Resumption of ovarian function during lactational amenorrhoea in breastfeeding women with polycystic ovarian syndrome: endocrine aspects

T. Sir-Petermann1,6, L. Devoto2, M. Maliqueo1, P. Peirano3, S.E. Recabarren4 and L. Wildt5

1 Division of Endocrinology, Department of Internal Medicine, School of Medicine, University of Chile, 2 Institute of Maternal and Child Research, University of Chile, 3 Laboratory of Sleep and Functional Neurobiology, INTA, University of Chile, Santiago, 4 Laboratory of Animal Physiology and Endocrinology, School of Veterinary Medicine, University of Concepción, Chillán, Chile and 5 Division of Gynecological Endocrinology and Reproductive Medicine, Department of Obstetrics and Gynecology, University of Erlangen, Erlangen, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of this study was to evaluate the changes in gonadotrophin concentrations and the dynamics of the episodic fluctuations of circulating LH during night-time, in fully breastfeeding normal women and in those with polycystic ovarian syndrome (PCOS) during lactational amenorrhoea and after weaning, in order to provide insights into the onset of this syndrome. Additionally, ovarian activity was evaluated by ultrasound examination and steroid concentrations. METHODS: Twelve lactating PCOS (LPCOS) women and six normal lactating (NL) women of similar age were selected. On the 4th and 8th week postpartum (PP) and eight weeks after weaning, blood samples were collected every 10 min (10.00–20.00h). Gonadotrophin concentrations were determined in all samples. Steroid hormones were measured in one fasting sample and ovarian morphology was assessed by ultrasound. RESULTS: On the 8th week PP, LH pulse frequency was higher and FSH concentrations were lower in LPCOS women compared with NL women, and steroid hormone concentrations remained low, except for androstenedione which was higher in LPCOS patients. After weaning, similar differences were observed between both groups. PCOS patients also showed enlarged ovaries with a PCOS pattern in the three study periods. CONCLUSIONS: The enlarged ovaries associated with higher androstenedione concentrations suggest that PCOS is a primary ovarian defect, making it difficult to establish if the abnormal LH pattern observed in these women is primary or secondary to the ovarian dysfunction.

Key words: LH/lactation/PCOS


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine-metabolic disorder in women, of unknown cause, with well-described abnormalities in hypothalamic, pituitary and ovarian function. Abnormality in the gonadotrophin-secretory pattern, characterized by increased LH, pulse amplitude and frequency, and normal to slightly decreased FSH secretion, has been observed in PCOS patients, compared with ovulatory control women ( Yen et al.1970Go; Rebar et al.1976Go; Kazer et al.1987Go; Berga et al.1993Go). However, the underlying aetiological basis of the gonadotrophin abnormalities is still under debate ( Rosenfield et al.1997). Although abnormal ovarian steroidogenesis probably contributes to the characteristic pattern of gonadotrophin secretion observed in this syndrome, altered gonadotrophin secretion explains, at least in part, the characteristic ovarian defects, making it difficult to establish if central or ovarian dysfunction is primary. It has been described that teenage girls with PCOS had abnormal LH profiles compared with normal pubertal girls (Zumoff et al.1983Go). Whereas normal pubertal girls had LH secretion surges concomitant with their nocturnal sleep period, PCOS pubertal girls exhibited LH secretion surges during the daytime that were grossly desynchronized from their sleep period. According to the authors, this finding shows that the central nervous system may be the probable locus contributing to the pathophysiology of PCOS.

