these sheep during the scanning period to monitor plasma P concentrations, which were used to confirm CL presence and function. All procedures were approved by the institutional animal care and use committee of University of Michigan and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
RIA
Plasma P concentrations were measured with a well-validated assay using a solid phase kit (Coat-a-Count P; Diagnostic Products Corp., Los Angeles, CA) as previously described (44). Duplicate unextracted plasma samples of 100 µl were used in the assay. The sensitivity of this assay was 0.025 ± 0.003 ng/ml (n = 49 assays; mean ± SEM). The intraassay coefficients of variation, based on three quality control pools measuring 0.13 ± 0.004, 1.97 ± 0.04, and 13.47 ± 0.18 ng/ml, were 12.6 ± 1.3%, 7.4 ± 0.6%, and 7.9 ± 0.6%, respectively. The interassay coefficients of variation for the same quality control pools were 18.1%, 14.7%, and 9.4%, respectively.
Statistical analysis
Age at puberty was defined as the first day of the first progestogenic cycle and was analyzed by ANOVA. A progestogenic cycle was defined as a plasma P concentration greater than or equal to the baseline plus twice the assay sensitivity that remained at 0.5 ng/ml or more for at least two consecutive twice-weekly time points. The duration of each progestogenic cycle was calculated from the day of P rise greater than or equal to baseline plus twice assay sensitivity to the day when the P concentration fell below this value. A short luteal cycle was defined as a progestogenic cycle with duration less than 2 SD below the mean duration of the control cycles. A subluteal cycle was defined as a progestogenic cycle with peak plasma P concentration less than 2 SD below the mean peak P concentration of control cycles. Treatment effects on proportions of ewes with short luteal and subluteal cycles in T60 and TP animals were analyzed by Fisher’s exact test. The effect of prenatal T treatment on duration and peak P was analyzed for each year using a one-way ANOVA, and the effect of prenatal T on number of cycles was analyzed using a Poisson regression using generalized estimating equations (45) to account for the correlations among observations of the same animal.
For studying follicular dynamics, all follicles 3 mm or larger and observed for more than 1 d were included in the analysis as reported by Ginther et al. (46) and validated by Schrick et al. (47). Follicles larger than 2 mm and less than 3 mm were counted only for estimation of the total number of follicles, but not for other variables. Follicles were grouped into three size classes (3–4, more than 4–8, and more than 8 mm) that correspond to gonadotropin-dependent recruited follicles selected to become ovulatory size follicles and follicles larger than ovulatory size follicles (48, 49, 50). Duration of follicular presence from 3 mm through growth and regression back to 3 mm was determined for all follicles 3 mm or larger. However, it was not always possible to observe each follicle from the first day on which it achieved a 3-mm size until the last day when it regressed to 3 mm. To obtain the estimated duration of follicle presence from 3 mm to 3 mm for follicles that were 4 mm or larger on the first or last day of scanning, a constant growth or regression rate of 1 mm/d was employed based on previous studies (46, 47). A parametric survival analysis with left- and right-truncated data was also carried out, without assigning specific durations to the follicles that were not observed for the entire 3 mm to 3 mm duration. Follicles were described as persistent if they were observed on the ovary for 12 or more days (51), and their number, size, and duration were computed. Likewise, the number, size, and duration of nonpersistent follicles (present on the ovary for <12 d) were computed. The proportion of ewes that ovulated as well as the size and duration of the ovulatory follicles were calculated for the yr 2 data. The duration of the ovulatory follicles during yr 2 was calculated from the time when they were 3 mm until ovulation and was confirmed by CL formation and plasma P levels above 0.5 ng/ml. Lastly, the number, size, and duration of CL as well as the proportion of ewes with CL were calculated. Daily plasma P measurements were taken into account for determining the presence or absence of a functional CL.
