Clinical Endocrinology (1976) 5, 643-650.
PROLACTIN LEVELS D U R I N G THE MENSTRUAL CYCLE* P. F R A N C H I M O N T , C. D O U R C Y , J. J. LEGROS,? A. REUTER, Y. VRINDTS-GEVAERT, J. R. VAN C A U W E N B E R G E A N D u. G A S P A R D Institute of Medicine-Immunology Laboratory, lnstitut National des Radio-elements-Fleurur, and Department of Gynaecology and Obstetrics, University of Liege (Receiced 22 January 1976;revised 25 June 1976;accepted 30 June 1976)
SUMMARY
The levels of prolactin, FSH, LH, oestradiol and progesterone were measured daily during fourteen ovulatory cycles. The behaviour of FSH, LH, oestradiol and progesterone was classical. Non-systematic changes occurred in prolactin levels during the course of the menstrual cycle with the highest level being either during the ovulatory period or during the luteal phase. However, the mean level of prolactin was significantly higher during the ovulatory and luteal phases than during the follicular phase. A direct relationship between oestradiol and prolactin levels was noted, although there was no correlation between prolactin on the one hand and FSH, LH and progesterone on the other. Changes in prolactin levels during the menstrual cycle still remain very controversial. Certain investigators (Hwang et al., 1971; Friesen et al., 1972) have failed to observe any systematic fluctuation on prolactin levels during the cycle while the group of L'Hermite (L'Hermite et al., 1972; Robyn et al., 1973) have found an increase during the late follicular phase and the ovulatory period. The later investigators have also observed that prolactin levels during the luteal phase are higher than during the follicular phase. In the study of McNeilly & Chard (1974) carried out in seventeen female volunteers, changes in prolactin levels were irregular and not constant during the cycle, although certain women had increased levels at the time of the ovulatory LH surge and had values higher than those of the follicular phase during the luteal phase. We have re-investigated this problem carrying out daily assays of prolactin in fourteen normally cycling women and subjecting the results to appropriate statistical analysis correlating the changes with well-known changes in the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol and progesterone. Supported by Grants from the Fonds Cancerologiques de la Caisse d'Epargne et de Retraite and from the F.R.S.M. 20305. 7 Charge de recherches of the FNRS. Correspondence: Dr P. Franchimont, Institute of Medicine-Immunology Laboratory, The University, Liege, France.
643
644
P. Franchimont et al. MATERIAL A N D METHODS
Subjects investigated Fourteen women aged 22-35 years were bled daily throughout an ovulatory cycle between 08.00 and 10.00 hours for the measurement of FSH, LH, prolactin, oestradiol and progesterone. In all cases the cycles investigated were characterized by a duration of 26-30 days, by a peak of FSH and LH just prior to the shift in base1 body temperature and by levels of progesterone greater than 10 ng/ml during the luteal phase which lasted at least 12 days and by biphasic behaviour of oestradiol. Assay methods 1. FSH and LH were assayed using the method of Franchimont (1968, 1973). The results are expressed in miu/ml of preparation 68/39 for FSH and 68/40for LH (Medical Research Council). 2. Oestradiol and progesterone were measured by the radioimmunoassay methods described by Malvano et 01. (1974) using kits supplied by CEA-IRE Sorin. 3. Prolactin was measured by a homologous radioimmunoassay (Reuter et al., 1976). We shall only record the data for this technique. Labelling ofprolactin. 5 pg of human prolactin (Calbiochem)were labelled with 1 mCi’’’1 using the method of Greenwood et al. (1963). 