It is apparent that puberty and the postpartum period share some common neuroendocrine features in the initiation and reactivation of menstrual cyclicity. Increments in LH secretion during night-time are a feature of postpartum, analogous to those observed during early puberty (Liu and Park, 1988Go), thus resembling a `miniature puberty' (Yen, 1998Go). This `miniature puberty' could serve as a neuroendocrine model in exploring the link of central and peripheral influences in the reactivation of the gonadal axis in normal conditions and in reproductive dysfunctions such as PCOS. Thus, the observation of different patterns of gonadotrophin secretion during night-time in the postpartum period of lactating PCOS patients could provide a clue to establish whether this is a primary hypothalamic defect. The aim of this study was to evaluate the changes in gonadotrophin concentrations and the dynamics of the episodic fluctuations of circulating LH during night-time in fully breastfeeding normal and PCOS women during the postpartum period (lactational amenorrhoea) and after weaning. Additionally, ovarian activity was evaluated by ultrasound examination and serum steroid concentrations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Twelve lactating PCOS (LPCOS) women with normal term pregnancies were selected for the study from patients attending the Unit of Reproductive Medicine, University of Chile. They were aged 26.9 years (range 19–34) and had a body mass index (BMI) of 29.53 kg/m2 (range 22.4–35.2). Preconceptional diagnosis of PCOS was made if the following clinical and endocrine criteria were present: chronic oligo-or amenorrhoea, hirsutism, plasma testosterone concentration >0.6 ng/ml or free androgen index (FAI) >5.0 and characteristic ovarian morphology on ultrasound based on previously described criteria (Adams et al.1985Go). A normal LH/FSH ratio was not considered an exclusion criterion. All women were amenorrhoeic and anovulatory according to progesterone measurements and ultrasound examinations. Hyperprolactinaemia, androgen-secreting neoplasm, Cushing's syndrome and attenuated 21-hydroxylase deficiency, as well as thyroid disease, were excluded by appropriate tests.

PCOS patients included in the study were selected from a group of 20 PCOS patients attending the Unit of Reproductive Medicine who desired fertility, and were placed on a 6 month diet and exercise programme which consisted of a 1000 kcal low-fat diet and a daily walk of 30 min. During this programme, six patients ovulated and became pregnant. The other six patients became pregnant after pharmacological induction of ovulation with Clomiphene citrate 100 mg/day from cycle days 5–9.

By design, six normal lactating (NL) women of similar age 25.4 years (range 20–34) and BMI 26.4 kg/m2 (range 20.8–30.0) acted as the control group. Each one had a history of regular 28–32 day menstrual cycles, absence of hirsutism and other manifestations of hyperandrogenism, absence of galactorrhoea, thyroid dysfunction and family history of diabetes. All were healthy, were not receiving any drug therapy and had a normal term pregnancy and vaginal delivery of a healthy child. These women were recruited from the maternity unit of the same hospital.

Both groups of lactating women maintained exclusive breastfeeding for at least 3 months and all were in amenorrhoea during this period. Prior to the study, informed consent was obtained from all subjects. This study was approved by the local ethical committee. These two groups of lactating women were also included in a study examining the metabolic aspects of the resumption of ovarian function during lactational amenorrhoea (Maliqueo et al., 2001Go ).

Study protocol
The women were admitted to the Clinical Research Center on the evening before the study in the 4th and 8th weeks postpartum and again eight weeks after weaning. During the first two study periods, infants accompanied their mothers, and breastfeeding was continued ad libitum. The study was initiated at the 4th week postpartum to avoid the effect of placental steroid and peptide hormones.

During a 12 h period (10.00–20.00 h), blood samples were collected every 10 min, using a sampling device that allowed the continuous withdrawal of blood through a heparinized catheter (Sir-Petermann et al.1995Go). A 24 h sampling period was not possible for ethical reasons. Serum LH and FSH concentrations were determined in all samples. Serum total testosterone, oestradiol and androstenedione were determined in one morning fasting sample and prolactin (PRL) was measured in samples taken before and 30 min after each suckling episode and every hour after weaning.

During blood sampling, meal times and sleep patterns were recorded. The presence of apparent sleep was noted by the attending staff. In most patients, sleep/wake patterns were also recorded by means of an actigraph (Mini Motionlogger, Advanced Model; Ambulatory Monitoring Inc., Ardsley, NY, USA) placed on the nondominant wrist (Sadeh et al.1994Go,1995Go). In short, this device weighing 57 g, set for 1 min recording bins and zero crossing mode (a measure of movement frequency), accumulates one count each time movement causes the sensor signal to cross a fixed reference signal. An amplified setting 18 was used for this device, which yields a sensitivity of 0.05 g in a frequency range of 2–3 Hz. The actigraph data were downloaded into the computer for off-line analyses. Sleep/wake measurements were estimated from actigraphic data using the validated ASA algorithm (Sadeh et al.1994Go).