Ultrasonographic data in yr 2 were complete for only five of eight T60 ewes scanned, and therefore, only complete data from five T60 ewes were included in the analysis. We considered the three T60 ewes with incomplete data only for including the qualitative information of the number of T60 ewes that had follicles larger than 8 mm and the presence of CL. The size and duration of follicles were analyzed within the first breeding season (T vs. C) and during the second breeding season using one-way ANOVA with Dunnett’s adjustment for post hoc comparisons (T vs. C and CP; TP vs. C and CP). The number of follicles within each size and size class during the 8-d (yr 1) and 21-d scanning periods (yr 2) for all ewes was analyzed by Poisson regression using as an offset the log of the number of days scanned to take into account the differing number of days scanned for each ewe. All analyses were carried out using SAS for Windows release 9.1.3 (SAS Institute, Inc., Cary, NC).
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Results |
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. The mean ± SE of dates of onset of puberty were September 18, 2002 (±5 d), and September 28, 2002 (±9 d) for C and T60 sheep, respectively, and they did not differ (n = 16 and 14; P = 0.31). The corresponding ages at puberty were 25.5 ± 0.7 and 27.6 ± 1.3 wk, which also were not statistically different (P = 0.15). All the C sheep in yr 1 (16 of 16) and C (nine of nine) and CP (seven of seven) sheep in yr 2 showed repetitive progestogenic cycles. The end of cycles for the first season occurred on February 15, 2003 (±4 d) and February 25, 2003 (±8 d) for C and T60 sheep, respectively, and they did not differ (P = 0.19).
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, top left), but in the other T60 ewes, progestogenic cycles were either absent or severely disrupted (five of 14, 35.71%). In yr 2, of the eight T60 animals, six (75%) showed absent or severe disruption of cycles (P < 0.01; Fig. 3, top right). Of the six TP animals, four of five animals (80%) either cycled normally (three of five) or showed improved cycles (one of five) in yr 2 (one died at the start of second year). The proportion of TP ewes with normal cycles was not different from that of C or CP ewes (P = 0.2381 and 0.1515). The proportion of ewes that had short luteal cycles in T60 group (one of 14) did not differ from C group (P > 0.05). The proportion of ewes with subluteal cycles in yr 1 was higher in T60 (six of 14) than in C sheep (zero of 16; Fig. 3; P = 0.0051). In yr 2, the proportions of ewes with subluteal cycles in T60 (based on those that cycled) and TP sheep did not differ and were only marginally higher than those in controls (P = 0.0783; Fig. 3).
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). There was no difference in peak plasma P concentrations achieved in T60 and C sheep (P > 0.05; C: 5.30 ± 0.22 vs. T60: 4.73 ± 0.81 ng/ml). In the anestrous season, as expected, neither C nor T60 sheep presented any progestogenic cycles. In CP and TP sheep, CIDR implantation led to progestogenic increases in duration similar to control cycles (cycle duration in days: C, 13.8 ± 0.8; CP, 14.2 ± 0.9; TP, 13.9 ± 0.7). The mean peak P plasma concentrations achieved during anestrus were only 28% and 23% in C and T60 groups, respectively, of the concentrations found in control sheep during the first breeding season. There were no differences in mean peak P concentrations achieved between CP and TP sheep (P = 0.19).
In the second breeding season, as in yr 1, the number of progestogenic cycles was reduced (Fig. 3; P = 0.0090), and cycle duration was longer (Fig. 3; P = 0.0341) in T60 sheep compared with C sheep. There was no difference in peak concentrations of P between the two groups (P > 0.05). Unlike T60 sheep, the number and duration of progestogenic cycles of TP sheep during the second breeding season were similar to those of C sheep, indicating recovery of cyclic function after cyclic P treatment (Fig. 3; P > 0.05).
Follicular dynamics in C, T60, CP, and TP sheep
Figure 4 summarizes patterns of follicular dynamics from two C and two T60 females during the 8 d of scanning in yr 1. The sizes of the largest follicles and their durations were greater in T60 females compared with C females. Figure 5 shows follicular dynamics from two each of C, CP, T60, and TP females during 21 d of scanning in yr 2. Similar to yr 1, the sizes of the largest follicles and their durations were increased in T60 sheep compared with the C group. The sizes of the largest follicles and their duration in CP ewes were similar to those in C ewes. The sizes of the largest follicles were larger in some, but not all, TP sheep.