10 pg of chloramine T was used for 5 pg of hormone. Separation of labelled immunoreactive prolactin on the one hand and the radioactive ions and degraded polymerized fractions on the other, was by means of gel filtration on Sephadex G75 (90/1 cm) using Sorenson phosphate buffer 005 M pH 7.5 containing 0.1 % of bovine albumin and 0.05% sodium azide. Incubation. The incubation volume consisted of 0-35ml of phosphate buffer 0.05 M pH 7-5 containing 0.05% sodiuh azide and 0.5% bovine albumin. It contained 0.2 ng of labelled prolactin, prolactin antiserum at a final dilution of 1/30OOO and unlabelled hormone in progressivelyincreasing doses, or 0 0 5 ml of the serum to be assayed. Incubation was for 2 days at 18°C 0 5 ml of anti-rabbit y-globulin immunoadsorbent was then added (Reuter et al., 1973). After 5 h of incubation at room temperature with continuous rotation the immunoadsorbent was separated by centrifugation at 2500 g, washed and counted. With this method, the assay of prolactin is highly specific as there is no cross reaction with pituitary and placental hormones. The sensitivity of the assay is 0.1425 ng (or 4-10 pu) per tube. The coefficient of variation is less than 3% within assay and less than 12% between assay. Reference material. We used the preparation of the Medical Research Council 71/222, the potency of which by radioimmunoassayis 40 units for 1 mg of the preparation provided by NIH (NIH-V-L-S No 1). On all occasions samples from an individual cycle were measured in triplicate within the same assay. Statistical analysis For each of the hormones the mean level was determined for the days of, before and after the day of the ovulatory LH peak. Further, for each woman the mean levels of FSH, LH, prolactin, oestradiol and progesterone were calculated for the follicular, ovulatory and luteal phases as defined by Judd & Yen (1973), the ovulatory period corresponding to the 2 days prior to and the 2 days following the LH peak. The means of the individual means for
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these hormones were then calculated for the three phases of the cycle and the differences
. analysed by Students’ ‘ t y test for paired samples (Snedecor & Cochran, 1967) using as a reference the values for either the follicular or ovulatory phases. To assess the statistical significance, Wilcoxon test was also applied (Siegel, 1956). Further correlations were sought between the prolactin levels on the one hand and the levels of FSH, LH, oestradiol and progesterone. In ten of the cycles where the assays of the five hormones were carried out under these conditions, a correlation was sought between the increases in the levels of oestradiol progesterone and prolactin during the ovulatory and luteal phases, as compared with the mean levels observed during the follicular phase. RESULTS Fig. l(a) shows the classic course of the levels of FSH, LH, oestradiol and progesterone during the menstrual cycle. In all cases, a pre-ovulatory peak of FSH and LH was seen as were elevated levels of FSH at the commencement of the cycle. Likewise, oestradiol showed two peaks during the cycle. The pre-ovulatory peak occurred prior to that of LH. Progesterone levels were low or unmeasurable during the follicular phase. They increased significantly at the time of the pre-ovulatory LH peak and plateaued during the luteal phase. In contrast to these systematic hormonal changes, prolactin levels changed from one day to the next in a non-systematic fashion; the highest levels were seen in seven cases during the ovulatory phase either on the day of the LH peak (four), on the 2 days before that (two), on the day following (one) and in seven other cases during the last 8 days of the luteal phase. The highest mean level was Seen on the day of the pre-ovulatory LH peak (Fig. 1b). When one compares the mean levels obtained in each of the fourteen women during the three periods of the cycle, it is apparent that the mean levels of prolactin increased during the ovulatory period in all cases and in the luteal phase in twelve of fourteen, as compared with
%
Folliiulor phose
Luteal phorc
180 160
140 I 20
100
80
s-
60
40
p c 001 20
FIG.2. Individual changesof mean prolactinlevels expressedas a percentageof the mean values in the reference period as 100%. On the left of the graph the reference period is the follicular phase whereas on the right of the graph it is the ovulatory phase.
