LH pulses and the corpus luteum: The luteal phase deficiency (LPD)

VITAMINS AND HORMONES, VOL. 63

LH Pulses and the Corpus Luteum: The Luteal Phase Deficiency (LPD) W. WUTTKE, L. PITZEL, D. SEIDLOVA-WUTTKE, ANDB. HINNEY Division of Clinical and Experimental Endocrinology, Department of Obstetrics and Gynecology, University of G~ttingen, D-37075 G6ttingen, Germany I. Introduction II. The Short Luteal Phase as a Cause of Infertility III. Serum Progesterone Levels in Infertile Subjects IV. What Is "Normal'~ A. Corpus Luteum Insufficiency V. Treatment of Luteal Phase Deficiencies References

The proper function of the GnRH pulse generator in the hypothalamus is essential for normal ovarian function, hence also for proper function of the corpus luteum. During the luteal phase LH pulses stimulate progesterone release, which is essential for normal endometrial transformation. Approximately one-half of all luteal phase deficiencies (LPD) are due to improper function of the GnRH pulse generator. Obviously, following ovulation the increased serum progesterone levels oversuppress the GnRH pulse generator, resulting in too few LH pulses and therefore improper luteal function. Also, latent hyperprolactinemia may lead to an LPD which can be effectively treated with plant extracts containing dopaminergic (prolactin-suppressing) compounds. Our increasing knowledge of auto- and paracrine mechanisms between nonsteroidogenic and steroidogenic cells now allow subclassification of LPDs of ovarian origin. The so-called small luteal cells are LH-responsive. If they develop improperly the regularly occurring LH pulses are unable to stimulate progesterone secretion from the small luteal cells, which results in what we call the small luteal cell defect. In addition, there is also evidence that the large luteal cells may function improperly. Hence, basal progesterone release is too low while LH-stimulated progesterone release from the small luteal cells appears to be intact. This subclassification of luteal phase deficiency results in the suggestion of different treatments. In cases where the corpus luteum is LH-responsive, such as the hypothalamic corpus luteum insufficiency and the large luteal cell defect, HCG treatment or 131

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0083-6729/01 $35.00

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w. WUTTKEETAL. pulsatile treatment with GnRH is advisable. In the case of LH/hCGunresponsive small luteal cell defect a progesterone substitution is suggested. ©2001AcademicPress.

I. INTRODUCTION

It has been almost 50 years since Jones (1949) first described luteal phase deficiency (luteal phase insufficiency, luteal phase defect, or corpus luteum insufficiency). This disease is defined as a defect of the corpus luteum (CL) to secrete progesterone in high enough amounts or for too short a duration. In the latter case, the term "short luteal phase" is commonly used. This results in an inadequate or out-of-time transformation of the endometrium and as a result implantation of the trophoblast (nidation) is often impossible. Therefore LPD is believed to be a common factor in infertility. As a clinical entity, LPD is poorly characterized and the matter is confused further by the observations that LPD may not be chronic and may also appear in women with proven fertility. When reviewing the literature, it is evident that two factions of gynecological endocrinologists exist, one of which claim that the actual hight of serum progesterone levels may be meaningless as long as the transformation of the endometrium is optimal and in phase with the development of the trophoblast (Annos et al., 1980; Balasch, 1987; Balasch et al., 1985; for review see McNeely and Soules, 1988), while the other faction claims that only progesterone levels above a certain threshold guarantee optimal endometrial transformation and that therefore measurement of progesterone is the ultimate (and easiest) choice for diagnosing LPD (Israel et al., 1972; Shepard and Senturia, 1977; Hecht et al., 1990; Jordan et al., 1994). In this chapter we focus primarily on the latter alternative and elaborate on the normal function of the CL, the frequency of occurrence of occasional and chronic LPD, and on their possible etiologies. II. THE SHORT LUTEAL PHASE AS A CAUSE OF INFERTILITY

Lenton et al. (1984) reported that 5.2% of women with apparently normal ovulatory cycles have indeed a short luteal phase--shorter than 9 days. Such short luteal phases were commonly observed in women younger than 24 and older than 45 years of age. In a population of 95 patients with unexplained infertility, however, the same group (Smith et al., 1984) found no differences in the length of the luteal phase when

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compared to 92 control subjects with normal ovulatory cycles. Whether luteal phases shorter than 9-11 days are indeed the reason for reduced fertility remains unclear at present.