Breastfeeding took place on demand, and the time of onset of suckling and the duration of each feed were noted in detail.

Ultrasound examination
Ovarian morphology was assessed by ultrasound examination of the ovaries one day before the pulse studies in the 4th and the 8th week postpartum, and again eight weeks after weaning. Scans were made using a 5.0 MHz vaginal probe (Aloka, Tokyo, Japan). The mean ovarian volume was considered to be representative of both ovaries in each woman.

Hormone assays
Serum LH, FSH and oestradiol were determined by electrochemiluminiscence (Roche, Basel Switzerland; range: 0.1–200 IU/l for LH and FSH and 10–4000 pg/ml for oestradiol). The intra- and interassay coefficients of variation respectively were 1.1 and 2.1% for LH; 1.67 and 3.7 % for FSH; 2.7 and 5% for oestradiol.

Testosterone, androstenedione and prolactin were measured by radioimmunoassay using commercial kits (DPC, Los Angeles, CA, USA). Steroid hormones were measured in the same assay. The intra- and interassay coefficients of variation respectively were 7.0 and 11% for testosterone; 3.2 and 6.1% for androstenedione and 1.1 and 1.6% for PRL.

LH pulse analysis and statistical evaluation
For pulse analysis, the computerized version of the cluster pulse algorithm (Veldhuis and Johnson, 1986Go) was used. We selected a cluster configuration of 1x2 (one sample for the test peak and two for the test nadir), and a t-value of 2.1/2.1 to constrain the likelihood of false positive pulse determination to <5%. The following mean properties of pulsatile LH concentrations were analysed: pulse frequency (number of significant peaks/12 h), pulse height and pulse amplitude. They were evaluated by analysis of variance (ANOVA) followed by Newman–Keul's multiple range tests. Comparisons between the groups were performed using the Mann–Whitney test. The significance level was set at 5% (P < 0.05 ). Results are expressed as means and ranges.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Gonadotrophin concentrations and LH pulse analysis
Table IGo shows the serum gonadotrophin concentrations and LH pulse characteristics at the 4th and 8th week postpartum and eight weeks after weaning.


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Table I. Serum gonadotrophin concentrations and luteinizing hormone (LH) pulse characteristics in normal lactating (NL) women and lactating PCOS (LPCOS) women at the 4th and 8th week postpartum and after weaning
 
In NL women, mean serum LH concentrations increased significantly between the 4th week postpartum and after weaning, this was associated with an increase in LH pulse amplitude and pulse height. Mean serum FSH concentrations remained unchanged during the study and were higher than mean serum LH concentrations on the 4th and 8th week postpartum. After weaning, however, mean serum LH and FSH concentrations were similar.

In LPCOS women, mean serum LH concentrations also increased significantly between the 4th week postpartum and after weaning, concomitantly with an increase in pulse amplitude, pulse height and pulse frequency. Mean serum FSH concentrations decreased significantly during the three study periods but remained significantly higher than LH concentrations at the 4th and 8th week postpartum. After weaning, mean LH and FSH concentrations were similar.

Comparing both groups of lactating women, on the 4th week PP LH pulse characteristics and gonadotrophin concentrations were similar between both groups. At the 8th week postpartum, LH pulse frequency was significantly higher and FSH concentrations were significantly lower in LPCOS women compared with NL women. After weaning, LH pulse frequency was significantly higher and LH pulse amplitude significantly lower than in NL women. Mean serum FSH concentrations remained significantly lower in the PCOS group compared with the control group.

