Plasma prolactin/oestradiol ratio at 38 weeks gestation predicts the duration of lactational amenorrhoea

Carmen Campino1, Claudia Torres5, Alonso Rioseco2, Andrés Poblete2, Edda Pugin4, Verónica Valdés3, Silvia Catalán3, Cristián Belmar2 and María Serón-Ferré5,6

1 Departments of 1Endocrinología, 2 Obstetricia y Ginecología, 3 Pediatría, 4 Centro de Lactancia, Facultad de Medicina and 5 Department of Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Fully breastfeeding women experience an amenorrhoea of variable duration. Our aim was to identify in pregnancy, endocrine markers that could predict the duration of subsequent lactational amenorrhoea. METHODS: We studied 17 healthy women at 34 and 38 weeks gestation, and 1 and 3 months post-partum. The women fully breastfed until 6 months post-partum. During pregnancy, prolactin (PRL), oestrogens (total oestradiol, unconjugated oestrone, unconjugated oestriol), sex hormone binding globulin (SHBG), dehydroepiandrosterone sulphate (DHEA-S), progesterone and placental lactogen, and during post-partum PRL, oestrogens and SHBG, were measured. Free oestradiol in pregnancy and post-partum was calculated. RESULTS: Ten women experienced long (>6 months) and seven experienced short (<6 months) lactational amenorrhoea. At 38 weeks gestation, the women who experienced a long lactational amenorrhoea had twice as much PRL, about half the total oestradiol, lower SHBG concentration (P < 0.05, Student's t-test, Bonferroni modification) and similar free oestradiol concentration, compared with those who experienced short lactational amenorrhoea. The difference in PRL concentration persisted in post-partum postsuckling samples. CONCLUSION: At 38 weeks gestation, the ratio PRL/oestradiol identified all individual women according to the subsequent duration of their lactational amenorrhoea, suggesting that duration of lactational amenorrhoea is conditioned during pregnancy.

Key words: duration of amenorrhoea/lactational amenorrhoea/oestradiol/prolactin


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Lactation induces infertility, providing the basis of the lactational amenorrhoea method (LAM) (Pérez et al., 1992Go) for family planning. However, lactational amenorrhoea duration varies in different women and countries. In Chile, only about half of the women who fully breastfeed have a post-partum amenorrhoea which lasts >6 months (Díaz et al., 1991Go). Women who will experience a long lactational amenorrhoea (defined as lasting >6 months) have higher prolactin (PRL) concentrations and a greater PRL response to suckling than those women whose lactational amenorrhoea lasted <6 months (Díaz et al., 1991Go; Campino et al., 1997Go). Oestradiol concentration also differed between these groups and the concentration of PRL relative to oestradiol was greater in the long than in the short lactational amenorrhoea women when studied at 3 months post-partum (Díaz et al., 1991Go; Campino et al., 1994Go, 1997Go).

The endocrine environment of pregnancy prepares the mammary gland and the pituitary for the ensuing lactation (Ingram et al., 1999Go) and suppresses the hypothalamus–pituitary–ovarian axis (Donnet et al., 1990Go). Hormones such as PRL and total and free oestradiol, oestrone, oestriol, progesterone and placental lactogen (hPL) are elevated during pregnancy, whereas dehydroepiandrosterone sulphate (DHEA-S) is decreased (Ylikorkala et al., 1973Go; O'Leary et al., 1991Go). We postulate that during pregnancy, common endocrine mechanisms prepare women for lactation and lactational amenorrhoea. Our aim in this study was to identify prospectively whether the concentration of these hormones during pregnancy related to duration of lactational amenorrhoea and could therefore serve as a marker for predicting the duration of lactational amenorrhoea in women fully breastfeeding.


    Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
A total of 22 volunteers were recruited using procedures approved by the Institutional Ethical Committee of the Pontificia Universidad Católica de Chile at ~32 weeks gestation. All women gave their consent in writing and expressed willingness to fully breastfeed and not use hormonal contraception for at least 6 months post-partum. Of the 22 women, 17 completed the protocol until 6 months post-partum. Five women were excluded: one for hysterectomy at delivery, one for delivery of a small for gestational age newborn, one for early weaning and two for use of hormonal contraception in the post-partum. The women were healthy, 21–39 years old, parity 1–2, with appropriate fetal size, non-smokers and only received prenatal vitamins during pregnancy. All women were followed during pregnancy, delivery and up to 6 months post-partum or until restarting the menstrual cycle. Their infants were seen at the Paediatrics Unit of our hospital, certified as a Baby Friendly Hospital in 1992 by United Nations Children's Fund (UNICEF). Lactation support is provided to all women delivering in the hospital. In the present study, a nurse midwife from the Lactation Centre interacted with the women during pregnancy, upon delivery and at each subsequent clinic visit, providing lactation support. Women were instructed to fully breastfeed for providing liquids and nutrients during the first 6 months post-partum, except for the administration of vitamin drops. Breastfeeding characteristics were recorded by the same nurse midwife at each clinic visit. Women were divided into two groups on the basis of having their menses before 6 months post-partum (mean ± SEM = 119.9 ± 16.7 days, range 64–169, n = 7) and were classified as short lactational amenorrhoea group. The other 10 women remained in amenorrhoea for >6 months and were classified as long lactational amenorrhoea group. Of these, eight of the women had the first menses between 195–395 days post-partum (mean ± SEM = 249.8 ± 20.1) whereas the remaining two women initiated steroid contraception after months 6 post-partum.

Study protocol
Women were studied at 34–35 and 38–39 weeks gestation and at 1 month (between 3–5 weeks) and 3 months post-partum (12–14 weeks). Eight women of the long lactational and six of the short amenorrhoea group completed the pregnancy protocol. Nine women in the long and all of the women in the short lactational amenorrhoea group completed the post-partum protocol. During each appointment, a butterfly needle was inserted into an antecubital vein between 1500–1530 h and a 20 ml heparinized blood sample was taken at 1600 h (basal sample during pregnancy and lactation). Blood sampling collection time was selected based on our previous study in non-ambulatory conditions in fully breastfeeding women (Campino et al., 1997Go). Post-partum blood withdrawal was adjusted so that the 1600 h sample (basal sample) was drawn at least 90 min after the end of a breastfeeding episode. As soon as the basal sample was taken, the mother nursed the baby, 10 min from each breast. A postsuckling blood sample was drawn 30 min after initiation of suckling. The blood samples were centrifuged, and the plasma was stored at –20°C until assayed.

Laboratory assays
Plasma PRL and pregnancy total oestradiol concentrations were measured by radioimmunoassay (RIA) using the reagents and methodology of the World Health Organization (WHO) Programme for the Provision of Matched Assay Reagents for the RIA of the Hormones in Reproductive Physiology Programme (World Health Organization, 1994Go).

PRL concentration
We used a PRL antibody (donated to WHO by Dr B.Morris, Guildford, UK) and purified pituitary PRL (WHO 75/504, biological potency 32.5 mIU/µg) as standard. Interassay coefficient of variation was 9.5% for the 27.7 µg/l plasma pool.

Total oestradiol concentration
Pregnancy: samples diluted 50–200 fold in PBS buffer were extracted with diethyl ether. Several dilutions were assayed to ascertain linearity. Interassay coefficient of variation was 10.8% for the 1619 pmol/l plasma pool. Post-partum: undiluted samples were measured using the automated chemiluminescence system (ACS; Centaur, Bayer, NY, USA). Interassay coefficient of variation was 13.4% for the 236 pmol/l plasma pool.

Plasma sex hormone binding globulin concentration
Sex hormone binding globulin (SHBG) was measured using the automated chemiluminescence system (Immulite, Diagnostic Products Corporation, CA, USA). Interassay coefficient of variation was 7.0% for 68.5 nmol/l plasma pool.

Free oestradiol concentration
Free oestradiol was calculated as described by Vermeulen (Vermeulen et al., 1999Go) for free serum testosterone, using the values of 4.0x108 mol/l–1 and 3.7x104 mol/l–1 for the association constants of SHBG and albumin for oestradiol (Rosenfield and Moll, 1983Go) respectively and assuming an albumin concentration of 5x10–4 mol/l (3.5 g/l).

