Human Reproduction, Vol. 18, No. 7, 1428-1431,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Evidence that oxytocin is a physiological component of LH regulation in non-pregnant women
Department of Obstetrics and Gynaecology, Christchurch School of Medicine and Health Sciences and New Zealand Centre for Reproductive Medicine, Christchurch, New Zealand
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Christchurch Women’s Hospital, Private Bag 4711, Christchurch, New Zealand. e-mail: john.evans{at}chmeds.ac.nz
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Abstract |
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BACKGROUND: Regulation of the LH surge is central to the functioning of the female ovulatory cycle. In animal models, oxytocin has been shown to alter LH activity. Oxytocin advanced the LH surge and, conversely, oxytocin receptor antagonists inhibited full production of the LH surge in rats. Few data exist on the possibility that oxytocin modulates LH in women. METHODS: Ten non-pregnant women participated in this study over two menstrual cycles. One cycle was a control cycle, and the other a trial cycle; the two were separated by at least one cycle. When the diameter of an ovarian follicle was >15 mm, a subject was allocated at random into either a control or treatment group. In a control cycle, volunteers received normal saline; in a treatment cycle, volunteers received an oxytocin antagonist (atosiban). RESULTS: For treatment cycles, the maximum LH concentration was significantly less than that in control cycles (42.1 ± 6.2 versus 60.3 ± 8.3 IU/l respectively; P < 0.05). Maximum FSH and estradiol concentrations were not significantly different between the two groups. CONCLUSIONS: The results indicated that inhibition of endogenous oxytocin affects the endocrinology of the ovulatory cycle in women, and strongly suggest that oxytocin has a role in the physiological processes of LH regulation.
Key words: atosiban/LH/ovulatory cycle/oxytocin
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Introduction |
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Regulation of the LH surge is central to the functioning of the female ovulatory cycle. Understanding of the processes underlying the production of the LH surge may enable the development of methods to modify fertility—either suppression for contraception, or enhancement in cases where there is suboptimal functioning. It is known that stimulation of the pituitary by GnRH is a crucial component in the increase in LH levels at midcycle. The administration of exogenous GnRH induces a rapid rise in LH levels. Conversely, GnRH receptor antagonists will inhibit LH secretion. Steroids modulate the effect of GnRH on the pituitary, and cyclical changes in estradiol and progesterone are important in ensuring correct timing of the LH surge. However, it is becoming increasingly clear that the physiological regulation of LH also involves other factors (Kerrigan et al., 1995
), and several peptides which act at the pituitary are believed to alter the behaviour of LH (Evans, 1999). In animal models, oxytocin has been shown to have a number of potential roles, including activity on GnRH neurones in the hypothalamus (O’Byrne et al., 1990; Johnston et al., 1992), direct action on the pituitary as a releasing factor (Evans et al., 1992), induction of LH synthesis in the pituitary (Robinson et al., 1992), interaction with GnRH in a synergistic manner to elicit augmented LH release (Evans and Tulloch, 1995; Evans et al., 1995), and modulation of long-term effects via c-fos (Abbas and Evans, 2000). The use of oxytocin antagonists has shown that endogenous oxytocin is indeed crucial to full production of the LH surge in rats (Johnston and Negro-Vilar, 1988; Robinson and Evans, 1990). These data led to the consideration that oxytocin might also be active in the regulation of LH in women. In fact, peripheral oxytocin concentrations vary during the ovulatory cycle, with maximum levels near midcyle (Amico et al., 1981; Mitchell et al., 1981; Kumaresan et al., 1983; Shukovski et al., 1989). Indeed, the administration of oxytocin to women in the late follicular phase induced an advancement of the LH surge (Hull et al., 1995). Thus, the decision was made to investigate further the role of oxytocin in the regulation of LH levels during the pre-ovulatory period in women. In the present study, an oxytocin antagonist [atosiban; 1-deamino-2-D-Tyr(OEt)-4-Thr- 8-Orn-oxytocin] was administered to women, and the effect on the LH surge monitored. |
Materials and methods |
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Ten volunteers participated in this study during two different menstrual cycles, with at least one month between the study cycles. At the time of the study, no volunteers were receiving medications that would affect the hypothalamic–pituitary–gonadal axis, in particular an oral contraceptive pill. A pre-assessment vaginal scan was performed, and women with any signs of ovarian pathology were excluded. Volunteers who were attempting pregnancy were excluded, while women who were sexually active at the time of the study were advised to ensure adequate non-hormonal contraception. The mean (± SEM) age of the women was 28.1 ± 1.4 years (range 20.3–35.1), and the mean menstrual cycle length was 27.8 ± 1.1 days (range 23–31). For each woman, one studied menstrual cycle served as a control, and the other as a trial cycle. The protocol was approved by the Canterbury Ethics Committee, and the women provided their written informed consent.