The nocturnal pattern of LH secretion differed between NL and LPCOS women at the 8th week postpartum (Figures 1 and 2GoGo) . All NL women presented a nocturnal LH surge between 23:00 and 04:00 h, while PCOS women exhibited absence (n = 4 ) or a shift (n = 8 ) of the nocturnal LH surge to the following hours (05:00–10:00 h). Three representative cases for each group are shown in Figures 1 and 2GoGo. At the 8th week postpartum, in neither group was there any clear relationship between apparent sleep and the nocturnal LH surge. After weaning, the nocturnal LH surge was not detected and both groups of lactating women exhibited, during this sampling period, regular LH pulses, but the number of LH pulses was significantly different (P = 0.036 ) between groups (Table IGo).



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Figure 1. 12 h pulsatile LH patterns in three normal lactating women at the 8th week postpartum (A) and 8 weeks after weaning (B). Sleep periods are marked by bars, and prolactin concentrations in each suckling episode by asterisks.

 


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Figure 2. 12 h pulsatile LH patterns in three lactating PCOS women at the 8th week postpartum (A) and 8 weeks after weaning (B). Sleep periods are marked by bars, and prolactin concentrations in each suckling episode by asterisks.

 
Sex hormone concentrations and ultrasound examination
Serum steroid concentrations and ovarian volume in LPCOS and NL women at the 4th and 8th week postpartum and eight weeks after weaning are shown in Table IIGo. In both groups, on the 4th and 8th week postpartum, steroid concentrations remained low, except for androstenedione concentrations which were higher in LPCOS patients compared to NL women at the 8th week postpartum. After weaning, androstenedione concentrations remained significantly higher (P = 0.020 ) and oestradiol concentrations significantly lower (P = 0.013 ) in the PCOS group compared to the control group.


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Table II. Serum steroid concentrations and ovarian volume in normal lactating women (NL) and lactating PCOS (LPCOS) women at the 4th and 8th week postpartum and after weaning
 
The ultrasound ovarian morphology of all patients showed a PCOS pattern with the distribution of follicles limited to the ovarian periphery, and increased ovarian stroma. Ovarian volume was significantly higher in the PCOS compared with the normal group at the 4th and 8th week postpartum and after weaning.

Lactating episodes and prolactin values
The number and duration of the lactating episodes were similar in both groups during the first two sampling periods. Post-suckling prolactin (PRL) concentrations were significantly higher (P < 0.05 ) than basal PRL concentrations in both groups at the 4th and 8th week postpartum. Basal PRL concentrations were significantly lower in LPCOS women at the 8th week postpartum compared with NL women (Table IIIGo) . After weaning, PRL concentrations were not significantly different between both groups [NL: 18.99 (7.66–31.4); LPCOS: 14.04 (4.87–43.6)].


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Table III. Nursing episodes and serum prolactin concentrations in normal lactating women (NL) and lactating PCOS (LPCOS) women at the 4th and 8th week postpartum
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we evaluated the changes in gonadotrophin concentrations and the dynamics of the episodic fluctuations of circulating LH during night-time in a group of PCOS women during lactational amenorrhoea and after weaning. Gonadotrophin concentrations and the secretory pattern of LH differ from those observed in age- and weight-matched normal lactating women.

Several neuroendocrine aberrations were observed in these women, in accordance with those described in adolescent girls with PCOS (Zumoff et al.1983Go; Apter et al.1995Go; Porcu et al.1997Go), namely an increased LH pulsatility, a relative suppression of FSH secretion and disturbance of the nocturnal rhythm of LH secretion, suggesting a deranged gonadotrophin-releasing hormone (GnRH)–gonadotrophin activity.

With respect to the rapid GnRH pulse frequency, a critical questions is whether it represents a primary hypothalamic defect or is secondary to the elevated plasma oestrogen, androgen, or insulin concentrations present in this syndrome.

Many studies have tested the hypothesis that sex steroid feedback could modulate the gonadotrophin abnormalities observed in PCOS patients. It has been proposed that hypersecretion of androgens, either directly or as a consequence of the aromatization of oestrogens, causes the abnormal gonadotrophin dynamic which characterizes this syndrome (Rebar et al.1976Go; Yen, 1980Go). In some studies, oestrogen concentrations have been positively correlated with gonadotrophin dynamics (Chang et al.1982Go; Waldstreicher et al.1988Go; Urban et al.1991Go), in others, such a correlation has not been established (Dunaif et al.1985Go; Dunaif, 1986Go). Nevertheless, in our postpartum model, in contrast to the above described studies and those in adolescent girls with PCOS (van Hooff et al.1999Go), ovarian activity evaluated as oestradiol synthesis was low, making it difficult to attribute this phenomenon to an abnormal oestrogen feedback.

