Gynecol. Ettdocrinol. 5 (1991) 15-32
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Thyrotropin-releasing hormone (TRH) and metoclopramide testing in infertile women I. Gerhard, W . Eggert-Kruse, K. Merzoug, K. Klinga and B. I2.50 ng/nil 2.5-1 3 0 pg/1nl
Testo\tcrone
Serono
< 3 0 0 pg/nll
DHEAS TSH
Iliagn. Prod. Corporation
< 4000 ng/1nl
Uehrinpvcrkc
hJ5'1l
Progcwroiie
l h g i i . l'rod. Corporation
> 1 0 ng/iIll 111 Iuteal phaw
SHK*
0-0 lllU/I
10-400 nmol/l
*IRMA
At the time of the female patients' endocrine evaluation, the male partners had an andrological examination which included a sperm analysis. In the cycles whiih followed the endocrinological work-up, the uterine and tuba1 factors were studiiod by hysterosalpingography and/or laparoscopy, and the speriIi-tiiucus interaction was assessed with the Sims-Huhner postcoital test (PCT).When the PCT \vas negative ( 1I propulsive motile spermatozoon per high-power field (HPF)), or poor (2-6 propulsive motile spermatozoa), it was repeated during the following cycle after pretreatment with ethinylestradiol (80 pgper day from the 3rd to 9th day ofcycle). The cervical mucus was assessed using a score described by Insler'. The same regimen was used in evaluating
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18
Gerhard et al.
anovulating patients. In the presence of 2 normal Insler score (> 10) and a reduced number of motile spermatozoa (< 7/HPF), the examination was extended to include various in vitro sperm penetration tests'".". After completion of the diagnostic procedures, most women went on to receive individual treatment: compensated and subclinical hypothyroidism with levothyroxine 100 pg/day ( n = 103); hyperprolactinemia (basal prolactin < 20 ng/ml and/or prolactin 30 min after metoclopramide > 250 ng/ml) with a dopamine agonist (bromocriptine or lisuride, n = 121); hyperandrogenemia (basal testosterone > 500 p g / d and/or DHEAS > 4000 ng/ml) with dexamethasone 0.5-0.75 mg/day ( n = 123); luteal insufficiency (endometrial biopsy out ofphase for more than 2 days, or mean luteal phase progesterone value < 10 ng/ml) with progesterone supplementation or clomiphene (n = 50), and anovulation, without the above described hormonal disorders, with clomiphene, gonadotropins or pulsatile gonadotropin releasing hormone infusions ( n = 57). Some of the women, with proven tuba1 patency on at least one side and a normal postcoital test, were assigned to one of three randomized treatment protocols: hyperandrogenemia was treated with dexamethasone or clomiphene ( n = 80), luteal insufficiency and unexplained infertility had therapy with bromocriptine, clomiphene or progesterone suppositories ( n = 85) (2 x 25 mg intravagmally over 10 days according to Jones'?). When patients had multiple abnormal hormone concentrations, therapy occurred in 3-month time kames, ordered as follows: thyroid substitution, dopamine agonist, dexamethasone, antiestrogen therapy. Treatment was interrupted for 1-5 months between the various therapy stages. If prolactin determinations during the luteal phase of clomiphene- or gonadotropin-stimulated cycles identified elevated concentrations, dopamine agonists were administered during the following treatment cycles. The number of patients receiving medication was smaller than the frequency with which hormonal disorders were seen in our patient population, previously described', since several women ( n = 98) conceived spontaneously, were lost from follow-up, or were not compliant. Thus, the population of this paper included 191 women who had been treated with dopamine agonists at some point (Group B), while 577 women never received this substance (Group A). Patients' data were encoded and processed at the University Computer Center, Heidelberg. SAS was used for the statistical evaluation. Since there was no normal distribution of hormone values, non-parametric tests were applied: the Kruskal-Wallis test, the Wilcoxon test for dependent samples, the Wilcoxon test for independent samples, the x' test, and the Spearman correlation coefficent.
Results Thyroid function disturbances Out ofa total of834 TRH stimulation tests, 666 (80%)were normal (Table 2). A total of 65 patients were already receiving thyroid hormones. Six patients
19
TRH and metoclopramidc testing in infertile women
Table 2 Frequency of various obtcrved thyroid function disturbances identified by TKH testing of 834 women with sterility TRH test
fl
666 65
79.8
Blocked
6
0.7
Compensated hypothyroid
3
0.4
94
11.3
NOrIlld Under levothyroxine
Subclimcal hypothyroid Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 12/06/14 For personal use only.
1%
7.8
ZOO
834
Total
displayed an insufficient TSH increase following T K H . Hyperthyroidism was confirmed in these patients through internal examination. Compensated hypothyroidism was found in three women; subclinical hypothyroidism was suspected in 04 women. Thus, 20% of all patients had thyroid disorders, twothirds being diagnosed through T K H testing. In order to determine the effect which thyroid disorders have on fertility, the basal and stimulated TSH levels were compared to various serum hormones in both the follicular and the luteal phase (Table 3 ) . Significant correlations were only noted for the prolactin levels in the fohcular phase and following metoclopramide in the luteal phase. The correlation coefficients were, however, very low. In addition, stimulated TSH levels correlated positively with the progesterone concentration, arid with the progesterone-estradiol-17p ratio in the luteal phase. It was not possible to demonstrate a relationship for any of the other hormones (gonadotropins, androgens, SHBG, estradiol). Premenstrual endometrial biopsies were obtained in 62 women with hypothyroidism, and in 384 women with euthyroidism. The histological dating was out of phase by
Table 3 Significant correlations (Spearman coefficient) observed between basal and stimulated TSH levels, and different hormone concentrations determined during the cycle. P/E,, progesterone-estradiol-17P ratio; 15'TRH and 30' TRH, levels 15 min and 30 min post-TKH stiniulation; 30'met., 30 miii post-metoclopramide stimulation (p < 0.01) Follintlar pitas?
TSH
Basal 30' TKH
ATSH
Luted phase
Prolactin
Prolactin 30'met.
Progesterone
PIE,
Basal
l5'TRH
30'TRH
0.100
0.102 0.225
0.080
-
-
-
-
0.090
0.729
0.161 0.167
0.032
-
0.252 0.261
0.082
0.088
20
Gerhard et al.
Table 4 Relationship between T R H testing and postcoital testing. (Pathological: fewer than 1 propulsive motile spermatozoa per high power field; normal: 2 and more propulsive motile spermatozoa per high power field.) Pathological postcoital tests were more frequent among women with hypothyroidism (p < 0.05) ~
Thyroidfunction
Postcoital test* Normal
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Total
Pathological
n
%
n
%
Hypothyroid Euthyroid
69 532
44 400
64 75
25 132
36 25
Total
60 1
444
74
157
26
* 8-12
h following intercourse, cervical index > 10 (Insler score)
more than 2 days in 56% of the hypothyroid, and in 61% of the euthyroid patients (not significant). When non-hormonal causes of sterility (tubal-uterine factor, cervical, male) were matched to thyroid function test results, we failed to note any significant differences between women with normal and pathological thyroid function. However, the PCT yielded significantly poorer results in women with hypothyroidism, when compared to women with euthyroidism (Table 4, p < 0.05). Pregnancy occurred in 39% of the women with euthyroidism, and in 32% of those with hypothyroid function (Table 5). Two of the three patients with compensated hypothyroidism, and 29 of the 94 women with subclinical hypothyroidism, conceived. Out of six hyperthyroid patients three became pregnant following therapy. O f the patients with thyroid disorders, 6% had spontaneous pregnancies if they failed to receive substitution therapy. In comparison, spontaneous pregTable 5 Pregnancy and abortion rates observed during a 2-year period in women with subclinical hypothyroidism. Results are compared with euthyroidism. A = abortion rate Thyroid Junction
Pregnancy None
Hypothyroid (n = 97) Euthyroid ( n = 666) Total (n = 763)
Therapy
Spontaneous
n
O/o
n
66
68
6
%
6
n
25
(A = 17%)
404
61
109
16
62
115
15
26 (A = 18%)
153
(A = 28%)
470
94
23 (A = 21%)
178
23
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TRH and merodoprarnide testing in infertile women
21
nancies occuired in 16% ofthe euthyroid patients. Out of the 97 hypothyroid patients 11 blxame pregnant following single thyroxine substitution therapy, while 14%ofthe wonien went on to pregnancy after the additional administration of clomiphene or hMG. The rate of abortion was 17% for hypothyroid and 28% for euthyroid women, which did not represent a significant difference. To explain why a low pregnancy rate existed in compensated and subclinical hypothyroid patients despite thyroxine substitution, the remaining fertility inhibiting factors were examined. It was shown that women who failed to conceive displayed significantly poorer tubal factors, and their partners had both significantly low sperm counts, and lower percentages of niotile spermatozoa (x' test, p < 0.01, Figure 1; for definitions of tubal and andrologcal factor see Gerhard et al.').
