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Original Studies |
Department of Obstetrics and Gynecology and The Center For Reproductive Sciences, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, 630 West 168 street, New York, New York 10032. E-mail: mf8{at}columbia.edu
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Abstract |
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Introduction |
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The present study uses a relevant nonhuman primate model to investigate the cyclic endocrine effects imposed by a moderate short-term stress episode in the follicular phase. The stress selected is a 5-day inflammatory/immune-like challenge produced by the administration of endotoxin. Bacterial endotoxin, through the release of endogenous cytokines and other mediators, induces a physiopathological response similar to a bacterial infection (8, 9). Data in several species have shown that acute endotoxin or cytokine administration results both in the activation of the HPA axis and in a rapid decrease in tonic gonadotropin secretion (8, 9, 10, 11, 12, 13, 14). The use of peripheral injections of endotoxin, rather than central administration of interleukin-1 in the present study, insures that physiopathological (rather than pharmacological) amounts of cytokines are released. Because, in nonhuman primate experimentation, restraint procedures may add to the overall stress stimulus, the experiments were performed in unrestrained and untethered monkeys within their cage, so that the data obtained would relate more specifically to the inflammatory stress challenge.
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Materials and Methods |
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Eight adult (BW, 5.0–7.5 kg) female rhesus monkeys (macaca mulatta), showing regular ovulatory cycles, were used in 10 experiments. They were housed in individual cages and in temperature- and light-controlled rooms (23–25 C; lights on, 0730–1930 h). The animals were fed a high-protein daily diet (PNI Feeds Inc., St Louis, MO), supplemented by fruits or vegetables, and had unlimited access to water. Blood samples were obtained by venipuncture (a process to which the animals had previously been habituated). Menstruation was checked for by daily vaginal swabbing.
Experimental protocols
The experimental protocols were conducted in accordance with the NIH guide for the care and use of laboratory animals and approved by the institutional animal care and use committee of Columbia University. The experimental objective was to study the effects of a 5-day inflammatory/immune stress-like episode and activation of the HPA axis on menstrual cyclicity. Experimental protocols were performed between the months of September and April. To simulate an inflammatory/immune stress challenge, lipopolysaccharide (LPS) (W E. coli 055:B5; Difco Laboratories, Detroit, MI) was administered iv twice daily (at 0900 and 1700 h) for 5 days. The lyophilized material was dissolved in saline, and aliquots were kept at -80 C until use. Each injection contained 150 µg in 0.5 mL of saline.
To account for potential individual cyclic endocrine variations, the protocols include a documentation of several menstrual cycles. As controls, the two ovulatory cycles before treatment were documented. The 5-day LPS treatment (n = 10) was started on days 2–8 of the follicular phase (day 1 = menstruation). To monitor possible delayed effects of the treatment, the two post-LPS treatment cycles also were documented. In the two animals in which the experiment was repeated, a minimum of six ovulatory cycles separated the two protocols. To monitor the control cycles and to investigate the effects of LPS treatment on the menstrual cycle, daily blood samples were obtained throughout the control period, during the treatment cycle and during the two post-LPS treatment cycles for LH, FSH, estradiol, and progesterone measurements. To document the HPG and HPA axes responses during LPS treatment, additional blood samples were obtained 3 and 8 h after the morning LPS injection on each day of the 5-day treatment for LH, FSH, estradiol, cortisol, and progesterone measurements. For comparison and to control for circadian fluctuations and possible changes related to meals and the stress of venipuncture, blood samples also were obtained at similar times of day and of the cycle from control cycles (n = 20) for cortisol measurement.
Assays and statistical analysis
Blood samples were centrifuged, and sera were kept at -20 C until assay. Estradiol, progesterone, and cortisol were measured using a solid-phase 125I RIA (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay CVs (coefficients of variation) were 3.0% and 4.0%, and 2.9% and 6.1%, respectively, for estradiol and cortisol. Progesterone measurement required prior extraction with petroleum ether (Aldrich Chemical Co., Milwaukee, WI); the extraction recovery rate was 94.2 ± 2.1%. Intra- and interassay CVs were 4.8 and 9.1%. LH and FSH levels were measured with recombinant homologous RIAs previously described (15). Intra- and interassay CVs were 7.0% and 13.1% and 5.0% and 6.1%, for LH and FSH, respectively.