Plasma unconjugated oestrone, unconjugated oestriol, progesterone, DHEA-S, hPL and thyroid stimulating hormone concentrations
Unconjugated plasma oestrone and oestriol were measured by RIA using commercial kits (Diagnostic Systems Laboratories, TX, USA). Pregnancy plasma was diluted in multidiluent 3 (ACS; Centaur). This solution did not interfere in the assay. Interassay coefficient of variation was 9.1% for the 1184 pmol/l unconjugated oestrone plasma pool and 11.2% for the 9.1 nmol/l unconjugated oestriol plasma pool. Progesterone was measured by immunoassay using ACS. Pregnancy samples with progesterone concentration >127.2 nmol/l (40 ng/ml) were diluted in multi-diluent 3 and reassayed. Interassay coefficient of variation was 7.8% for the 3.93 nmol/l plasma pools. DHEA-S was measured by immunoassay using the Immulite automated chemiluminescence system. Interassay coefficient of variation was 8.0% for the 5780 nmol/l plasma pool. Plasma hPL was measured by RIA using rabbit anti hPL antiserum (NIDDK anti hPL-2 AFP-C1801010) at a final dilution of 1:400 000. Standard hPL and 125I-hPL were purchased from Diagnostic Products Corporation. Samples were diluted 100–200 fold to fall within the standard curve. The interassay coefficient of variation was 14.3% for the 8.1 µg/l plasma pool. Thyroid stimulating hormone (TSH) concentration was measured by immunoassay in ACS, using TSH standard 2nd IRP 80–558 reference material from WHO. Interassay coefficient of variation was 6.0% for the 2.0 mU/l pool.

Data analysis
Data was analysed using Prism 3.0 software. Values are presented as mean ± SEM for plasma concentration of PRL, total oestradiol, free oestradiol, unconjugated oestrone, unconjugated oestriol, progesterone, DHEA-S, SHBG, hPL and TSH, which showed Gaussian distribution. Means within groups were compared by analysis of variance (ANOVA) with repeated measures and Newman–Keul's test as a post-hoc comparison test. Missing values (two in the long and one in the short lactational amenorrhoea group at 38 weeks gestation and one in the long lactational amenorrhoea group at 3 months post-partum) were replaced by the mean of the group. Comparisons of means between groups during pregnancy (two comparisons) and during post-partum (four comparisons for PRL and two for the other hormones) were performed by unpaired Student's t-test with Bonferroni modification. Means of PRL/oestradiol ratios were compared after logarithmic transformation. Comparison of PRL response to suckling at 1 and 3 months post-partum was performed by paired Student's t-test. Correlation coefficients were calculated using the Pearson coefficient of correlation and compared by likelihood analysis (Mardia et al., 1982Go). Differences were considered significant when P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Women of the long (>6 months) and the short (<6 months) lactational amenorrhoea groups had similar characteristics during pregnancy and lactation (Table IGo). All the women participating in the study were euthyroid at 38 weeks gestation. TSH concentration (mean ± SEM) were 1.55 ± 0.17 and 1.10 ± 0.13 mU/l, for the long and short amenorrhoea group respectively (P = not significant). All the women had fetuses of appropriate size for gestational age and experienced normal pregnancy ending at 38–40 weeks gestation in vaginal delivery of a child of normal weight and Apgar score. Parity, length of gestation, infant weight at birth, infant growth and breastfeeding pattern also were similar in both groups of women.


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Table I. Mother and infant characteristics
 
Plasma PRL concentration
PRL concentration related to the duration of lactational amenorrhoea in pregnancy and post-partum (Figure 1Go). At 38 weeks gestation and in the postsuckling samples at month 1 and 3 of post-partum, women who subsequently experienced a long lactational amenorrhoea had twice as much PRL compared with those who would experience a short lactational amenorrhoea (P < 0.05). Basal plasma PRL fell following delivery in both groups of women (Figure 1, PGo < 0.05). In each group of women PRL increased after suckling (P < 0.05). At month 1 post-partum, post suckling PRL concentration was similar to that found at 38 weeks gestation.



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Figure 1. Changes in prolactin (PRL) concentrations from the last weeks of gestation to months 1 and 3 post-partum in women who subsequently experienced long or short lactational amenorrhoea. Filled bars: long amenorrhoea. Open bars: short amenorrhoea. Stippled bars: postsuckling samples. *Long versus short amenorrhoea women, P < 0.05. {dagger} Pre versus postsuckling, P < 0.05.

 
Plasma total oestradiol, SHBG and free oestradiol concentrations
At 38 weeks of pregnancy, the women who subsequently experienced a long lactational amenorrhoea had about half the total oestradiol concentration when compared with those who would experience a short lactational amenorrhoea (P < 0.05, Table IIGo).