L a t ~ n thyperlirolactine~iia Serum prolactin concentrations were determined for 759 patients in the early follicular phase following T R H , and during the same cycle in the luteal phase following metoclopramide administration. Figure 2 shows the averages, medians, percentiles and the ranges for the entire population. Basal prolactin levels were significantly higher in the luteal phase (median 9 ng/ml) than in the early follicular phase (median 7.2 ng/ml, p < 0.01). Futhermore, stimulated prolactin levels were 3-4 times higher in the h e a l phase following metoclopramide application (median 162 ng/ml) than after use of T K H (median 51 ng/nd) in the early follicular phase. There was a significant negative correlation ( Y = -0.42) between the stimulated prolactin levels and the body mass index (kg body weight per m3 body surface) (p < 0.001). As expected a significant correlation existed between the various prolactin concentrations (Figure 3). The basal prolactin level in the follicular phase had the lowest correlation to prolactin concentrations after metoclopramide stimulation. The lowest correlation of basal prolactin level in the luteal phase was with the level of prolactin following metoclopramide stimulation. Basal prolactin concentrations in the early follicular phase, as well as in the luteal phase, exhibited very weak positive correlations (p < 0.01) to the concentrations of DtIEAS ( r = 0.12), estradiol-17P ( r = 0.18), testosterone/SHBG ratio ( r = 0.13) and estradiol (r = 0.15). The correlation was negative for the progesterone values ( r = - 0.16) in the early h e a l phase. Stimulated prolactin concentrati'ons after T R H showed positive correlations to FSH (r = 0.12), estradiol-1r'P ( r = 0.13) and SHBG ( Y = 0.14), and to estradiol-17P in the luteal phase (r = 0.15). A positive correlation between prolactin and progesterone was only found in the late luteal phase ( r = 0.1 1). Metoclopranlide-stimulated prolactin levels exhibited a significant negative correlation to the LH/FSH ratio ( r = - 0.14) of the follicular phase, and had a positive correlation to progesterone ( r = 0.19) and estradiol-17P ( r = 0.31) in the luteal phase. All the other correlation coefficients were below 0.1 1. The norrnal range for prolactin concentrations following T K H stimulation did not exist, and different levels were reported in the literature for meto-
n
c
I
lxesm
m
46 18
r w e g m
9
lxesm
p < 0.01
m
22
tuba1 patency
pregnant
p < 0.01
I
22
m
r w e g m
sperm count
WM
I
poor normal
0.very poor
1 I
56
p * 0.01
sperm count
Figure 1 Non-hormonal causes of sterility in women with subclinical hypothyroidism, who either became pregnant through levothyroxine substitution or remained sterile.X?testlevel ofsignificance is p e n . Results ofall diagnostic procedures were not available for all women, so that the number ofpatients vaned between the groups
0
20
40
60
80
100
%
result of resutt of hysterosalpingography chromolaparoscopy
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?
Q
& 2
P 3-
-
Basal
WIOG
Follicular phase
15'TRH
X
>1m
30'TRH
X
X
Basal
X
W
#/
r/
I 30'meto
X
25%
mh
10%
mean medii
75%
+ -
Luteal phase
1+1
X
I 90%
rnax
I
0
50
100
150
:
-
- 250 - 200
- 300
Figure 2 Prolactin concentrations observed i n the early follicular phase 15 i i m and 30 min after ctimulation with T l l H ( I S'TRH and 30'TllH), and during the luteat phase 30 inin after stimulation with inetoclopramidc (30'meto.). Listed are thc means, medians, percentiles and highest (max) and lowest (min) levels
n = 759
0
40'
60-
ao
100-
x
528 x
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-. 3
a
3
n
24
Gerhard et al.
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prolactin
basal1
I
TRH 15'
TRH 3 0
basal 2
meto 30.
folllcular phase
Figure 3 Correlation coefficients, according to Spearman, between the basal prolactin level in the early follicular phase (prolactinl) (empty bars), the basal prolactin level in the luteal phase (prolactin 2) (hatched bars), and the stimulated prolactin level. The lowest, though still significant correlations, were found to exist between the basal levels and the metoclopramide stimulated levels
clopramide-stimulated prolactin. Therefore we decided to use values greater than the 90th percentile of the collective as being abnormal. Borderline levels for stimulated prolactin were: 15 min following TRH, 97 ng/ml; 30 min following T R H , 76 ng/ml, and 30 min followingmetoclopramide, 254 ng/ml. Values equal to and below the 90th percentiles were considered normal. Elevated prolactin concentrations were present in 2-4% of the patients. A total of 287 (39%) of 745 women became pregnant during the 2-year period of observation (14% spontaneous, 25% medically induced). Table 6 shows the rate of pregnancy relative to the results of the prolactin tests. The overall pregnancy rate did not significantlydiffer between the five prolactin test groups, while being significantly influenced by uterine, tuba1 and andrological factors, as previously de~cribed'~. The spontaneous pregnancy rate, however, was greatly reduced when both the T R H and the metoclopramide test identified elevated prolactin levels, relative to the remaining groups (p < 0.05) without an adequate explanation by additional non-hormonal infertility factors.
25
T R H and wietoclopramide testirig in if!fertile women Table 6 l'regnincy rate compared t o prolactin levels
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~~~
~
All normal Basally raiccd TKH and meto. raised TRH raircd only M e t o raised only Total
~
522 76
28 54 65 745
~
70 10
198 26
38 34
14 12
24 22
62
4
12 25 26 287
43 46 40 39
4
13 14 14
39 33 20 25
57 54 60 61
7 c)
100
66
meto. = nictoclnpratnidc
W e direct attention to the therapy of women with medically induced pregnancies. Table 7 differentiates between monotherapy with dopamine agonists, con-lbination therapies (bromocnptine combined with clomiphene, hMG, dexaniethasone or progesterone), and therapeutic approaches without dopamine agonists. It, therefore, was not surprising that the group with basal prolactin concentrations above the 90th percentile had significantly more pregnancies when treated with dopamine agonists, than with any other therapy (x' test, p < 0.01). The same applied to elevated prolactin levels in the luteal phase (p < 0.001). O n the other hand, stimulated prolactin levels failed to correlate to the type of therapy used. A total of 21 patients with elevated
Table 7
Medical trcatint'tit resulting in conception, correlated with the prolactin levels of all pregnant woiiit'n ( n 1 186). Frequency '% is g v e n ; test was applied I'rolactiri
x'
1rilc.l
Dopamine
aSqi~tiists
Other thrrup y
I'
Basal > 90. pen:.
23
30
47
0.006
15'TKH > 90. perc.
15
11
74
ns*
3O'TKH > 90. perc. Luteal > 90. perc. 30'Meto > 90. perc. All normal
12
11
77
11s
28 23
28 14
44
0.001
64
ns
11
7
82
11s
Total
14
9
77
11s
* ns, not significant Perc. = percentilc
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26
Gerhard et al.
prolactin values 15 min post-TRH injection had a significantly higher rate of pregnancy after treatment, than those not requiring therapy. However, when therapy forms were evaluated for these 21 women, the analysis showed that seven became pregnant due to sole or combined treatment with dopamine agonists, four after thyroid substitution treatment (single or in combination), and ten after other forms of therapy (i.e. three patients, clomiphene; two patients, clomiphene-dexamethasone; two patients, gonadotropins; two patients, clomiphene-gonadotropins; one patient, progesterone substitution). The sterility and pregnancy rates of bromocriptine patients (Group B, n = 191) and of women who never received dopamine agonists (Group A, n = 577) are shown in Figure 4. Pregnancies which occurred after therapy are listed, according to the type of therapy (bromocriptine monotherapy, bromocriptine combination therapy, other). Note that in patients with elevated basal prolactin levels ( only six patients displayed concentrations over 30 ng/ ml), the spontaneous pregnancy rate was higher in Group A than in Group B (p=0.003). The male and the tuba1 factors were better for this elevated prolactin group than for the other prolactin groups (p < 0.01). The highest basal prolactin level at which a spontaneous pregnancy occurred was 26 ng/ml, while the level required to induce pregnancies was above 30 ng/ml. The pregnancy rate was highest when substances other than bromocriptine were used in patients with raised prolactin levels following initial T R H and metoclopramide stimulation, and there was a lower infertility rate in Group A than in Group B (p < 0.01). Where prolactin was raised only in the TRH or metoclopramide tests, pregnancies were induced with equal frequency with brornocriptine, or other medication. In patients with normal prolactin levels, both basal and following stimulation, sterility was higher in Group A than in Group B, due to the higher rate oftherapy-induced pregnancies in Group B (p < 0.001), and the frequency of reduced sperm counts in Group A.