~
FSH (miulml) (68/39) 6.8f0.67 LH (miulml) (68/40) 5.47-10.7 Prolactin (pu/ml) 264k30 (71/222) 163.8-113.3 Oestradiol (pg/ml) Progesterone (nglml) 0.29k0.18
MeanfSEM
261.6-116.8 159 244-1 1.3 703
100
100 100
129 297 132
%
8.8k0.86 16.24+2 349-149
MeanfSEM
228.8-122.7 139 12.58f5.2 4337
%
4.32
PROLACTIN LEVELS D U R I N G THE MENSTRUAL CYCLE* P. F R A N C H I M O N T , C. D O U R C Y , J. J. LEGROS,? A. REUTER, Y. VRINDTS-GEVAERT, J. R. VAN C A U W E N B E R G E A N D u. G A S P A R D Institute of Medicine-Immunology Laboratory, lnstitut National des Radio-elements-Fleurur, and Department of Gynaecology and Obstetrics, University of Liege (Receiced 22 January 1976;revised 25 June 1976;accepted 30 June 1976)
SUMMARY
The levels of prolactin, FSH, LH, oestradiol and progesterone were measured daily during fourteen ovulatory cycles. The behaviour of FSH, LH, oestradiol and progesterone was classical. Non-systematic changes occurred in prolactin levels during the course of the menstrual cycle with the highest level being either during the ovulatory period or during the luteal phase. However, the mean level of prolactin was significantly higher during the ovulatory and luteal phases than during the follicular phase. A direct relationship between oestradiol and prolactin levels was noted, although there was no correlation between prolactin on the one hand and FSH, LH and progesterone on the other. Changes in prolactin levels during the menstrual cycle still remain very controversial. Certain investigators (Hwang et al., 1971; Friesen et al., 1972) have failed to observe any systematic fluctuation on prolactin levels during the cycle while the group of L'Hermite (L'Hermite et al., 1972; Robyn et al., 1973) have found an increase during the late follicular phase and the ovulatory period. The later investigators have also observed that prolactin levels during the luteal phase are higher than during the follicular phase. In the study of McNeilly & Chard (1974) carried out in seventeen female volunteers, changes in prolactin levels were irregular and not constant during the cycle, although certain women had increased levels at the time of the ovulatory LH surge and had values higher than those of the follicular phase during the luteal phase. We have re-investigated this problem carrying out daily assays of prolactin in fourteen normally cycling women and subjecting the results to appropriate statistical analysis correlating the changes with well-known changes in the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol and progesterone. Supported by Grants from the Fonds Cancerologiques de la Caisse d'Epargne et de Retraite and from the F.R.S.M. 20305. 7 Charge de recherches of the FNRS. Correspondence: Dr P. Franchimont, Institute of Medicine-Immunology Laboratory, The University, Liege, France.
643
644
P. Franchimont et al. MATERIAL A N D METHODS
Subjects investigated Fourteen women aged 22-35 years were bled daily throughout an ovulatory cycle between 08.00 and 10.00 hours for the measurement of FSH, LH, prolactin, oestradiol and progesterone. In all cases the cycles investigated were characterized by a duration of 26-30 days, by a peak of FSH and LH just prior to the shift in base1 body temperature and by levels of progesterone greater than 10 ng/ml during the luteal phase which lasted at least 12 days and by biphasic behaviour of oestradiol. Assay methods 1. FSH and LH were assayed using the method of Franchimont (1968, 1973). The results are expressed in miu/ml of preparation 68/39 for FSH and 68/40for LH (Medical Research Council). 2. Oestradiol and progesterone were measured by the radioimmunoassay methods described by Malvano et 01. (1974) using kits supplied by CEA-IRE Sorin. 3. Prolactin was measured by a homologous radioimmunoassay (Reuter et al., 1976). We shall only record the data for this technique. Labelling ofprolactin. 5 pg of human prolactin (Calbiochem)were labelled with 1 mCi’’’1 using the method of Greenwood et al. (1963). 