Cycle parameters (such as the length of the follicular and luteal phase), hormone concentrations, and integrated progesterone values in the luteal phase were compared in the control, LPS-treated, and post-LPS cycles. For luteal progesterone evaluation, the areas under the luteal phase progesterone curves were calculated by trapezoidal analysis. Comparisons between control and experimental cycles were made by multiple ANOVA, followed by the Tukey test. The level of significance was established at P < 0.05.
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Results |
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LPS administration during the early-midfollicular phase resulted
in a significant lengthening of the follicular phase in all animals.
Subsequent analysis revealed that, when applying specific criteria
regarding cycle lengthening (number of days between the end of the LPS
challenge and the estradiol preovulatory peak: <6 days vs.
>10 days) and estradiol levels during the challenge and in the days
following it (no decrease vs. decrease to early follicular
phase levels), the animals could be subdivided into two distinct and
nonoverlapping groups. In group 1 (n = 5), the mean length of the
follicular phase in the LPS-treated cycle was significantly increased
from 10.2 ± 0.2, in control cycle 2, to 30.8 ± 4.3 days
(±SE) (Table 1
). In one
monkey, not included in this mean, LPS induced a 4-month amenorrheic
period followed by an ovulatory cycle. In group 2 (n = 5), the
length of the follicular phase significantly increased but did not
exceed the duration of the LPS treatment (9.7 ± 1.1
vs. 13.6 ± 1.2). There were no differences, in either
group, in the length of the follicular phase between control and the
two post-LPS cycles. However, the monkey from group 1, in which a long
amenorrheic period occurred after treatment, had again a prolonged (4
months) anovulatory period during post-LPS cycle 2. No differences were
observed in the two groups, in regard to the length of the luteal phase
between control, LPS, and post-LPS cycles.
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Daily estradiol concentrations, in response to LPS treatment in
the follicular phase, are compared with control values (upper
panel) in Fig. 1. In group 1
(lower left panel), a marked decrease in estradiol occurred
within 24–48 h of the first LPS injection (except in the long-term
amenorrheic monkey, in which estradiol decreased only at the end of the
5-day LPS treatment). Mean (±SE) estradiol concentrations
were significantly lower on day 5 of LPS, as compared with the morning
of the first LPS day (16.2 ± 6.5 vs. 34.8 ± 5.5
pg/mL). Estradiol concentrations remained suppressed for 8–120 days.
In group 2, estradiol levels remained stationary throughout the 5-day
LPS treatment [25.6 ± 3.9 vs. 26.0 ± 6.5, NS
(not significant)]. After the respective suppression periods in both
groups, estradiol increased in a pattern similar to that seen in the
late follicular phase of the control cycles. Peak estradiol
concentrations in both groups were within the control mean (197.3
± 42.9, controls; 161.6 ± 31.6, group 1; 193.4 ± 32.7,
group 2; NS). Patterns of estradiol increases during the follicular
phase of the post-LPS cycles seemed similar to controls.
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. Of interest, there were significant
differences between control cycles of groups 1 and 2 (group 1,
36.5 ± 1.5; group 2, 47.5 ± 2.6). In group 1, there were no
significant effects of LPS on progesterone during the treatment and two
post-LPS cycles. In contrast, in group 2, luteal progesterone was
significantly decreased, in post-LPS cycle 1, to 36.0 ± 4.4
(P < 0.05 vs. control, LPS-, and post-LPS
cycle 2, NS vs. all cycles in group 1).