III. SERUM PROGESTERONE LEVELS IN INFERTILE SUBJECTS In the past, most researchers have studied serum progesterone levels in relation to infertility on the basis of one or perhaps a few more measurements (Annos et al., 1980; Hensleigh and Fainstat, 1979; for review see McNeely and Soules, 1988). In view of the large fluctuations due to pulsatile progesterone release (see below), such an approach appears to be problematic. Few authors have determined serum progesterone levels in women with unexplained infertility and compared them to levels found in women with normal ovulatory or conception cycles on the basis of daily blood sampling during the luteal phase. In one of these studies the authors defined normality as values within the 95% confidence limits and calculated that progesterone levels <5 ng/ml for 5 or more days were abnormal (Landgren et al., 1980; Olive, 1991). On the basis of literature surveys, Van Zonneveld et al. (1994), however, came to the conclusion that abnormal progesterone values are those below 10 ng/ml. Over the past few years we investigated a group of 543 patients with patent tubes and andrologically unconspicuous partners. They had a 2-year or longer history of infertility and 461 (90.2%) had normal spontaneous cycles and ovulated as approved by vaginal ultrasound. Fifty of the patients (9.8%) had unovulatory cycles and 32 (5.9%) were amenorrhoic. In the course of our investigation 4 patients (0.8%) became pregnant in the cycle under investigation. Of the 457 remaining patients who had ovulatory cycles, the distribution of serum progesterone levels determined during the midluteal phase are shown in Fig. 1. The distribution shows two peaks, one at 7 ng/ml, the other at 11 ng/ml; therefore (and for reasons indicated below) we chose our lower range of normality as progesterone levels >8 ng/ml. On this basis 292 (63.9%) of the patients had progesterone levels in the normal range, whereas 165 (36.1%) had "abnormally" low progesterone values 221. From the 292 patients with apparently normal progesterone values and 123 patients with abnormally low progesterone values could be studied during the next three cycles. During this time 7.2% of patients with normal progesterone values in the preceding investigational cycle became pregnant; this compares to 3.6% of the patients with abnormally low progesterone values in the preceding investigational cycles. A life table

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analysis (Fig. 2) of patients with low (<8 ng/ml) or normal progesterone levels (>8 ng/ml) in the monitoring cycle also indicates a significantly (P < 0.001) reduced fecundity in those patients with too-low progesterone levels. It should be noted, however, that over a period of 24 months 10% of those patients with too-low progesterone levels conceived compared to 21% of the patients with normal serum progesterone levels. On this basis and for other reasons discussed below, we feel that the cutoff between for too-low progesterone levels should be 8 ng/ml. It should be emphasized that all patients with apparent LPD had ovulated. This is in striking contrast to data published by Hamilton et al. (1987), who found that more than 70% of their patients with progesterone levels < 10 ng/ml failed to ovulate and suffered from luteinized unruptured follicle (LUF) syndrome. Less dramatically, van Zonneveld et al. (1994) found LUF syndrome in 28% of their patients and their cutofffor too-low progesterone levels was also 10 ng/ml. In a meta-analysis of their data Jordan et al. (1994) determined sensitivity and specificity of commonly used diagnostic methods including serum progesterone and endometrial biopsies. When using a cutoff of 10 ng/ml, measured during the midluteal phase, both sensitivity and specificity were well above 80%. Therefore a single factor, progesterone level, was superior

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to any other parameter including endometrial biopsy. In this retrospective analysis they found no LUF syndrome in their patients suffering from LPD.