Discussion
Thyroid function disturbances were observed in 23% of the sterile patients of this study by means of T R H testing. Only one-third of these patients had received levothyroxine due to thyroid illness. This corresponds with the suspected frequency of thyroid disease (20-30%) in the female population of southern Germany, and correlates with the proven goiter frequency of men (21%) in this region. The previously assumed etiologic role of thyroid dysfunction in infertile women may thus have been overestimated'. A possible role ofsubclinical hypothyroidism in infertility was suggested since thyroid antibodies were seen more frequently in women with overshooting responses during T R H testing, than in patients with a normal TRH responseI4. Furthermore, it was shown that asymptomatic patients with thyroid antibodies had pathologcal results more frequently during T R H testingI5.Lowered free thyroxine concentrations were measured in 60% ofthe women with preclinical
(n)
0
10
20
30
40
50
brornocriptine Induced pregnancy with brornocriptine in combination
Induced pregnancy with
2
2
z-
2
\
,-
E
-.. -..
-. 3
2
3
Induced pregnancy (different drugs)
"
z
G
Spontaneous pregnancy
Figure 4 Pregnancy rate compared to the prolactin concentration of women w h o received broniocriptnie at wine point (Group 13, i t = 1 O l ) , and ie ~nfertilityr a e higher 111 A than 111 U OJ < O.O(I1). those w h o were iiever treated with doparnine dgotiists (Group A, ! I = 577). Normal b a s ~ ~ l i tprolactin: I'rolactin bayally raised: ypoiitaiieou~pregnancy ratc higher in A than in 13, infertility rate higher in I3 t h x i in A (JJ = 0.0003). Elevatcd prolactin aftcr
5
a
C
* 0
E
Q, c
-8
60
70
80
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28
Gevhavd et al.
In this study, various hormones were determined together with TSH, permitting the direct comparison between TSH and gonadotropins, estradiol, androgens and prolactin. However, only TSH and prolactin concentrations were positively correlated. As in compensated thyroxine deficiency, the pituitary sensitivity for T R H is increased. Since T R H increases prolactin release, compensated hypothyroidism might influence ovarian action via hyperprolactinemia. Patients with subclinical hypothyroidism had a significantly poorer postcoital test than women with normal thyroid function. Neither group differed with respect to the cervical index, or the partner’s spermiogram, so that immunological dysfunction can be assumed. While Pencea et al.” found sperm antibodies in 6.7% of the examined women with euthyroidism, and in 12% of his patients with thyroid function disturbances, we failed to identify a relationship between thyroid antibodies and an immunological factorT4. The spontaneous pregnancy rate was lower in hypothyroid women (6%)than in euthyroid (16%) patients. Pregnancies occurred in 12% of the women after levothyroxine substitution only. Bohnet et aL3 reported a pregnancy rate of lo%, after therapy with only 50 pg levothyroxine, during a treatment period of 1 month. Louvet et al.”, on the other hand, achieved a 54%pregnancy rate in 37 women with anovulation. The low pregnancy rate in our study is due to the fact that non-hormonal causes of sterility did not represent criteria for exclusion from therapy. While patients with thyroid dysfunction have normal basal prolactin levels, the TRH-stimulated prolactin release is exaggerated in hypothyroidism, and decreased in hyperthyroidism’9. Following levothyroxine replacement therapy in subclinical hypothyroid patients, the plasma prolactin and TSH secretion is inhibited when metoclopramide and T R H are gwenT.s.T R H synthesis is regulated by levothyroxine by way of a negative feedback system. In hyperprolactinemic women, the mean basal TSH concentrations were found to be similar to those in normal women, but the incremental TSH response to T R H stimulation was considerably greater”’. The association between hyperprolactinemia and disturbed ovulatory function may in part be explained by activation of the prolactin-dopamine shortloop feedback. This activation increases dopaminergic activity in the medial basal hypothalamus, and inhibits gonadotropin-releasing hormone (GnRH) release”. Under certain in vitvo conditions, human prolactin can inhibit human chorionic gonadotropin (hCG)-stimulated production of estradiol and progesterone by human granulosa cells obtained from preovulatory
follicle^".'^. Since the TSH and prolactin responses to T R H fail to be affected by the menstrual cyclez4,we chose the early follicular phase to carry out T R H testing, and assess the basal values of androgens, gonadotropins and estradiol. Thus, the interval between T R H and metoclopramide testing was adequate to exclude an influence of one test on the next. The metoclopramide test was performed during days 20-23 of the cycle, as proposed by LeidenbergerZs,who observed the greatest prolactin response to metoclopramide at this time. Moreover,
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TRH and metoclopramide testing in infertile women
29
Bohnet’ observed a significant correlation between the spontaneous nocturnal prolactin peaks in infertile women, and the peak concentrations of prolactin after metoclopramide. In our patients the prolactin response to metoclopraniide was about 4 tinies higher than after T K H . This was due in part to the submaximal T R H dose administered, since 500 pg T R H are required to produce a maximum prolactin secretion. O n the other hand, estradiol concentrations were higher in the luteal than in the follicular phase, and it is known that estrogen or estrogen treatment increases the :prolactin response to T R H and to meto~lopramide~~~”. Basal prolactin concentrations were positively correlated to DHEAS as well as the testosterone/SHBG ratio, suggesting stimulation. McKenna et al. ”, described ele.vated prolactin concentrations in women with hirsutism and elevated androgens. According to Lob0 et al.”’, prolactin modulates the secretion of DHEAS. An increase in prolactin concentrations is associated with elevated concentrations of DHEAS, whereas a decrease is followed by a fall in DHEAS. All stimulated prolactin concentrations were negatively correlated with the body mass index. This corresponds to data presented by Cavagini et al.””, who observed significantly lower prolactin secretion after T R H injection in obese patients, than in nornial subjects. We were able to demonstrate in an earlier study3’that luteal phase progesterone concentrations are inversely related to the body mass index. This result fits in with the observation that the response of prolactin to stimulation is exaggerated in the presence of elevated progesterone and estradiol concentrations in the luteal phase. In amenorrhea, the prolactin and TSH response to inetoclopramide was significantly less pronounced than in eumenorrhea, suggestive of raised dopaminergic tone in the former group3’. Though a correlation existed between all prolactin concentrations, both baseline and stimulated, the link between the baseline values and prolactin concentrations after metoclopramide was lowest. Therefore, metoclopramide testing was considered to give maximum additional information. However, conflicting d,ata have been reported for the borderline region, where values ranged from 160 ng/nll to 300 ng/rnP6. Therefore, the 90th percentile was chosen for TRH, as well as for the metoclopramide test. Treatment with dopamine agonists was recommended to all patients w h o had raised basal prolactin concentrations. Furthermore, those with prolactin values above 250 n g / d after rnetoclopramide stimulation received the same treatment. Not all women accepted treatment, however. In the course of randomized studies, women with a normal prolactin pattern received dopamine agonists, as well. The overall pregnancy rate was similar for all prolactin groups. The spontaneous pregnancy rate, however, was lowest in women with an exaggerated response to T R H and metoclopramide. The evaluation of the data for this group failed to identifi a particularly advantageous medication”. In the past:, latent hyperprolactinemia was felt to contribute to infertility. Better response rates to bromocriptine therapy were observed in patients with excessive. as well as borderline, exaggerated responses of prolactin to TRH or nietoclopraniide administration than in patients with normal responses’.’.
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30
Gerhard et al.
We did not succeed in inducing a higher pregnancy rate with dopamine agonist treatment in women with a pathological T R H or metoclopramide test result. The infertility rate was lower in women with normal prolactin tests who had received dopamine agonists, than in normals who had never received bromocriptine. This result confirms a previous study which demonstrated the superiority of bromocriptine treatment over clomiphene or progesterone substitution in women with unexplained infertility or luteal i n s ~ f i c i e n c y ~ ~ , irrespective of their prolactin tests. It is possible that some infertile women experience transient prolactin elevations at midcycle, which are preventable by bromocriptine. In this context, a study by Harrison34is of interest: in couples with unexplained infertility, one-third had high stress profile scores, and intermittent prolactin spikes. The pregnancy rate in such women was clearly higher with a combination of clomiphene and bromocriptine than with placebo. In comparison, results from active therapy were no more effective than placebo in women with normal stress scores and non-spiking prolactin. There were no significant differences in the T R H releasing hormone tests. Harrison's3' results, as well as our own, indicate that disturbances of prolactin secretion exist which respond to dopamine agonist treatment, and that the prolactin response to T R H or metoclopramide does not permit a prognostic differentiation.