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Table II. Mean ± SEM hormone concentrations at the end of gestation in women that subsequently experienced long or short lactational amenorrhoea
 
Both groups increased their concentration of total oestradiol between 34–38 weeks of pregnancy (P < 0.05, Table IIGo). In the post-partum, oestradiol fell to similar values in both groups at 1 month. At 3 months post-partum, total oestradiol concentration was lower in the women who would experience a long lactational amenorrhoea than in the other group of women (P < 0.05, Table IIIGo). In this latter group, three of the seven women had already reinitiated menses.


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Table III. Mean ± SEM hormone concentrations during post-partum in fully nursing women that subsequently experienced long or short lactational amenorrhoea
 
At 38 weeks gestation, long lactational amenorrhoea women had lower SHBG concentration than short lactational amenorrhoea women (P < 0.05, Table IIGo). Prior to then, SHBG increased between 34–38 weeks only in the short amenorrhoea women. After delivery, SHBG concentration decreased to similar values in both groups of women (Table IIIGo). Calculated free oestradiol concentrations were similar and increased between 34–38 weeks of gestation (P < 0.05, Table IIGo) and decreased markedly in post-partum in both groups of women (Table IIIGo).

There was a correlation between total oestradiol and SHBG concentrations when considering all values obtained during pregnancy and post-partum. Correlations fitted similar regression equations in both groups of women; SHBG = 7.24x10–3 total oestradiol + 108.2 (n = 37, r = 0.80, P < 0.01) and SHBG = 7.15x10–3 total oestradiol + 127.3 (n = 27, r = 0.8356, P < 0.01) for long and short lactational amenorrhoea groups respectively.

Unconjugated oestriol, unconjugated oestrone, DHEA-S, progesterone and hPL
There were no differences between long and short amenorrhoea women in any of these hormones during gestation (Table IIGo). In both groups of women there was an increase in unconjugated oestriol, unconjugated oestrone and progesterone with gestational age. In the post-partum, unconjugated oestrone decreased to similar values in both groups (Table IIIGo) whereas oestriol concentration was below the sensitivity of the assay (data not shown).

Plasma PRL/oestradiol ratio
At 34 and 38 weeks gestation, the mean PRL/oestradiol ratio (both hormones expressed in µg/l) was higher in women who would subsequently experience a long lactational amenorrhoea than in those who would experience a short one (Figure 2Go). Long amenorrhoea women also had a higher mean postsuckling PRL/oestradiol ratio at 3 months post-partum (data not shown).



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Figure 2. Prolactin (PRL) ratios at 34 and 38 weeks gestation in individual women who subsequently experienced long (filled circles) or short lactational amenorrhoea (open circles). *Long versus short amenorrhoea women, P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study shows that at 38 weeks gestation, PRL and oestradiol plasma concentrations are predictive markers for the duration of lactational amenorrhoea. Women who subsequently experienced a long lactational amenorrhoea (>6 months) had almost twice the concentration of PRL and about half the concentration of oestradiol compared with those who experienced a shorter one (<6 months). Differences in PRL concentration between long and short lactational amenorrhoea women continued during post-partum, as was reported by us previously (Díaz et al., 1991Go; Campino et al., 1994Go, 1997Go). The endocrine differences between both groups occurred despite similarities between the women in age, weight and infant characteristics and in breastfeeding pattern and infant growth. Furthermore, at 38 weeks gestation, the ratio of PRL/oestradiol was able to identify all the individual women who subsequently would have a long or short lactational amenorrhoea.

The difference in total plasma oestradiol concentration between long and short lactational amenorrhoea women may represent differences in oestradiol synthesis or clearance. In pregnancy, oestradiol is synthesized by placental aromatization of maternal and fetal DHEA-S (Siiteri and MacDonald, 1966Go). We did not find differences in maternal DHEA-S concentration, placental weight, progesterone or hPL between long and short amenorrhoea women. The difference in oestradiol concentration between the women was established between 34–38 weeks, gestational ages at which there is a marked increment in fetal adrenal growth and a marked increase in cord blood DHEA-S concentration (Parker et al., 1982Go). On the other hand, the increase in oestradiol in the short amenorrhoea women coincided with an increase in SHBG. Oestradiol induces SHBG (Odlind et al., 1982Go) and oestradiol binding to SHBG reduces oestradiol clearance (Ridgway et al., 1975Go). Clarifying the source of the larger oestradiol concentration in short amenorrhoea women requires further investigation.