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. The total number of follicles and the number of follicles in the size class larger than 4–8 mm decreased in T60 sheep vs. C sheep in yr 2, but not in yr 1. The number of 3- to 4-mm follicles was lower in T60 sheep compared with C sheep in both years. Only one of the eight C ewes scanned in yr 1 and one of eight C ewes scanned in yr 2 (five C and three CP) had follicles more than 8 mm in size. The proportions of ewes with follicles larger than 8 mm were significantly higher in T60 compared with C ewes in both years (P < 0.01). Thirteen of the 14 T60 animals scanned in yr 1, and five of five T60 animals scanned in yr 2 had follicles more than 8 mm in size. Sixty percent of TP ewes scanned (three of five) had follicles larger than 8 mm. The proportion of TP ewes with follicles larger than 8 mm did not differ from that of C ewes (P > 0.05). Cyclic P treatment increased the total number of follicles, the number of 3- to 4-mm follicles, and the number of follicles larger than 4–8 mm.
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. Overall, follicles stayed longer in T60 than in C sheep in both years (yr 1, P = 0.0003; yr 2, P = 0.0058). In general, this was reflected across all size classes. Follicles in the 3- to 4-mm size range stayed longer in T60 than C sheep in yr 2 (P < 0.05), but not in yr 1. In contrast, follicles larger than 4–8 mm stayed longer in T60 compared with C sheep in both years (yr 1, P < 0.0003; yr 2, P = 0.04). Follicles larger than 8 mm remained on the ovaries for approximately 17 d (estimated) in yr 1 and approximately 20 d in yr 2 in T60 animals. Cyclic P treatment failed to reduce follicular duration to that of C females in most classes. Survival analysis found that overall, across all follicular size ranges, the estimated life spans for the follicles of ewes in T60 and TP groups were 1.37 and 1.23 times longer than those for ewes in the control group (P < 0.01).
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. None of eight C sheep in yr 1 and one of eight C sheep in yr 2 had persistent follicles. In contrast, all T60 ewes (14 of 14 in yr 1 and five of five in yr 2) for which complete data were available had persistent follicles (the other three ewes in yr 2 had missing data points, precluding determination of persistence). The proportions of ewes with persistent follicles in T60 (100%) and TP (100%) groups were greater than that in the C group (0% in yr 1 and 12.5% in yr 2; P < 0.01). Cyclic P treatment did not reduce the number of animals with persistent follicles. There was no difference in the number, size, or duration of persistent follicles between T60 and TP sheep.
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. There was no difference in the number of nonpersistent follicles in yr 1 between C and T60 sheep. However, a reduction in number of nonpersistent follicles was evident in T60 sheep in yr 2 (P < 0.01). Cyclic P treatment prevented this decline in nonpersistent follicles in TP sheep (P > 0.05). The nonpersistent follicles of T60 sheep were larger (P < 0.05) in size and remained longer (P < 0.01) in yr 1. There were no differences in the size or duration of nonpersistent follicles among C, T60, and TP sheep in yr 2 (P > 0.05).
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, ovulations were detected in all C ewes (eight of eight) that previously had a luteal phase (presence of CL and plasma P >0.5 ng/ml). Complete ultrasound data were available for five of the eight T60 ewes (due to scanning difficulties, three T60 sheep had incomplete data) and five of six TP ewes (one died before scanning). Only three of the five T60 and 3 of 5 TP sheep had definable luteal phases. None of these 3 (0%) T60 sheep and two of three (66%) TP sheep with a preceding luteal phase had ovulatory follicles during the scanning period. The proportion of ewes with ovulatory follicles in the T60 group was thus lower than that in C sheep (P < 0.01), but the proportion of ewes with ovulations in the TP group was not different from that in C ewes (P = 0.2727). The size (5.6 ± 0.4 and 6.5 ± 1.5 mm) and duration (4.8 ± 0.6 and 5.5 ± 1.5 d) of ovulatory follicles did not differ between C and TP sheep, respectively (P > 0.05).
Proportion of ewes with CL and number of CL
There were no differences in the proportions of ewes with CL between C and T60 groups in yr 1 (100% vs. 78.57%, respectively; P = 0.2727). In yr 2, 100% of C sheep had normal CL, but only 66% of T60 sheep had CL (three T60 ewes had normal CL, one T60 ewe had a luteinized cyst for 3 d; no scan data were available for the remaining two due to scanning difficulties). Eighty percent of TP sheep (four of five) had CL. The number of CL per ewe was reduced in T60 sheep (1.3 ± 0.2 and 0.0 ± 0.0) compared with C sheep (1.9 ± 0.1 and 2.0 ± 0.0) in both yr 1 and 2 (P = 0.0313 and P < 0.0001, respectively). The number of CL per ewe also was reduced in TP sheep (1.0 ± 0) compared with C sheep (P < 0.0001).