Acknowledgements The authors thank the pharmaceutical companies Henning Berlin and KaliChemie, both of West Germany, for financial support of this study. They are grateful to the laboratory staff for performing the hormone assay, and to Mrs Mahrla for expert secretarial assistance.
References 1. Gerhard, I., Eggert-Kruse, W. and Runnebaum, B. (1988). Diagnosis and treatment of subclinical hormonal disorders in infertile women with special regard to thyroid dysfunction. Aktrrelle Endokrinologie 11. Stofwechsel, 4, 200 2. Peillon, F., Vincens, M., Cesselin, F., Doumith, R. and Mowszowicz, I. (1982). Exaggerated prolactin response of thyrotropin-releasing hormone in women with anovulatory cycles: possible role of endogenous estrogen and effect of bromocriptine. Fertil. Steril., 37,530 3. Bohnet, H. G., Fiedler, K. and Leidenberger, F. A. (1981). Subclinical hypothyroidism and infertility. Lancet, 2, 1278 4. Lombardi, G., Iodice, M., Miletto, P., Merola, B., Panza, N. and Annunziato, L. (1986). Prolactin and TSH-response to T R H and metoclopramide before and after therapy in subclinical hypothyroidism. Neuroendocrinology, 43, 676 5. Bohnet, H. G. (1980). Gelbkorper-Schwache als Prolaktin-bedingte Storung des Menstruationszyklus. Fortschr. d . Medizin, 98, 1618 6. Merzoug, K., Gerhard, 1. and Runnebaum, B. (1990). Incidence and
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conditiom €or therapy independent pregnancies in patients under treatment for riterility. Gebirrtsh. Frauenheilk, 50, 177 7. Noyes, K. W., Hertig, A. T . and Rock, J. (1950). Dating the endometrial biopsy. Fertil. Steril., 1, 3 8. Gerhard, I., Bechthold, E., Eggert-Kruse, W., Heberling, D. and Kunnebaum, B. (1990). Value of endometrial biopsies and serum hormone determinations in luteal insufficiency. Hum. Reprod., in press 9. Insler, V., Melmed, H., Eichenbrenner, I., Serr, D. M . and Lunenfeld, B. (1972).The cervical score. A simple semiquantitative method for monitoring of the menstrual cycle. Int. /. Gyrzaecol. Obrtet., 10, 223 10. Eggert-Kruse, W., Gerhard, I., Hofmann, H., Kunnebaum, B. and Petzoldt, D. (1987). Influence of microbial colonization of sperm-mucus interaction in vivo and in vitro. Hum. Reprod., 2, 301 11. Eggert-Kruse, W., Gerhard, I., Tilgen, W. and Kunnebaum, B. (1989). Clinical significance of crossed in vitro sperrn-cervical mucus penetration test in infertility investigation. Fertil. Steuil., 52, 1032 12. Jones, G. S. (1976). The luteal phase defect. Fertil. Steril., 27, 351 13. Gerhard, I., Keischmann, T., Eggert-Kruse, W. and Runnebaum, B. (1 990). Factors contributing to success rate of hormonal treatment in infertile women. 1. Patients history and clinical parameters. 11. Hormonal disorders and treatment. Fertilitiit, 6, 123 and 136 14. Gerhard, I., Becker, T., Eggert-Kruse, W. and Runnebaum, B. (1991). Thyroid and ovarian function in infertile women. Hirr?.~. Reprod., in press 15. Betterle, C., Callegari, G., Presotto, F., Zanette, F., Pedini, B., Kampazzo, T., Slack., K. S., Girelli, M. E. and Busnaro, U. (1987). Thyroid autoantibodies: a good marker for the study of symptomless autoimmune thyroiditis. A r t a Endocrinol., 114, 321 16. Pacchiarotti, A., Martino, E., Bartalena, L., Aghini-Lombardi, F., Grasso, L., Buratti, L., Falcone, M. and Pinchera, A. (1986). Serum free thyroid hormonses in subclinical hypothyroidism. _I. E n d o r r i d . Iiivert., 9, 315 17. Pencea, C., Zbranca-Toporas, E., Thirer, O., Moscovivi, K. and Davidescu, I>. (1979). Incidence of antispermatic antibodies in sterile women with thyroid insufficiency. RW. Roirm. Med. Etidocritiol., 17, 55 18. Louvet, J . P., Gouarre, M., Salandini, A. M. and Boulard, C. L. (1979). Hypothyroidism and anovulation. Lancet, 1, 1032 19. Ferrari, C., Parachi, A., Parisio, E., Codecasa, F., Mucci, M., Boghen, M., Gerevini, G. and Rampini, P. (1987). Serum free thyroid hormones in different degrees of hypothyroidism and in euthyroid autoimmune thyroiditis. Acta Endorritiol. Copenh., 14, 559 20. Seki, K. and Kato, K. (1985). Increased thyroid-stimulating hormone response to thyrotropin-releasing hormone in hyperprolactinemic women. /. Cliri. Errdocrid. 121etab., 61, 1138 21. Hagen, C., Andersen, A. N . and Djursing, H. (1984). Evidence ofaltered doparninergic moduldtion ofPrl, LH, FSH, G H and TSH secretion during chronic partial dopamine receptor blockade in normal women. Acta Etrdacritiol., 106, 1
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Gerhard et al.
22. Uilenbroek, J. T. J. and van den Linden, R . (1984). Effects ofprolactin on follicular oestradiol production in the rat. J . Endocrinol., 102, 245 23. Sjogren, A., Hillensjo, T., Roos, P. and Hamberger, L. (1988). Prolactin and gonadotrophin interactions on progesterone formation in cultured human granulosa cells. Hum. Reprod., 3, 601 24. Savin, C. T., Hershman, J. M., Boyd, A. E., Longcope, C. and Bacharach, P. (1978). The relationship of changes in serum estradiol and progesterone during the menstrual cycle to the thyrotropin and prolactin responses to thyrotropin-releasing hormone. J . Clin. Endocrinol. Metab., 47, 1296 25. Leidenberger, F. (1983). In praxi hat die Hormondiagnostik rationell, d.h. zielgerichtet und trennscharf zu erfolgen. HUF Verlag Essen, Gyne, 4 26. Franks, S., Mason, H. D., Shennan, K. I. J. and Sheppard, M. C. (1984). Stimulation of prolactin secretion by oestradiol in the rat is associated with increased hypothalamic release of thyrotrophin-releasing hormone. J . Endocrinol., 103, 257 27. Martin, R . H. (1983). Metoclopramide challenge: a measure of human lactotroph activity. Obstet. Gynecol., 62, 691 28. McKenna, T. J., Loughlin, T., Daly, L., Smyth, P. A., Culliton, M. and Cunningham, S. K. (1984). Variable clinical and hormonal manifestations of hyperandrogenemia. Metab: Clin.Exp., 33, 714 29. Lobo, R. A., Letzky, 0.A., Kaptein, E. M. and Goebelmann, U. (1980). Prolactin modulation of dehydroepiandrosterone sulfate secretion. A m .J . Obstet. Gynecol., 138, 632 30. Cavagnini, F., Maraschini, C., Pinto, M., Dubini, A. and Polli, E. E. (198 1). Impaired prolactin secretion in obese patients.J. Endocrinol. Invest., 4, 149 31. Gerhard, I., Schatanek, U., Klinga, K., Eggert-Kruse, W. and Runnebaum, B. (1989). Determination of sex hormone binding globulin as part of the endocrinologcal diagnostic procedure in sterility. Fertilitat, 5 , 6 32. Hagen, C., Petersen, K., Djursing, H. and Andresen, A. N. (1984). Evidence of altered dopaminergic modulation of Prl, LH and TSH secretion in patients with normoprolactinaemic amenorrhoea. Acta Endocrinol., 106, 8 33. Gerhard, I., Weidenkopf, K., Eggert-Kruse, W. and Runnebaum, B. (1988). Hinweise zur gezielten Therapie der Lutealinsuffizienz und funktionellen Sterilitat aufgrund einer randomisierten Studie. In Schirren, C. and Semm, K. (eds.) Fortschritte der Fertilitiitsforschung, Nr. 16, p. 82, KongreBbericht Bonn 1987. Berlin: Grosse-Verlag 34. Harrison, R. F. (1988). Stress spike of hyperprolactinaemia and infertility. Hum. Reprod., 3, 173
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Thyrotropin-releasing hormone (TRH) and metoclopramide testing in infertile women I. Gerhard, W . Eggert-Kruse, K. Merzoug, K. Klinga and B. I2.50 ng/nil 2.5-1 3 0 pg/1nl
Testo\tcrone
Serono
< 3 0 0 pg/nll
DHEAS TSH
Iliagn. Prod. Corporation
< 4000 ng/1nl
Uehrinpvcrkc
hJ5'1l
Progcwroiie
l h g i i . l'rod. Corporation
> 1 0 ng/iIll 111 Iuteal phaw
SHK*
0-0 lllU/I
10-400 nmol/l
*IRMA
At the time of the female patients' endocrine evaluation, the male partners had an andrological examination which included a sperm analysis. In the cycles whiih followed the endocrinological work-up, the uterine and tuba1 factors were studiiod by hysterosalpingography and/or laparoscopy, and the speriIi-tiiucus interaction was assessed with the Sims-Huhner postcoital test (PCT).When the PCT \vas negative ( 1I propulsive motile spermatozoon per high-power field (HPF)), or poor (2-6 propulsive motile spermatozoa), it was repeated during the following cycle after pretreatment with ethinylestradiol (80 pgper day from the 3rd to 9th day ofcycle). The cervical mucus was assessed using a score described by Insler'. The same regimen was used in evaluating
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18
Gerhard et al.