10 pg of chloramine T was used for 5 pg of hormone. Separation of labelled immunoreactive prolactin on the one hand and the radioactive ions and degraded polymerized fractions on the other, was by means of gel filtration on Sephadex G75 (90/1 cm) using Sorenson phosphate buffer 005 M pH 7.5 containing 0.1 % of bovine albumin and 0.05% sodium azide. Incubation. The incubation volume consisted of 0-35ml of phosphate buffer 0.05 M pH 7-5 containing 0.05% sodiuh azide and 0.5% bovine albumin. It contained 0.2 ng of labelled prolactin, prolactin antiserum at a final dilution of 1/30OOO and unlabelled hormone in progressivelyincreasing doses, or 0 0 5 ml of the serum to be assayed. Incubation was for 2 days at 18°C 0 5 ml of anti-rabbit y-globulin immunoadsorbent was then added (Reuter et al., 1973). After 5 h of incubation at room temperature with continuous rotation the immunoadsorbent was separated by centrifugation at 2500 g, washed and counted. With this method, the assay of prolactin is highly specific as there is no cross reaction with pituitary and placental hormones. The sensitivity of the assay is 0.1425 ng (or 4-10 pu) per tube. The coefficient of variation is less than 3% within assay and less than 12% between assay. Reference material. We used the preparation of the Medical Research Council 71/222, the potency of which by radioimmunoassayis 40 units for 1 mg of the preparation provided by NIH (NIH-V-L-S No 1). On all occasions samples from an individual cycle were measured in triplicate within the same assay. Statistical analysis For each of the hormones the mean level was determined for the days of, before and after the day of the ovulatory LH peak. Further, for each woman the mean levels of FSH, LH, prolactin, oestradiol and progesterone were calculated for the follicular, ovulatory and luteal phases as defined by Judd & Yen (1973), the ovulatory period corresponding to the 2 days prior to and the 2 days following the LH peak. The means of the individual means for
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646
P. Franchimont et al.
these hormones were then calculated for the three phases of the cycle and the differences
. analysed by Students’ ‘ t y test for paired samples (Snedecor & Cochran, 1967) using as a reference the values for either the follicular or ovulatory phases. To assess the statistical significance, Wilcoxon test was also applied (Siegel, 1956). Further correlations were sought between the prolactin levels on the one hand and the levels of FSH, LH, oestradiol and progesterone. In ten of the cycles where the assays of the five hormones were carried out under these conditions, a correlation was sought between the increases in the levels of oestradiol progesterone and prolactin during the ovulatory and luteal phases, as compared with the mean levels observed during the follicular phase. RESULTS Fig. l(a) shows the classic course of the levels of FSH, LH, oestradiol and progesterone during the menstrual cycle. In all cases, a pre-ovulatory peak of FSH and LH was seen as were elevated levels of FSH at the commencement of the cycle. Likewise, oestradiol showed two peaks during the cycle. The pre-ovulatory peak occurred prior to that of LH. Progesterone levels were low or unmeasurable during the follicular phase. They increased significantly at the time of the pre-ovulatory LH peak and plateaued during the luteal phase. In contrast to these systematic hormonal changes, prolactin levels changed from one day to the next in a non-systematic fashion; the highest levels were seen in seven cases during the ovulatory phase either on the day of the LH peak (four), on the 2 days before that (two), on the day following (one) and in seven other cases during the last 8 days of the luteal phase. The highest mean level was Seen on the day of the pre-ovulatory LH peak (Fig. 1b). When one compares the mean levels obtained in each of the fourteen women during the three periods of the cycle, it is apparent that the mean levels of prolactin increased during the ovulatory period in all cases and in the luteal phase in twelve of fourteen, as compared with
%
Folliiulor phose
Luteal phorc
180 160
140 I 20
100
80
s-
60
40
p c 001 20
FIG.2. Individual changesof mean prolactinlevels expressedas a percentageof the mean values in the reference period as 100%. On the left of the graph the reference period is the follicular phase whereas on the right of the graph it is the ovulatory phase.
~
FSH (miulml) (68/39) 6.8f0.67 LH (miulml) (68/40) 5.47-10.7 Prolactin (pu/ml) 264k30 (71/222) 163.8-113.3 Oestradiol (pg/ml) Progesterone (nglml) 0.29k0.18
MeanfSEM
261.6-116.8 159 244-1 1.3 703
100
100 100
129 297 132
%
8.8k0.86 16.24+2 349-149
MeanfSEM
228.8-122.7 139 12.58f5.2 4337
%
4.32