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Discussion |
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Effects of prenatal T on reproductive cyclicity and follicular dynamics
Findings from this study using Suffolk sheep extend earlier findings of the effects of excess T during fetal life on reproductive dysfunction in Dorset and Merino sheep (20, 21), rats (52), and monkeys (19) and document that the disruptive effects of prenatal T excess are seen at several levels, such as ovulatory failure, subluteal or absent progestogenic cycles, and disruption of follicular dynamics. The longitudinal nature of the study over a 2-yr period helped document that prenatal T excess results in continued disruption of the reproductive axis similar to that found in two other breeds of sheep (20, 21). However, the pace of this disruption appears to be slower in the Suffolk than in the Dorset (21) breed of sheep. Although 100% of T60 Dorset sheep ceased to cycle in yr 2 of life (21), not all T60 sheep stopped cycling in yr 2. Comparison of progestogenic cycles in Suffolk (this study) and Dorset (21) sheep indicates that the Suffolk breed is less sensitive to prenatal T treatment. It should, however, be noted that the complete loss of cyclicity found in the first study with Dorset sheep was not evident in the second study (28), suggesting that other environmental or experimental factors may also play a role. The variability in the severity of reproductive defects and the timing of onset of reproductive perturbation programmed by prenatal T excess between breeds and within individuals of the same breed highlight the interaction between genetics and environment.
From an ovarian perspective, this is the first study that has addressed in detail the effects of prenatal T excess on follicular dynamics and ovulation. In this regard, the ease with which one can track follicular development in sheep sequentially over multiple days with ultrasonography proved to be a great asset. Our findings provide evidence that prenatal T excess leads to a progressive reduction in total number of follicles larger than 2 mm and an increase in the number and duration of persistent follicles. These findings are indicative of exertion of dominance by the persistent follicle and parallel the reduction in follicle number and prolonged life span of the largest follicle seen in a low P environment (53, 54). Alternatively, the reduction in follicular number seen in yr 2, but not yr 1, may reflect decreased recruitment due to a decline in ovarian reserve. Our previous studies on fetal d 140 provide evidence in support of reduced ovarian reserve and increased (not decreased) recruitment in T60 females (32). Because ultrasonography with the Aloka 500 scanner did not provide the resolution to count smaller follicles, the number estimates are restricted to follicles 2 mm or larger. Morphometry at various time points is required to assess to what extent recruitment differences contribute to the reduced number of emerging follicles. Other studies have reported protection from atresia and increased life span of the penultimate wave of dominant follicles under a low P concentration and increased LH pulse frequency (55) (Devonish, E. H., and E. K. Inskeep, unpublished observations). Low P has been found to reduce atresia of recruited follicles in ewes (56).
In addition to alterations in follicular dynamics, prenatal T excess caused a reduction in the number of ovulations or culminated in anovulation. Oligo- or anovulation is a diagnostic criterion in PCOS women (57), the features of whom the T60 sheep mimic (30, 31). Ovulatory defects in T60 females may be the result of LH surge defects or follicular persistence. Our recent studies found that T60 Suffolk females (same breed used in this study) have delayed and severely dampened LH surges (24). The fact that P treatment restores cyclicity in some animals without correcting follicular persistence in TP animals questions the contributory role of follicular persistence in development of the anovulatory condition. However, follicular environment and oocyte quality are likely to be compromised in persistent follicles. Previous studies indicated that persistent follicles can ovulate (58), but result in reduced pregnancy rates.
Contribution of follicular persistence to development of polycystic ovarian morphology
In previous studies we had documented the presence of polycystic ovaries in T60 females (29) and the possible contribution of increased recruitment in the development of polycystic morphology (32). Findings from this study complement those studies and document that regression failure, the means by which follicular persistence develops, might contribute to development of polycystic morphology. Previous studies in rhesus monkeys, although documenting the impact of prenatal T treatment on the development of polycystic ovarian morphology (30), did not address how polycystic ovarian morphology develops. Follicular arrest is also believed to be the cause of polycystic ovarian development in women with PCOS (36, 37).