The effect of androgens in increasing LH secretion in PCOS women has also been investigated with conflicting results (Dunaif, 1986Go; Couzinet et al.1989Go; De Leo et al.1998Go). Previous studies by our group (Sir-Petermann et al.1993Go), have demonstrated that in hyperandrogenaemic women, short term androgen receptor blockade with flutamide reduced LH pulse amplitude without affecting LH pulse frequency, suggesting a direct effect of androgens at the pituitary level rather than at the hypothalamus, and therefore making it difficult to support a role for the elevated levels of androgens observed in our lactating PCOS women, in enhancing LH pulse frequency.

Regarding the effect of insulin, although LH responsiveness to GnRH may be increased by hyperinsulinaemia (Adashi et al.1981Go), few data support the notion that elevated levels of insulin result in a rapid frequency of GnRH secretion (Poretsky et al.1988Go). On the other hand as discussed in our previous study (Maliqueo et al., 2001Go ), during lactation insulin concentrations are decreased in PCOS women, suggesting that elevated levels of insulin are probably not involved in increasing LH pulse frequency in these patients. Another metabolic signal that modulates hypothalamic–pituitary–gonadal axis function is leptin. However, recently we were able to establish (Sir-Petermann et al., 2001Go ), that during lactational amenorrhoea, circulating leptin is probably not involved as a primary signal in promoting pulsatile LH secretion in normal and in PCOS lactating women.

In the present study, PCOS patients showed lower basal prolactin levels associated with an increased LH pulse frequency at the 8th week postpartum, as compared with normal lactating women. One possible explanation for the enhanced LH pulse frequency observed in our lactating PCOS group could be a decrease in prolactin levels, which might lead to a reduction in dopaminergic activity and to an earlier recovery of the ovarian axis (Díaz et al.1989Go; Nagy et al.1992Go). However, after weaning, LH pulse frequency remained higher in the PCOS group compared with the normal group, while the prolactin levels were similar in both groups, making it difficult to explain this association of events simply by a reduction of dopaminergic activity as previously suggested (Cumming et al.1984Go).

The increase in LH pulse frequency observed in our PCOS patients provides clear evidence of an increase in GnRH pulse frequency, and therefore a hypothalamic defect.

Nevertheless, it cannot be established whether this is a primary hypothalamic defect or if it simply reflects the long-term effects of elevated androgens, oestrogens or other hormones on the hypothalamus. In this respect, studies in prenatally androgenized female Rhesus monkeys demonstrate that androgenized females exhibit ovarian and endocrinological features that closely resemble those found in women with PCOS (Abbott et al.1998Go). Therefore, prenatal androgen excess may provide one possible mechanism to explain the occurrence of PCOS.

In the present study, increased LH pulse frequency was associated with a relative suppression of FSH in PCOS lactating women. This observation is in agreement with clinical and experimental studies that demonstrate that LH and FSH synthesis and secretion are dependent on the pattern of GnRH pulse stimulation, with rapid frequencies favouring LH and slower pulses, FSH synthesis and secretion (Wildt et al.1981Go; Clarke et al.1984Go; Spratt et al.1987Go; Dalkin et al.1989Go; Haisenleder et al.1991Go). This observation is also in agreement with other studies in adult women and adolescent girls with PCOS (Waldstreicher et al.1988Go; Apter et al.1994Go), in which it has been proposed that partial pituitary desensitization, secondary to increased frequency of GnRH secretion, may exist in women with PCOS, with increased LH pulse frequency associated with a relative suppression of FSH.