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illustrates daily estradiol and
progesterone concentrations throughout the two control, the
LPS-treated, and the two post-LPS cycles in a representative monkey of
group 1 (upper panel) and of group 2 (lower
panel). In monkey 79T50 (group 1), estradiol secretion was
suppressed for 24 days. Integrated progesterone secretion during the
luteal phase for control cycles 1 and 2, LPS cycle, and post-LPS cycles
1 and 2, respectively, were 32.8, 28.7, 37.6, 31.5, and 31.9 in monkey
no. 79T50 (group 1) and 52.3, 48.2, 58.3, 32.0, and 54.0 in monkey no.
85N045 (group 2).
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FSH (upper panel) and LH (lower panel)
concentrations in both groups, measured at hour 3 and hour 8 after each
LPS morning injection during the 5-day treatment, are compared with
mean (±1 SD) concentrations in control cycles
(shaded area) in Fig. 4.
Compared with control values, most LH and FSH values were above the
mean values. On day 2 of LPS treatment, for FSH, 16 of 20 samples were
significantly higher than controls (P < 0.05
vs. control), whereas for LH, 15 of 20 samples were higher
(P < 0.05 vs. control). On days 3–5 of
treatment, all LH and FSH values, except in 1 monkey of group 2, were
above the mean values of control monkeys in the mid-late follicular
phase before the ovulatory surge. After cessation of the LPS treatment,
LH and FSH levels returned to the control follicular phase range. In
the control cycles, both LH and FSH levels started to increase on day 9
of the cycle to reach the preovulatory peak (LH, 7.6 ± 1.1; FSH,
2.4 ± 0.3 ng/mL; mean ± SE) on day 10 of the
cycle. These gonadotropin surges were delayed by LPS treatment for a
varying length of time, according to each group (peak LH, group 1:
5.4 ± 1.1, group 2: 8.3 ± 1.2; peak FSH, group 1: 2.2
± 0.5, group 2: 4.0 ± 0.5).
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Figure 5 illustrates the 5-day
adrenal steroid response to LPS treatment. Because data were similar in
both groups, the results were pooled. Cortisol (upper panel)
was significantly increased at hour 3 after each morning LPS injection.
However, the response decreased over the 5-day period, and on day 5,
the cortisol response at hour 3 was significantly lower than on day 1.
Cortisol was still elevated at hour 8 after LPS but only on day 1.
Cortisol values at 1200 h and 1655 h, on day 1 of LPS
treatment, were significantly higher than in the non-LPS control
follicular phase (0900 h, 26.6 ± 1.8; 1200 h, 20.4 ±
2.4; 1700 h, 12.4 ± 1.3 µg/dL). Progesterone (lower
panel) increased significantly by hour 3 after the first LPS
injection but remained unchanged after subsequent LPS
administration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms that are responsible for the inhibition of estradiol secretion, after endotoxin administration in the follicular phase, remain to be clarified. Because cytokines and endotoxin are known to inhibit pulsatile GnRH and LH release in several species (11, 12, 13, 14, 16), it would be logical to ascribe this effect to a hypothalamic inhibition of the HPG axis by the inflammatory-like stress challenge. Unexpectedly, however, the present data illustrate an inhibition of normal estradiol secretion, in the face of LH concentrations that are higher and not lower than control values. An alternative hypothesis for the disruption of normal estradiol secretion may then be that endotoxin exerts direct effects on ovarian steroid secretion. Studies in the rodent have suggested various actions of endotoxin and cytokines on the gonads (17, 18) and on the process of follicular atresia (19, 20). These compounds also have been shown to influence steroidogenesis (21, 22) and to inhibit LH-stimulated estrogen secretion by granulosa cells (23) and testosterone secretion by Leydig cells (24). Whether an ovarian action could explain our results entirely remains to be demonstrated, because in one of the two groups of animals, inhibition of estradiol secretion persists for a long period after the endotoxin treatment has ended.