anovulating patients. In the presence of 2 normal Insler score (> 10) and a reduced number of motile spermatozoa (< 7/HPF), the examination was extended to include various in vitro sperm penetration tests'".". After completion of the diagnostic procedures, most women went on to receive individual treatment: compensated and subclinical hypothyroidism with levothyroxine 100 pg/day ( n = 103); hyperprolactinemia (basal prolactin < 20 ng/ml and/or prolactin 30 min after metoclopramide > 250 ng/ml) with a dopamine agonist (bromocriptine or lisuride, n = 121); hyperandrogenemia (basal testosterone > 500 p g / d and/or DHEAS > 4000 ng/ml) with dexamethasone 0.5-0.75 mg/day ( n = 123); luteal insufficiency (endometrial biopsy out ofphase for more than 2 days, or mean luteal phase progesterone value < 10 ng/ml) with progesterone supplementation or clomiphene (n = 50), and anovulation, without the above described hormonal disorders, with clomiphene, gonadotropins or pulsatile gonadotropin releasing hormone infusions ( n = 57). Some of the women, with proven tuba1 patency on at least one side and a normal postcoital test, were assigned to one of three randomized treatment protocols: hyperandrogenemia was treated with dexamethasone or clomiphene ( n = 80), luteal insufficiency and unexplained infertility had therapy with bromocriptine, clomiphene or progesterone suppositories ( n = 85) (2 x 25 mg intravagmally over 10 days according to Jones'?). When patients had multiple abnormal hormone concentrations, therapy occurred in 3-month time kames, ordered as follows: thyroid substitution, dopamine agonist, dexamethasone, antiestrogen therapy. Treatment was interrupted for 1-5 months between the various therapy stages. If prolactin determinations during the luteal phase of clomiphene- or gonadotropin-stimulated cycles identified elevated concentrations, dopamine agonists were administered during the following treatment cycles. The number of patients receiving medication was smaller than the frequency with which hormonal disorders were seen in our patient population, previously described', since several women ( n = 98) conceived spontaneously, were lost from follow-up, or were not compliant. Thus, the population of this paper included 191 women who had been treated with dopamine agonists at some point (Group B), while 577 women never received this substance (Group A). Patients' data were encoded and processed at the University Computer Center, Heidelberg. SAS was used for the statistical evaluation. Since there was no normal distribution of hormone values, non-parametric tests were applied: the Kruskal-Wallis test, the Wilcoxon test for dependent samples, the Wilcoxon test for independent samples, the x' test, and the Spearman correlation coefficent.
Results Thyroid function disturbances Out ofa total of834 TRH stimulation tests, 666 (80%)were normal (Table 2). A total of 65 patients were already receiving thyroid hormones. Six patients
19
TRH and metoclopramidc testing in infertile women
Table 2 Frequency of various obtcrved thyroid function disturbances identified by TKH testing of 834 women with sterility TRH test
fl
666 65
79.8
Blocked
6
0.7
Compensated hypothyroid
3
0.4
94
11.3
NOrIlld Under levothyroxine
Subclimcal hypothyroid Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 12/06/14 For personal use only.
1%
7.8
ZOO
834
Total
displayed an insufficient TSH increase following T K H . Hyperthyroidism was confirmed in these patients through internal examination. Compensated hypothyroidism was found in three women; subclinical hypothyroidism was suspected in 04 women. Thus, 20% of all patients had thyroid disorders, twothirds being diagnosed through T K H testing. In order to determine the effect which thyroid disorders have on fertility, the basal and stimulated TSH levels were compared to various serum hormones in both the follicular and the luteal phase (Table 3 ) . Significant correlations were only noted for the prolactin levels in the fohcular phase and following metoclopramide in the luteal phase. The correlation coefficients were, however, very low. In addition, stimulated TSH levels correlated positively with the progesterone concentration, arid with the progesterone-estradiol-17p ratio in the luteal phase. It was not possible to demonstrate a relationship for any of the other hormones (gonadotropins, androgens, SHBG, estradiol). Premenstrual endometrial biopsies were obtained in 62 women with hypothyroidism, and in 384 women with euthyroidism. The histological dating was out of phase by
Table 3 Significant correlations (Spearman coefficient) observed between basal and stimulated TSH levels, and different hormone concentrations determined during the cycle. P/E,, progesterone-estradiol-17P ratio; 15'TRH and 30' TRH, levels 15 min and 30 min post-TKH stiniulation; 30'met., 30 miii post-metoclopramide stimulation (p < 0.01) Follintlar pitas?
TSH
Basal 30' TKH
ATSH
Luted phase
Prolactin
Prolactin 30'met.
Progesterone
PIE,
Basal
l5'TRH
30'TRH
0.100
0.102 0.225
0.080
-
-
-
-
0.090
0.729
0.161 0.167
0.032
-
0.252 0.261
0.082
0.088
20
Gerhard et al.
Table 4 Relationship between T R H testing and postcoital testing. (Pathological: fewer than 1 propulsive motile spermatozoa per high power field; normal: 2 and more propulsive motile spermatozoa per high power field.) Pathological postcoital tests were more frequent among women with hypothyroidism (p < 0.05) ~
Thyroidfunction
Postcoital test* Normal
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Total
Pathological
n
%
n
%
Hypothyroid Euthyroid
69 532
44 400
64 75
25 132
36 25
Total
60 1
444
74
157
26
* 8-12
h following intercourse, cervical index > 10 (Insler score)
more than 2 days in 56% of the hypothyroid, and in 61% of the euthyroid patients (not significant). When non-hormonal causes of sterility (tubal-uterine factor, cervical, male) were matched to thyroid function test results, we failed to note any significant differences between women with normal and pathological thyroid function. However, the PCT yielded significantly poorer results in women with hypothyroidism, when compared to women with euthyroidism (Table 4, p < 0.05). Pregnancy occurred in 39% of the women with euthyroidism, and in 32% of those with hypothyroid function (Table 5). Two of the three patients with compensated hypothyroidism, and 29 of the 94 women with subclinical hypothyroidism, conceived. Out of six hyperthyroid patients three became pregnant following therapy. O f the patients with thyroid disorders, 6% had spontaneous pregnancies if they failed to receive substitution therapy. In comparison, spontaneous pregTable 5 Pregnancy and abortion rates observed during a 2-year period in women with subclinical hypothyroidism. Results are compared with euthyroidism. A = abortion rate Thyroid Junction
Pregnancy None
Hypothyroid (n = 97) Euthyroid ( n = 666) Total (n = 763)
Therapy
Spontaneous
n
O/o
n
66
68
6
%
6
n
25
(A = 17%)
404
61
109
16
62
115
15
26 (A = 18%)
153
(A = 28%)
470
94
23 (A = 21%)
178
23
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TRH and merodoprarnide testing in infertile women
21
nancies occuired in 16% ofthe euthyroid patients. Out of the 97 hypothyroid patients 11 blxame pregnant following single thyroxine substitution therapy, while 14%ofthe wonien went on to pregnancy after the additional administration of clomiphene or hMG. The rate of abortion was 17% for hypothyroid and 28% for euthyroid women, which did not represent a significant difference. To explain why a low pregnancy rate existed in compensated and subclinical hypothyroid patients despite thyroxine substitution, the remaining fertility inhibiting factors were examined. It was shown that women who failed to conceive displayed significantly poorer tubal factors, and their partners had both significantly low sperm counts, and lower percentages of niotile spermatozoa (x' test, p < 0.01, Figure 1; for definitions of tubal and andrologcal factor see Gerhard et al.').