Although episodic secretion of LH was evaluated only during a 12 h period and therefore a circadian variation of LH could not be demonstrated, the periodicity of LH secretion during night-time differed between normal lactating and PCOS women. This phenomenon was previously described in teenage girls (Zumoff et al.1983Go) and adult women with PCOS (Venturoli et al., 1988 ), with higher LH values occurring in the afternoon, thus suggesting an abnormal circadian control of LH secretion. The fact that in this study LH concentrations and LH pulse amplitude appear to be lower in PCOS women during the night, as compared with those observed in normal lactating women, is explained through the same event. This observation is also in agreement with the study of Apter et al. (1994) who demonstrated that in hyperandrogenaemic adolescents, LH pulse frequency was higher during both waking and sleeping hours, but pulse amplitude was only increased during the day (Apter et al.1994Go). Moreover, in these studies in accordance with our study, no clear relationship between apparent sleep and changes in LH profiles could be established in PCOS patients. In our postpartum model, this could be the consequence of the abnormal circadian periodicity of LH secretion or the lactating activity that disturbs the sleep/wake pattern.

Thus, the association of abnormalities in both ultradian and circadian signals strongly points to the central nervous system as one site responsible for the onset and persistence of this syndrome.

Nevertheless, although LH and insulin concentrations during lactational amenorrhoea were still reduced, a higher androstenedione release was detected in PCOS patients, as compared with normal lactating women, probably reflecting adrenal steroidogenesis or some ovarian steroidogenic activity in the absence of aromatase effect. This assumption is in accordance with previous observations that have demonstrated that ovarian secretory abnormality occurs in PCOS patients in the absence of LH excess (Ehrmann et al.1995Go; Ibañez et al.1996Go), suggesting a primary abnormality of thecal cell steroidogenesis in this syndrome (Gilling-Smith et al.1997Go).

Another interesting observation of this study was that the ovarian volume of the PCOS patients was increased, with a PCOS pattern which was clearly distinguished ultrasonographically from the multifollicular pattern that accompanies anovulation associated with decreased GnRH secretion (Adams et al.1986Go). The presence of enlarged ovaries with stromal hyperplasia after delivery could indicate that these ovaries are persistantly stimulated during pregnancy, or that the low gonodotrophin and insulin concentrations observed during lactational amenorrhoea were able to maintain the function of intragonadal factors with mitogenic activity (Nestler et al.1998Go; Devoto et al.1999Go; Franks et al.1999Go).

In summary, we have demonstrated that in a state of natural ovarian inactivity, such as lactational amenorrhoea, the pattern of pulsatile LH secretion of lactating PCOS patients is different from that observed in normal lactating women. Whether these defects are primary, or secondary to other hormonal influences, or whether they represent a reprogramming of the regulation of the GnRH pulse generator by prenatal or prepubertal exposure to elevated androgen levels, remains to be determined. On the other hand, the increased ovarian size and higher androstenedione levels observed in these patients during lactational amenorrhoea might lead us to speculate that this is, in fact, a primary ovarian defect.

Taking all these observations into account, we agree with previous studies which proposed that PCOS is a neuroendocrine, an ovarian and a systemic disorder (Poretsky and Piper, 1994Go; Homburg, 1996Go; Rosenfield, 1997Go) and that these three main features probably co-exist from the onset of this syndrome (Apter et al.1995Go; Porcu et al.1997Go; Ibañez et al.1998Go, van Hoof et al., 1999 ). The postpartum model provides an interesting approach to further explore some aspects of this syndrome, especially in regard to prolactin secretion, intraovarian autocrine and paracrine regulators and the role of metabolic and other endocrine factors outside the reproductive axis, in the pathogenesis of this disorder.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors express their gratitude to Dr Soledad Díaz for her editorial comments and thank Ms Maria Eugenia Casado from ICMER and Ms Estela Gonzalez and Ms Isabel Leiva for their help with the nocturnal care of the patients. This work was supported by Fondecyt 1970291 grant and Alexander von Humboldt Foundation.


    Notes
 
6 To whom correspondence should be addressed at: Las Palmeras 299, Interior Quinta Normal Casilla 33052, Correo 33, Santiago, Chile. E-mail: tsir{at}entelchile.net Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 30, 2000; accepted on June 7, 2001.