In each cycle, follicular growth was monitored by serial vaginal scans and estradiol estimations between 12:00 and 13:00 from day 8 of the menstrual cycle. When a follicle was >15 mm diameter (measured in three dimensions), each subject was allocated at random to either a control or treatment group. In a control cycle, volunteers received a bolus of normal saline followed by 500 ml normal saline over a 2 h period. In a treatment cycle, volunteers received a bolus of 6.5 mg oxytocin antagonist (atosiban, Tractocile; Ferring AB, Malmo, Sweden), followed by an infusion at 300 µg/min in 500 ml normal saline over a 2 h period. The atosiban dose chosen has been shown to be safe and effective in studies in women with preterm labour (Goodwin et al., 1994; Moutquin et al., 2000). All infusions took place between 14:00 and 16:00 at Christchurch Women’s Hospital.
During all cycles, a blood sample was taken immediately before infusion (at the time of insertion of the i.v. access). Blood levels of LH were monitored at this time, after which the serum was removed and frozen for subsequent measurement of estradiol level. Blood LH measurements were carried out at 12 h intervals (20:00 and 08:00) in all women until a LH surge had been shown to occur. Transvaginal scans were undertaken to obtain evidence of ovulation.
The time from infusion to onset of the LH surge was quantified in two ways. First, a method based on a procedure widely used and validated in IVF clinics was employed. The standard logarithmic rate of LH increase (Hoff et al., 1983) was plotted and aligned through the first measured concentration of LH that was 50% above baseline. The intersect with the extended baseline was the time at which the surge was deemed to have started. Second, points along the rising phase of the surge were noted, and the rates of increase in LH concentrations calculated; the first point after a rate of increase in LH concentration over the previous 12 h of >1 IU/l per hour was deemed to be the first observed point of the LH surge. The actual rate of increase to this point over the 12 h period was noted, and called the ‘initial observed rate of increase’. The peak of the LH surge was confirmed by observation of descending concentrations of hormone. All hormone measurements were performed using immunoassays, with coefficients of variation <12%. The results of the control and treatment cycles were compared using a paired t-test or a one-way ANOVA as appropriate.
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Results |
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During the cycle in which atosiban was administered, the mean (± SEM) maximum LH level was less than that in control cycles (42.1 ± 6.2 versus 60.3 ± 8.3 IU/L; P < 0.05, paired t-test) (Figure 1). Infusion of atosiban did not significantly alter the time to onset of the LH surge in the two cycles (35.2 ± 4.2 versus 36.4 ± 6.1 h from the time of infusion). The rate of initial increase was lower in eight (80%) of the atosiban-treated women than in those infused with saline (1.8 ± 0.4 versus 2.3 ± 0.5 IU/l per hour), but this difference was not statistically significant. The maximum FSH level was apparently less sensitive than LH to the presence of atosiban; the mean maximum FSH level in atosiban cycles was modestly reduced (11.4 ± 1.7 versus 12.8 ± 1.1 IU/l) (Figure 2), but again the reduction was not statistically significant. Among the women as a group, the mean maximum estradiol level in atosiban cycles was similar to that in control cycles (1.3 ± 0.1 versus 1.4 ± 0.1 pmol/l; P = NS).