The mechanisms by which differences in PRL and oestradiol concentration relate to the duration of subsequent lactational amenorrhoea may reside at the pituitary or at the hypothalamic levels. In both groups of women a different, but maximal capacity to secrete PRL was already attained by 38 weeks gestation, since PRL concentrations were similar to those reached in response to suckling at 1 month post-partum. In pregnancy, lactotrophs increase by division of existing lactotrophs and conversion of mamosomatotrophs to lactotrophs (Porter et al., 1990Go; Scheithauer et al., 1990Go). The number of lactotrophs declines abruptly in those women who do not breastfeed (Scheithauer et al., 1990Go). Hypothalamic mechanisms, like dopaminergic tone, continue during pregnancy (Bohnet and Kato, 1985Go). Oestradiol is a mitogen for rat lactotrophs and stimulates PRL secretion by acting at the pituitary (Yamamoto et al., 1986Go) and hypothalamic levels (Bethea et al., 1996Go). However, at 38 weeks gestation, the women with higher PRL had the lower total oestradiol concentration, and a similar calculated amount of free oestradiol (readily available for biological action) to the women who had less PRL. An intriguing possibility is that the differences in PRL concentration between the women may reflect a different sensitivity of the hypothalamic–pituitary axis to oestradiol. Differences in the response of bone density to oestrogen were detected amongst post-menopausal women (Komulainen et al., 2000Go).

These data support the hypothesis that determination of the duration of lactational amenorrhoea in fully breastfeeding women occurs during pregnancy. Women experiencing a full term pregnancy reinitiate menses later than those who abort in the first trimester (Donnet et al., 1990Go), suggesting that exposure to mid/late gestation pregnancy hormones suppress the hypothalamus–pituitary–ovarian axis. The negative feedback of oestrogens is enhanced during lactation (Baird et al., 1979Go). A lower sensitivity to oestradiol, as suggested by the lower PRL concentration found at 38 weeks in the short amenorrhoea women, would be consistent with a less suppressive effect of oestradiol upon the hypothalamus–pituitary–ovarian axis, and an early recovery of menses during breastfeeding. Whether PRL also participates in such processes is unknown. PRL is an heterogeneous molecule, present in plasma as several isoforms of different bioactivity (Campino et al., 1999Go). There are differences in PRL bioactivity and in PRL isoform profile relating to duration of lactational amenorrhoea (Campino et al., 1999Go). Whether such differences are established during pregnancy is being investigated.

Notwithstanding that the mechanisms determining the duration of lactational amenorrhoea remain unknown, the ratio of PRL and oestradiol concentration at 38 weeks gestation provides an endocrine marker to predict in each women the length of their future lactational amenorrhoea. This ratio was also effective at 3 months post-partum when the same women were fully breastfeeding. This confirms our previous findings obtained in non-ambulatory conditions (Campino et al., 1997Go). At 38 weeks gestation, the ratio of PRL/oestradiol provides several advantages over the ratio in post-partum. One is time, since some women had already reinitiated menses by 3 months post-partum. A second advantage is that a basal sample against the situation during post-partum in which the blood sample needs to be obtained 30 min after a suckling episode separated at least 90 min from the preceding one (Campino et al., 1997Go). Our findings will permit health professionals to detect in advance women that will experience long or short lactational amenorrhoea, improving security of natural family planning methods based upon lactational amenorrhoea.


    Acknowledgements
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are indebted to Dr A.F.Parlow, Scientific Director of the National Hormone and Pituitary Program of the NIDDK for providing the hPL antiserum, to the investigators from the Laboratorio de Medicina Nuclear, Centro de Diagnóstico, Pontificia Universidad Católica de Chile: Dr Arnaldo Foradori and Ms Carmen Gloria Velásquez and Julia Soto for the measurements of SHBG, progesterone, TSH and post-partum plasma oestradiol and to Dr Lorna Moore from the University of Denver, Colorado and Dr G.J.Valenzuela from Arrowhead Medical Center, San Bernardino, CA for critical reviews of the manuscript. Grant support was received from project Fondecyt 1971–105 and WHO RMG/LABENDO2.


    Notes
 
6 To whom correspondence should be addressed at: Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile, Santiago, Chile. E-mail: mseron{at}genes.bio.puc.cl Back


    References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 11, 2001; accepted on September 6, 2001.