Mechanisms by which follicular persistence develops
Previous studies have implicated a low P milieu in the development of persistent follicles [sheep (53, 55) and cattle (54, 59)]. Mechanistically, low P will result in reduced negative feedback and a consequent increase in LH drive (60). Increased LH drive is likely to facilitate increased thecal androgen production (61). Androgens are known facilitators of follicular differentiation (62) and follicular arrest (63). Our findings in T60 females are consistent with this premise. T60 females not only exhibit severe luteal defects, manifested as a higher percentage of subluteal P cycles or absence of cycles, but also have reduced hypothalamic sensitivity to P (27) as well as estradiol feedback (25). An increase in LH frequency is evident in T60 females during both prepubertal and adult life (23, 25, 64). An increase in LH pulse frequency has been implicated in the development of persistent follicles (65). Although low circulating concentrations of androgens have precluded us from comparing circulating androgens in control and T60 females, that T60 females are functionally hyperandrogenic is supported by evidence for increased follicular recruitment (32), follicular persistence (this study), and decreased follicular activin expression (29). The ovarian phenotype is similar to the occurrence of multifollicular ovaries seen in hyperandrogenic states such as PCOS, which are corrected by antiandrogen treatment (13).
An additional contributor to the development of persistent follicles is the hyperinsulinemic condition of the T60 females (22, 31, 66). Hyperinsulinemia can exacerbate follicular persistence by enhancing the LH responsiveness of follicles (67) and increasing intraovarian hyperandrogenism (63).
Role of P in overcoming cycle and follicular defects
Our prediction was that cyclic P treatment would arrest continued disruption of the reproductive axis. There is support in the literature for this premise. For example, P treatment has been used in women with PCOS, whose features prenatal T sheep mimic, to normalize the LH drive, leading to normal follicular development and ovulation (15, 16). Similarly, normal follicular dynamics were restored in cystic cows after P administration (38, 39). However, our prediction proved to be only partially true for T60 females. Cyclic P treatment during anestrus prevented deterioration of some aspects of follicular dynamics, but not others. For example, cyclic P treatment prevented the decline in the number of follicles, but failed to overcome follicular persistence. The restoration of cyclicity in TP sheep in this study without correction of follicular persistence questions the causal role of follicular persistence in the etiology of PCOS.
Incomplete restoration of follicular and cycle dynamics in TP animals may relate to the low levels of P achieved during cyclic P treatment. Cyclic P treatment resulted in plasma P concentrations that were only a third of midluteal P concentrations of control cycles during the breeding season. This may be due to the alternative sites of CIDR implantation or a possible reduction in surface area due to removal of CIDR wings. As discussed previously, a low P milieu supports follicular persistence. Additional studies using higher concentrations of P are required to determine whether complete correction of ovarian deficits can be achieved. In women with PCOS, higher P concentrations than those seen in control women are required for achieving suppression of LH pulse frequency (68). It should be noted that both T60 sheep (27) and women with PCOS (3, 68) have reduced sensitivity to P negative feedback.
Clinical implications
Follicular persistence, when followed by ovulation, has been associated with lower pregnancy rates, reduced developmental competence of oocytes, reduced cleavage rates, and retarded early embryonic growth (58). Studies in prenatally T-treated rhesus monkeys found defects in embryo development after in vitro fertilization (69). A combination of increased follicular recruitment at fetal age (depletion) (32) and later persistent follicular development in adulthood (aging), such as that seen in T60 sheep (this study), can potentially reduce the number of quality oocytes available, thus impairing fertility. A reduction in the number and an increase in the length of progestogenic cycles in T60 ewes, resembling premenopausal changes in reproductive cycles in women (70), can potentially reduce the length of the fertile period.