L a t ~ n thyperlirolactine~iia Serum prolactin concentrations were determined for 759 patients in the early follicular phase following T R H , and during the same cycle in the luteal phase following metoclopramide administration. Figure 2 shows the averages, medians, percentiles and the ranges for the entire population. Basal prolactin levels were significantly higher in the luteal phase (median 9 ng/ml) than in the early follicular phase (median 7.2 ng/ml, p < 0.01). Futhermore, stimulated prolactin levels were 3-4 times higher in the h e a l phase following metoclopramide application (median 162 ng/ml) than after use of T K H (median 51 ng/nd) in the early follicular phase. There was a significant negative correlation ( Y = -0.42) between the stimulated prolactin levels and the body mass index (kg body weight per m3 body surface) (p < 0.001). As expected a significant correlation existed between the various prolactin concentrations (Figure 3). The basal prolactin level in the follicular phase had the lowest correlation to prolactin concentrations after metoclopramide stimulation. The lowest correlation of basal prolactin level in the luteal phase was with the level of prolactin following metoclopramide stimulation. Basal prolactin concentrations in the early follicular phase, as well as in the luteal phase, exhibited very weak positive correlations (p < 0.01) to the concentrations of DtIEAS ( r = 0.12), estradiol-17P ( r = 0.18), testosterone/SHBG ratio ( r = 0.13) and estradiol (r = 0.15). The correlation was negative for the progesterone values ( r = - 0.16) in the early h e a l phase. Stimulated prolactin concentrati'ons after T R H showed positive correlations to FSH (r = 0.12), estradiol-1r'P ( r = 0.13) and SHBG ( Y = 0.14), and to estradiol-17P in the luteal phase (r = 0.15). A positive correlation between prolactin and progesterone was only found in the late luteal phase ( r = 0.1 1). Metoclopranlide-stimulated prolactin levels exhibited a significant negative correlation to the LH/FSH ratio ( r = - 0.14) of the follicular phase, and had a positive correlation to progesterone ( r = 0.19) and estradiol-17P ( r = 0.31) in the luteal phase. All the other correlation coefficients were below 0.1 1. The norrnal range for prolactin concentrations following T K H stimulation did not exist, and different levels were reported in the literature for meto-
n
c
I
lxesm
m
46 18
r w e g m
9
lxesm
p < 0.01
m
22
tuba1 patency
pregnant
p < 0.01
I
22
m
r w e g m
sperm count
WM
I
poor normal
0.very poor
1 I
56
p * 0.01
sperm count
Figure 1 Non-hormonal causes of sterility in women with subclinical hypothyroidism, who either became pregnant through levothyroxine substitution or remained sterile.X?testlevel ofsignificance is p e n . Results ofall diagnostic procedures were not available for all women, so that the number ofpatients vaned between the groups
0
20
40
60
80
100
%
result of resutt of hysterosalpingography chromolaparoscopy
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?
Q
& 2
P 3-
-
Basal
WIOG
Follicular phase
15'TRH
X
>1m
30'TRH
X
X
Basal
X
W
#/
r/
I 30'meto
X
25%
mh
10%
mean medii
75%
+ -
Luteal phase
1+1
X
I 90%
rnax
I
0
50
100
150
:
-
- 250 - 200
- 300
Figure 2 Prolactin concentrations observed i n the early follicular phase 15 i i m and 30 min after ctimulation with T l l H ( I S'TRH and 30'TllH), and during the luteat phase 30 inin after stimulation with inetoclopramidc (30'meto.). Listed are thc means, medians, percentiles and highest (max) and lowest (min) levels
n = 759
0
40'
60-
ao
100-
x
528 x
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-. 3
a
3
n
24
Gerhard et al.
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prolactin
basal1
I
TRH 15'
TRH 3 0
basal 2
meto 30.
folllcular phase
Figure 3 Correlation coefficients, according to Spearman, between the basal prolactin level in the early follicular phase (prolactinl) (empty bars), the basal prolactin level in the luteal phase (prolactin 2) (hatched bars), and the stimulated prolactin level. The lowest, though still significant correlations, were found to exist between the basal levels and the metoclopramide stimulated levels
clopramide-stimulated prolactin. Therefore we decided to use values greater than the 90th percentile of the collective as being abnormal. Borderline levels for stimulated prolactin were: 15 min following TRH, 97 ng/ml; 30 min following T R H , 76 ng/ml, and 30 min followingmetoclopramide, 254 ng/ml. Values equal to and below the 90th percentiles were considered normal. Elevated prolactin concentrations were present in 2-4% of the patients. A total of 287 (39%) of 745 women became pregnant during the 2-year period of observation (14% spontaneous, 25% medically induced). Table 6 shows the rate of pregnancy relative to the results of the prolactin tests. The overall pregnancy rate did not significantlydiffer between the five prolactin test groups, while being significantly influenced by uterine, tuba1 and andrological factors, as previously de~cribed'~. The spontaneous pregnancy rate, however, was greatly reduced when both the T R H and the metoclopramide test identified elevated prolactin levels, relative to the remaining groups (p < 0.05) without an adequate explanation by additional non-hormonal infertility factors.
25
T R H and wietoclopramide testirig in if!fertile women Table 6 l'regnincy rate compared t o prolactin levels
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~~~
~
All normal Basally raiccd TKH and meto. raised TRH raircd only M e t o raised only Total
~
522 76
28 54 65 745
~
70 10
198 26
38 34
14 12
24 22
62
4
12 25 26 287
43 46 40 39
4
13 14 14
39 33 20 25
57 54 60 61
7 c)
100
66
meto. = nictoclnpratnidc
W e direct attention to the therapy of women with medically induced pregnancies. Table 7 differentiates between monotherapy with dopamine agonists, con-lbination therapies (bromocnptine combined with clomiphene, hMG, dexaniethasone or progesterone), and therapeutic approaches without dopamine agonists. It, therefore, was not surprising that the group with basal prolactin concentrations above the 90th percentile had significantly more pregnancies when treated with dopamine agonists, than with any other therapy (x' test, p < 0.01). The same applied to elevated prolactin levels in the luteal phase (p < 0.001). O n the other hand, stimulated prolactin levels failed to correlate to the type of therapy used. A total of 21 patients with elevated
Table 7
Medical trcatint'tit resulting in conception, correlated with the prolactin levels of all pregnant woiiit'n ( n 1 186). Frequency '% is g v e n ; test was applied I'rolactiri
x'
1rilc.l
Dopamine
aSqi~tiists
Other thrrup y
I'
Basal > 90. pen:.
23
30
47
0.006
15'TKH > 90. perc.
15
11
74
ns*
3O'TKH > 90. perc. Luteal > 90. perc. 30'Meto > 90. perc. All normal
12
11
77
11s
28 23
28 14
44
0.001
64
ns
11
7
82
11s
Total
14
9
77
11s
* ns, not significant Perc. = percentilc
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26
Gerhard et al.
prolactin values 15 min post-TRH injection had a significantly higher rate of pregnancy after treatment, than those not requiring therapy. However, when therapy forms were evaluated for these 21 women, the analysis showed that seven became pregnant due to sole or combined treatment with dopamine agonists, four after thyroid substitution treatment (single or in combination), and ten after other forms of therapy (i.e. three patients, clomiphene; two patients, clomiphene-dexamethasone; two patients, gonadotropins; two patients, clomiphene-gonadotropins; one patient, progesterone substitution). The sterility and pregnancy rates of bromocriptine patients (Group B, n = 191) and of women who never received dopamine agonists (Group A, n = 577) are shown in Figure 4. Pregnancies which occurred after therapy are listed, according to the type of therapy (bromocriptine monotherapy, bromocriptine combination therapy, other). Note that in patients with elevated basal prolactin levels ( only six patients displayed concentrations over 30 ng/ ml), the spontaneous pregnancy rate was higher in Group A than in Group B (p=0.003). The male and the tuba1 factors were better for this elevated prolactin group than for the other prolactin groups (p < 0.01). The highest basal prolactin level at which a spontaneous pregnancy occurred was 26 ng/ml, while the level required to induce pregnancies was above 30 ng/ml. The pregnancy rate was highest when substances other than bromocriptine were used in patients with raised prolactin levels following initial T R H and metoclopramide stimulation, and there was a lower infertility rate in Group A than in Group B (p < 0.01). Where prolactin was raised only in the TRH or metoclopramide tests, pregnancies were induced with equal frequency with brornocriptine, or other medication. In patients with normal prolactin levels, both basal and following stimulation, sterility was higher in Group A than in Group B, due to the higher rate oftherapy-induced pregnancies in Group B (p < 0.001), and the frequency of reduced sperm counts in Group A.