The reasons for the increase in tonic LH and FSH secretion during the stress episode remain to be investigated. Although it may be logical to ascribe this increase in LH release, in the ovary-intact animal, to decreased estradiol negative feedback activity, this may not be the case, because LH levels are also increased in the group in which estradiol remains stationary during endotoxin treatment. Rather, we believe that this phenomenon probably reflects a modulatory effect of the estrogenic milieu on the LH response to the stress challenge, possibly specific to the primate. Indeed, although we have demonstrated acute inhibitory effects of centrally-administered interleukin-1 or LPS on gonadotropin secretion in the ovariectomized monkey (10, 25), these inhibitory effects cannot be reproduced in the estrogen-replaced ovariectomized monkey or in the intact animal at that stage of the cycle (15, 26). Under these endocrine conditions, these same compounds always provoke an acute release of LH in the primate, an observation that contrasts with the rodent (27, 28). Interestingly, increases in LH have been reported also in cyclic women, in response to the stress of exercise (29, 30, 31). In previous acute experiments, we hypothesized that, in the presence of midfollicular phase levels of estradiol, the increase in LH reflects the activation of the HPA axis and the consequential release of adrenal progesterone, which may then synergize with estradiol to induce LH release (15, 26). Yet, although an acute increase in progesterone is detected 3 h after the first endotoxin injection in the present experiment, this increase is short-lived. This hypothesis may thus have to be revisited, in view of the prolonged elevation of LH that lasts through the stress episode. An alternative hypothesis may include potential effects of the endotoxin at the pituitary level, perhaps to modify pituitary sensitivity to GnRH. That LPS may affect the gonadotrope is alluded to in experiments in the sheep (13).
A long-term effect of the 5-day inflammatory stress challenge, observed in the group in which folliculogenesis seems to be less affected by the endotoxin treatment, is represented by a relative (but significant) decrease in luteal progesterone in the first (but not the second) posttreatment cycle. The responsible mechanism for this delayed effect is presently under study in the monkey. It is of interest to note that initiation of an exercise program in the human is also found to be associated with a higher incidence of luteal deficiency (32, 33). Whatever the cause, we believe that these results are of interest, because they provide, for the first time, a direct demonstration that decreased luteal progesterone secretion may represent a delayed sequel from a short-term stress episode taking place in the follicular phase of the previous cycle.
As expected, there is a significant increase in cortisol secretion in response to endotoxin treatment, reflecting the activation of the HPA axis after this inflammatory/immune-like stress challenge. Similar acute increases in HPA activity have been well documented after central cytokine administration in the monkey and other species (8, 9, 10, 11, 12, 13, 34). Elevated cortisol concentrations persist throughout the 5-day endotoxin treatment, but there is a progressive and significant decline in the response over time. Though maintenance of the HPA axis response to chronic stress seems to vary with the type of stress (35, 36, 37), the cortisol response in our animals probably reflects a desensitization of the pituitary ACTH response and/or adaptation of the neurohormonal response to the repeated stress challenge (38).
In summary, our data demonstrate that, in the nonhuman primate, a 5-day stress period (in this case, an inflammatory-like episode), during the follicular phase of the menstrual cycle, delays folliculogenesis. The results indicate that damage to the follicular phase is intensified in individuals who already demonstrate a subtle cyclic degradation, in the form of decreased progesterone secretion in the cycles preceding the stress episode. In other individuals with normal control cycles, the data indicate that long-term effects (i.e. in the cycle after that in which the stress episode occurs) may occur, also in the form of a decreased secretory potential of the corpus luteum. Thus, the data lead us to the conclusion that decreased progesterone secretion in the luteal phase may represent the first stage in the damage that a stress episode can inflict upon the normal menstrual cycle. They also support the notion that individuals with the established inadequate luteal phase syndrome (39, 40, 41) may be more susceptible to further cyclic damage by stress. Thus, overall, it is predicted that, even though they may be insufficient to arrest the cycle and to lead to the functional hypothalamic amenorrhea syndrome, moderate short-term stress episodes of the type tested here have the potential to subtly alter the process of cyclicity and perhaps to impact on fertility potential.
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Acknowledgments |
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Footnotes |
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Received January 28, 1998.
Revised March 11, 1998.
Accepted March 25, 1998.
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