The findings from this study also have implications for understanding the etiology of PCOS. Follicular arrest is considered to be the underlying cause of the polycystic ovarian morphology in women with PCOS (36, 37, 63). Because sequential monitoring of follicular dynamics has not been performed in women with PCOS, it is not possible to assess whether the so-called arrested follicles reflect follicular persistence or, alternatively, enhanced recruitment. Our findings in T60 females, by sequential monitoring of follicular development, provide evidence for defects in selection and regression as possible mechanisms for polycystic ovarian development. It should be noted that the follicular phenotype of T60 females resembles women with PCOS in certain attributes, but not others. The persistence of follicles in size classes less than the size of preovulatory follicle parallels that in size classes of follicles exhibiting follicular arrest in women with PCOS. Large follicles, such as those seen in T60 sheep, which parallel the large follicles reported in normoandrogenic women (5), have not been reported in women with PCOS. Considering 1) the role that androgens play in the development of follicular cysts (71) and 2) studies involving sequential daily ovarian ultrasonography of hyperandrogenic women with PCOS are not available during various time points in their life, it is unclear whether women with PCOS manifest an ovarian phenotype similar to that of T60 females during the developmental progression of PCOS. From a treatment perspective, our findings indicate beneficial effects of cyclic P.
In summary, findings from this study demonstrate that prenatal treatment of sheep with T adversely affects ovarian follicular development, culminating in follicular persistence and ovulatory and luteal defects. In addition, cyclic P treatment can provide a potential therapeutic option to overcome some of those defects.
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Acknowledgments |
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Footnotes |
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First Published Online December 22, 2005
Abbreviations: C, Control; CIDR, controlled internal drug-releasing device; CL, corpora lutea; P, progesterone; P1, period 1; PCOS, polycystic ovary syndrome; PGF2, prostaglandin F2; T, testosterone; T60, prenatally T treated.
Received October 21, 2005.
Accepted for publication December 15, 2005.
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M. Manikkam, R. C. Thompson, C. Herkimer, K. B. Welch, J. Flak, F. J. Karsch, and V. Padmanabhan Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and Postnatal Gonadotropin Regulation in Sheep Biol Reprod, April 1, 2008; 78(4): 648 - 660. [Abstract] [Full Text] [PDF]
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A. Veiga-Lopez, W. Ye, D.J. Phillips, C. Herkimer, P.G. Knight, and V. Padmanabhan Developmental Programming: Deficits in Reproductive Hormone Dynamics and Ovulatory Outcomes in Prenatal, Testosterone-Treated Sheep Biol Reprod, April 1, 2008; 78(4): 636 - 647. [Abstract] [Full Text] [PDF]
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L. Manneras, S. Cajander, A. Holmang, Z. Seleskovic, T. Lystig, M. Lonn, and E. Stener-Victorin A New Rat Model Exhibiting Both Ovarian and Metabolic Characteristics of Polycystic Ovary Syndrome Endocrinology, August 1, 2007; 148(8): 3781 - 3791. [Abstract] [Full Text] [PDF]
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T. Steckler, M. Manikkam, E. K. Inskeep, and V. Padmanabhan Developmental Programming: Follicular Persistence in Prenatal Testosterone-Treated Sheep Is Not Programmed by Androgenic Actions of Testosterone Endocrinology, July 1, 2007; 148(7): 3532 - 3540. [Abstract] [Full Text] [PDF]
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 A. J. King, N. Bari Olivier, P. S. Mohankumar, J. S. Lee, V. Padmanabhan, and G. D. Fink Hypertension caused by prenatal testosterone excess in female sheep Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1837 - E1841. [Abstract] [Full Text] [PDF]
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M. Savabieasfahani, K. Kannan, O. Astapova, N. P. Evans, and V. Padmanabhan Developmental Programming: Differential Effects of Prenatal Exposure to Bisphenol-A or Methoxychlor on Reproductive Function Endocrinology, December 1, 2006; 147(12): 5956 - 5966. [Abstract] [Full Text] [PDF]
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, C; 
, T60;
, TP. Shown are 1) the percentage of animals with normal cycles, 2) the percentage of animals with subluteal cycles, 3) the total number of progestogenic cycles during each breeding season, and 4) the average duration of progestogenic cycle during each breeding season.
TP. Follicles were counted only once during their life span. As a result, on any given day although more follicles were visualized in T60 animals, newly emerging cohorts of follicles were far fewer, contributing to the lower number.