Discussion
Thyroid function disturbances were observed in 23% of the sterile patients of this study by means of T R H testing. Only one-third of these patients had received levothyroxine due to thyroid illness. This corresponds with the suspected frequency of thyroid disease (20-30%) in the female population of southern Germany, and correlates with the proven goiter frequency of men (21%) in this region. The previously assumed etiologic role of thyroid dysfunction in infertile women may thus have been overestimated'. A possible role ofsubclinical hypothyroidism in infertility was suggested since thyroid antibodies were seen more frequently in women with overshooting responses during T R H testing, than in patients with a normal TRH responseI4. Furthermore, it was shown that asymptomatic patients with thyroid antibodies had pathologcal results more frequently during T R H testingI5.Lowered free thyroxine concentrations were measured in 60% ofthe women with preclinical
(n)
0
10
20
30
40
50
brornocriptine Induced pregnancy with brornocriptine in combination
Induced pregnancy with
2
2
z-
2
\
,-
E
-.. -..
-. 3
2
3
Induced pregnancy (different drugs)
"
z
G
Spontaneous pregnancy
Figure 4 Pregnancy rate compared to the prolactin concentration of women w h o received broniocriptnie at wine point (Group 13, i t = 1 O l ) , and ie ~nfertilityr a e higher 111 A than 111 U OJ < O.O(I1). those w h o were iiever treated with doparnine dgotiists (Group A, ! I = 577). Normal b a s ~ ~ l i tprolactin: I'rolactin bayally raised: ypoiitaiieou~pregnancy ratc higher in A than in 13, infertility rate higher in I3 t h x i in A (JJ = 0.0003). Elevatcd prolactin aftcr
5
a
C
* 0
E
Q, c
-8
60
70
80
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Gevhavd et al.
In this study, various hormones were determined together with TSH, permitting the direct comparison between TSH and gonadotropins, estradiol, androgens and prolactin. However, only TSH and prolactin concentrations were positively correlated. As in compensated thyroxine deficiency, the pituitary sensitivity for T R H is increased. Since T R H increases prolactin release, compensated hypothyroidism might influence ovarian action via hyperprolactinemia. Patients with subclinical hypothyroidism had a significantly poorer postcoital test than women with normal thyroid function. Neither group differed with respect to the cervical index, or the partner’s spermiogram, so that immunological dysfunction can be assumed. While Pencea et al.” found sperm antibodies in 6.7% of the examined women with euthyroidism, and in 12% of his patients with thyroid function disturbances, we failed to identify a relationship between thyroid antibodies and an immunological factorT4. The spontaneous pregnancy rate was lower in hypothyroid women (6%)than in euthyroid (16%) patients. Pregnancies occurred in 12% of the women after levothyroxine substitution only. Bohnet et aL3 reported a pregnancy rate of lo%, after therapy with only 50 pg levothyroxine, during a treatment period of 1 month. Louvet et al.”, on the other hand, achieved a 54%pregnancy rate in 37 women with anovulation. The low pregnancy rate in our study is due to the fact that non-hormonal causes of sterility did not represent criteria for exclusion from therapy. While patients with thyroid dysfunction have normal basal prolactin levels, the TRH-stimulated prolactin release is exaggerated in hypothyroidism, and decreased in hyperthyroidism’9. Following levothyroxine replacement therapy in subclinical hypothyroid patients, the plasma prolactin and TSH secretion is inhibited when metoclopramide and T R H are gwenT.s.T R H synthesis is regulated by levothyroxine by way of a negative feedback system. In hyperprolactinemic women, the mean basal TSH concentrations were found to be similar to those in normal women, but the incremental TSH response to T R H stimulation was considerably greater”’. The association between hyperprolactinemia and disturbed ovulatory function may in part be explained by activation of the prolactin-dopamine shortloop feedback. This activation increases dopaminergic activity in the medial basal hypothalamus, and inhibits gonadotropin-releasing hormone (GnRH) release”. Under certain in vitvo conditions, human prolactin can inhibit human chorionic gonadotropin (hCG)-stimulated production of estradiol and progesterone by human granulosa cells obtained from preovulatory
follicle^".'^. Since the TSH and prolactin responses to T R H fail to be affected by the menstrual cyclez4,we chose the early follicular phase to carry out T R H testing, and assess the basal values of androgens, gonadotropins and estradiol. Thus, the interval between T R H and metoclopramide testing was adequate to exclude an influence of one test on the next. The metoclopramide test was performed during days 20-23 of the cycle, as proposed by LeidenbergerZs,who observed the greatest prolactin response to metoclopramide at this time. Moreover,
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TRH and metoclopramide testing in infertile women
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Bohnet’ observed a significant correlation between the spontaneous nocturnal prolactin peaks in infertile women, and the peak concentrations of prolactin after metoclopramide. In our patients the prolactin response to metoclopraniide was about 4 tinies higher than after T K H . This was due in part to the submaximal T R H dose administered, since 500 pg T R H are required to produce a maximum prolactin secretion. O n the other hand, estradiol concentrations were higher in the luteal than in the follicular phase, and it is known that estrogen or estrogen treatment increases the :prolactin response to T R H and to meto~lopramide~~~”. Basal prolactin concentrations were positively correlated to DHEAS as well as the testosterone/SHBG ratio, suggesting stimulation. McKenna et al. ”, described ele.vated prolactin concentrations in women with hirsutism and elevated androgens. According to Lob0 et al.”’, prolactin modulates the secretion of DHEAS. An increase in prolactin concentrations is associated with elevated concentrations of DHEAS, whereas a decrease is followed by a fall in DHEAS. All stimulated prolactin concentrations were negatively correlated with the body mass index. This corresponds to data presented by Cavagini et al.””, who observed significantly lower prolactin secretion after T R H injection in obese patients, than in nornial subjects. We were able to demonstrate in an earlier study3’that luteal phase progesterone concentrations are inversely related to the body mass index. This result fits in with the observation that the response of prolactin to stimulation is exaggerated in the presence of elevated progesterone and estradiol concentrations in the luteal phase. In amenorrhea, the prolactin and TSH response to inetoclopramide was significantly less pronounced than in eumenorrhea, suggestive of raised dopaminergic tone in the former group3’. Though a correlation existed between all prolactin concentrations, both baseline and stimulated, the link between the baseline values and prolactin concentrations after metoclopramide was lowest. Therefore, metoclopramide testing was considered to give maximum additional information. However, conflicting d,ata have been reported for the borderline region, where values ranged from 160 ng/nll to 300 ng/rnP6. Therefore, the 90th percentile was chosen for TRH, as well as for the metoclopramide test. Treatment with dopamine agonists was recommended to all patients w h o had raised basal prolactin concentrations. Furthermore, those with prolactin values above 250 n g / d after rnetoclopramide stimulation received the same treatment. Not all women accepted treatment, however. In the course of randomized studies, women with a normal prolactin pattern received dopamine agonists, as well. The overall pregnancy rate was similar for all prolactin groups. The spontaneous pregnancy rate, however, was lowest in women with an exaggerated response to T R H and metoclopramide. The evaluation of the data for this group failed to identifi a particularly advantageous medication”. In the past:, latent hyperprolactinemia was felt to contribute to infertility. Better response rates to bromocriptine therapy were observed in patients with excessive. as well as borderline, exaggerated responses of prolactin to TRH or nietoclopraniide administration than in patients with normal responses’.’.
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We did not succeed in inducing a higher pregnancy rate with dopamine agonist treatment in women with a pathological T R H or metoclopramide test result. The infertility rate was lower in women with normal prolactin tests who had received dopamine agonists, than in normals who had never received bromocriptine. This result confirms a previous study which demonstrated the superiority of bromocriptine treatment over clomiphene or progesterone substitution in women with unexplained infertility or luteal i n s ~ f i c i e n c y ~ ~ , irrespective of their prolactin tests. It is possible that some infertile women experience transient prolactin elevations at midcycle, which are preventable by bromocriptine. In this context, a study by Harrison34is of interest: in couples with unexplained infertility, one-third had high stress profile scores, and intermittent prolactin spikes. The pregnancy rate in such women was clearly higher with a combination of clomiphene and bromocriptine than with placebo. In comparison, results from active therapy were no more effective than placebo in women with normal stress scores and non-spiking prolactin. There were no significant differences in the T R H releasing hormone tests. Harrison's3' results, as well as our own, indicate that disturbances of prolactin secretion exist which respond to dopamine agonist treatment, and that the prolactin response to T R H or metoclopramide does not permit a prognostic differentiation.