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Discussion |
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The results of the present study strongly suggest that the oxy tocin receptor antagonist, atosiban, affects the endocrinology of the ovulatory cycle in women, and so points to a role for oxytocin in the physiological processes of LH regulation. Atosiban competitively inhibits oxytocin at the peptide’s binding sites (Thornton et al., 1993
; Phaneuf et al., 1994), but because it may also interfere at vasopressin receptors, it is possible that vasopressin might be the mediating factor (Manning et al., 2001), though vasopressin is substantially less potent than oxytocin in eliciting LH from gonadotrophs (Evans and Catt, 1989). Atosiban does not prevent Ca influx through voltage-operated Ca channels, but does reduce Ca influx and the release of intracellular Ca stores (Thornton et al., 1993). Atosiban has a biological half-life of 18–39 min and a clearance of 
24–42 L/h (Lundin et al., 1993; Goodwin et al., 1995). In addition, it does not have persistent effects when its infusion is stopped (Phaneuf et al., 1994).
Among the present women there was a reduction in maximum LH levels following antagonist infusion, and this was consistent with results from animal studies in which oxytocin receptor antagonists inhibited production of the LH surge (Johnston and Negro-Vilar, 1988; Robinson and Evans, 1990). By contrast, the administration of oxytocin to rats at pro-estrus led to an advancement in the LH surge (Robinson and Evans, 1990). Similarly, when oxytocin was delivered to women during the late follicular phase, the onset of the LH surge was advanced (Hull et al., 1995). The present study provides evidence, for the first time, that the suppression of endogenous oxytocin activity in women can affect the ovulatory cycle. Thus, it is becoming increasingly apparent that oxytocin is a physiological component of the endocrinological system that regulates LH (Robinson and Evans, 1991; Evans, 1999; 2002).
Oxytocin is believed to have effects on LH release at both the hypothalamic (Johnston et al., 1990) and pituitary (Evans et al., 1995) levels. However, the protocols used in such studies would require the peptide antagonist to cross the blood–brain barrier in order to act hypothalamically. Although peptides may reach cells in circumventricular areas (Ermisch et al., 1985), observations suggest that oxytocin and vasopressin—and presumably also related peptides—are likely to cross the blood–brain barrier only to a very limited extent (Ermisch et al., 1985; Carter and Altemus, 1997; Kang and Park, 2000). Therefore, it seems most likely that the effect occurs directly at the pituitary.
A smaller effect of atosiban was seen on FSH, which might be less sensitive to oxytocin activity (Evans et al., 1989). The role of oxytocin in reproductive pathology is unclear, but it is possible that abnormalities in oxytocin regulation could be related to anovulation following follicular growth, as is occasionally seen in patients with polycystic ovary syndrome.
The place of oxytocin in LH regulation has been the subject of discussion for almost five decades (Shibusawa et al., 1955). Although most recent studies on this topic have been conducted in animal models, women demonstrate measurably higher levels of oxytocin in the peripheral blood at midcycle (Amico et al., 1981; Mitchell et al., 1981; Kumaresan et al., 1983; Shukovski et al., 1989). In the present investigation, an approach was used which had been shown previously to be successful in animal studies (Johnston and Negro-Vilar, 1988; Robinson and Evans, 1990), whereby endogenous oxytocin was inhibited by use of a receptor antagonist. Herein, for the first time, evidence is reported that endogenous oxytocin might be involved in LH regulation in women.
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Acknowledgements |
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The authors thank the women who participated in this study for their cooperation and willingness. They also thank Ferring AB, Malmo, Sweden for the generous gift of Tractocile.
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Submitted on July 12, 2002; resubmitted on March 4, 2003; accepted on April 2, 2003.
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