Acknowledgements The authors thank the pharmaceutical companies Henning Berlin and KaliChemie, both of West Germany, for financial support of this study. They are grateful to the laboratory staff for performing the hormone assay, and to Mrs Mahrla for expert secretarial assistance.
References 1. Gerhard, I., Eggert-Kruse, W. and Runnebaum, B. (1988). Diagnosis and treatment of subclinical hormonal disorders in infertile women with special regard to thyroid dysfunction. Aktrrelle Endokrinologie 11. Stofwechsel, 4, 200 2. Peillon, F., Vincens, M., Cesselin, F., Doumith, R. and Mowszowicz, I. (1982). Exaggerated prolactin response of thyrotropin-releasing hormone in women with anovulatory cycles: possible role of endogenous estrogen and effect of bromocriptine. Fertil. Steril., 37,530 3. Bohnet, H. G., Fiedler, K. and Leidenberger, F. A. (1981). Subclinical hypothyroidism and infertility. Lancet, 2, 1278 4. Lombardi, G., Iodice, M., Miletto, P., Merola, B., Panza, N. and Annunziato, L. (1986). Prolactin and TSH-response to T R H and metoclopramide before and after therapy in subclinical hypothyroidism. Neuroendocrinology, 43, 676 5. Bohnet, H. G. (1980). Gelbkorper-Schwache als Prolaktin-bedingte Storung des Menstruationszyklus. Fortschr. d . Medizin, 98, 1618 6. Merzoug, K., Gerhard, 1. and Runnebaum, B. (1990). Incidence and
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conditiom €or therapy independent pregnancies in patients under treatment for riterility. Gebirrtsh. Frauenheilk, 50, 177 7. Noyes, K. W., Hertig, A. T . and Rock, J. (1950). Dating the endometrial biopsy. Fertil. Steril., 1, 3 8. Gerhard, I., Bechthold, E., Eggert-Kruse, W., Heberling, D. and Kunnebaum, B. (1990). Value of endometrial biopsies and serum hormone determinations in luteal insufficiency. Hum. Reprod., in press 9. Insler, V., Melmed, H., Eichenbrenner, I., Serr, D. M . and Lunenfeld, B. (1972).The cervical score. A simple semiquantitative method for monitoring of the menstrual cycle. Int. /. Gyrzaecol. Obrtet., 10, 223 10. Eggert-Kruse, W., Gerhard, I., Hofmann, H., Kunnebaum, B. and Petzoldt, D. (1987). Influence of microbial colonization of sperm-mucus interaction in vivo and in vitro. Hum. Reprod., 2, 301 11. Eggert-Kruse, W., Gerhard, I., Tilgen, W. and Kunnebaum, B. (1989). Clinical significance of crossed in vitro sperrn-cervical mucus penetration test in infertility investigation. Fertil. Steuil., 52, 1032 12. Jones, G. S. (1976). The luteal phase defect. Fertil. Steril., 27, 351 13. Gerhard, I., Keischmann, T., Eggert-Kruse, W. and Runnebaum, B. (1 990). Factors contributing to success rate of hormonal treatment in infertile women. 1. Patients history and clinical parameters. 11. Hormonal disorders and treatment. Fertilitiit, 6, 123 and 136 14. Gerhard, I., Becker, T., Eggert-Kruse, W. and Runnebaum, B. (1991). Thyroid and ovarian function in infertile women. Hirr?.~. Reprod., in press 15. Betterle, C., Callegari, G., Presotto, F., Zanette, F., Pedini, B., Kampazzo, T., Slack., K. S., Girelli, M. E. and Busnaro, U. (1987). Thyroid autoantibodies: a good marker for the study of symptomless autoimmune thyroiditis. A r t a Endocrinol., 114, 321 16. Pacchiarotti, A., Martino, E., Bartalena, L., Aghini-Lombardi, F., Grasso, L., Buratti, L., Falcone, M. and Pinchera, A. (1986). Serum free thyroid hormonses in subclinical hypothyroidism. _I. E n d o r r i d . Iiivert., 9, 315 17. Pencea, C., Zbranca-Toporas, E., Thirer, O., Moscovivi, K. and Davidescu, I>. (1979). Incidence of antispermatic antibodies in sterile women with thyroid insufficiency. RW. Roirm. Med. Etidocritiol., 17, 55 18. Louvet, J . P., Gouarre, M., Salandini, A. M. and Boulard, C. L. (1979). Hypothyroidism and anovulation. Lancet, 1, 1032 19. Ferrari, C., Parachi, A., Parisio, E., Codecasa, F., Mucci, M., Boghen, M., Gerevini, G. and Rampini, P. (1987). Serum free thyroid hormones in different degrees of hypothyroidism and in euthyroid autoimmune thyroiditis. Acta Endorritiol. Copenh., 14, 559 20. Seki, K. and Kato, K. (1985). Increased thyroid-stimulating hormone response to thyrotropin-releasing hormone in hyperprolactinemic women. /. Cliri. Errdocrid. 121etab., 61, 1138 21. Hagen, C., Andersen, A. N . and Djursing, H. (1984). Evidence ofaltered doparninergic moduldtion ofPrl, LH, FSH, G H and TSH secretion during chronic partial dopamine receptor blockade in normal women. Acta Etrdacritiol., 106, 1
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22. Uilenbroek, J. T. J. and van den Linden, R . (1984). Effects ofprolactin on follicular oestradiol production in the rat. J . Endocrinol., 102, 245 23. Sjogren, A., Hillensjo, T., Roos, P. and Hamberger, L. (1988). Prolactin and gonadotrophin interactions on progesterone formation in cultured human granulosa cells. Hum. Reprod., 3, 601 24. Savin, C. T., Hershman, J. M., Boyd, A. E., Longcope, C. and Bacharach, P. (1978). The relationship of changes in serum estradiol and progesterone during the menstrual cycle to the thyrotropin and prolactin responses to thyrotropin-releasing hormone. J . Clin. Endocrinol. Metab., 47, 1296 25. Leidenberger, F. (1983). In praxi hat die Hormondiagnostik rationell, d.h. zielgerichtet und trennscharf zu erfolgen. HUF Verlag Essen, Gyne, 4 26. Franks, S., Mason, H. D., Shennan, K. I. J. and Sheppard, M. C. (1984). Stimulation of prolactin secretion by oestradiol in the rat is associated with increased hypothalamic release of thyrotrophin-releasing hormone. J . Endocrinol., 103, 257 27. Martin, R . H. (1983). Metoclopramide challenge: a measure of human lactotroph activity. Obstet. Gynecol., 62, 691 28. McKenna, T. J., Loughlin, T., Daly, L., Smyth, P. A., Culliton, M. and Cunningham, S. K. (1984). Variable clinical and hormonal manifestations of hyperandrogenemia. Metab: Clin.Exp., 33, 714 29. Lobo, R. A., Letzky, 0.A., Kaptein, E. M. and Goebelmann, U. (1980). Prolactin modulation of dehydroepiandrosterone sulfate secretion. A m .J . Obstet. Gynecol., 138, 632 30. Cavagnini, F., Maraschini, C., Pinto, M., Dubini, A. and Polli, E. E. (198 1). Impaired prolactin secretion in obese patients.J. Endocrinol. Invest., 4, 149 31. Gerhard, I., Schatanek, U., Klinga, K., Eggert-Kruse, W. and Runnebaum, B. (1989). Determination of sex hormone binding globulin as part of the endocrinologcal diagnostic procedure in sterility. Fertilitat, 5 , 6 32. Hagen, C., Petersen, K., Djursing, H. and Andresen, A. N. (1984). Evidence of altered dopaminergic modulation of Prl, LH and TSH secretion in patients with normoprolactinaemic amenorrhoea. Acta Endocrinol., 106, 8 33. Gerhard, I., Weidenkopf, K., Eggert-Kruse, W. and Runnebaum, B. (1988). Hinweise zur gezielten Therapie der Lutealinsuffizienz und funktionellen Sterilitat aufgrund einer randomisierten Studie. In Schirren, C. and Semm, K. (eds.) Fortschritte der Fertilitiitsforschung, Nr. 16, p. 82, KongreBbericht Bonn 1987. Berlin: Grosse-Verlag 34. Harrison, R. F. (1988). Stress spike of hyperprolactinaemia and infertility. Hum. Reprod., 3, 173