IV. WHAT Is "NORMAL"?

It has been observed that serum progesterone levels are subject to large fluctuations due to pulsatile hormone release (Knobil, 1980). In a number of studies in primates including human, it was demonstrated that the pituitary releases LH in pulses (for a review see Filicori et al., 1984). The pulse frequency is high (approximately one pulse per 90 rain) during the follicular phase. Under the influence of progesterone the pulse frequency is significantly reduced (one pulse per 3-6 h) depending on the age of the corpus luteum (Yen et al., 1972; Filicori et al., 1984; Hinney et al., 1995). It was also reported that the corpus luteum is unresponsive to LH pulses during the early luteal phase. Responsivity

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to LH develops between days 4 and 6 after ovulation (Filicori et al., 1984; Hinney et al., 1995): Such pulsatility studies can only be done on the basis of frequent (every 10 min) blood samplings over an extended period of time and we have performed such studies in women with normal ovulatory cycles (Hinney et al., 1995). A representative example of LH and progesterone fluctuations during the follicular and the mid-/ late luteal phase of one subject with a normal ovulatory cycle is shown in Fig. 3. In agreement with the literature, we found in all subjects with normal ovulatory cycles high LH pulse frequencies in the follicular phase, which were significantly reduced during the midluteal phase (days 7-10). In the case of the subject shown in Fig. 3, an LH episode preceded the beginning of the blood sampling period and a second ocurred shortly after 15:00 h. Both episodes stimulated progesterone levels; however, prior to the beginning of the latter episode progesterone levels had decreased into a below-normal range. Such observations were made quite often in women with normal ovulatory cycles, including three who conceived during the cycle under investigation. This led us to calculate medians + the 25-75, 10-90, and 5-95% quartiles. The resulting box plots are illustrated for the serum progesterone values from 21 subjects analyzed so far (Fig. 4). The area below our "normal" (i.e., <8 ng/ml) in this figure is shaded and it is quite clear that in a number of cases subnormal values can be measured in subjects with normal ovulatory cycles. These subnormal values were always seen prior to the occurrence of the next LH episode. It thus appears that the next LH episode is due to a weakening negative feedback action of progesterone into the hypothalamus, which supports suggestions made earlier (Veldhuis et al., 1988; Rossmanith et al., 1990; Hinney et al., 1995). Hence, it can be concluded that on the basis of a single progesterone determination during the midluteal phase a false LPD may be diagnosed quite often

FIG. 3. Serum LH, estradiol, and progesterone levels in a woman with a normal ovulatory cycle. Note the occurrence of small LH pulses at a high frequency (seven during the 8-h investigation period) during the follicular phase (FP, left panel). This is in contrast to the occurrence of LH pulses during the midluteal phase (LP, right panel), where much higher LH pulses occur at a lower frequency. Note also the below-normal (i.e., <8 ng/ml) progesterone levels prior to the occurrence of the LH episode and the dramatic increase under the influence of the LH episode. Prior to the beginning of the investigation period another LH episode had apparently occurred (as evidenced by the decreasing LH levels) which had stimulated serum progesterone levels into the normal range over a period of approximately 5 h.

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subject FIG. 4. Box plots of 21 subjects with normal and regular ovulatory cycles. At days 7, 8, or 9 blood samples were withdrawn over an 8-h period at 10-min intervals. The box plots show the medians (narrowest part of the box), the 25-to-75% quartile (sloping part of the box), the 10-to-90% quartile (vertical part of the box), and the 5-to-95% quartile (flags above and below each box). Note that in a number of cases serum progesterone levels may be in the below-normal range (i.e., below 8 ng/ml). The area below 8 ng/ml is shaded.

(~15%). Since such low progesterone concentrations are regularly observed only prior to the occurrence of an LH episode, the probability is high that within the next 3 h an LH episode will have stimulated luteal progesterone secretion into the normal range. It is therefore advisable to withdraw at least two or three blood samples within this period of time for progesterone determination; this will reduce the probability of a falsely diagnosed LPD to between 2 and less than 0.5%. If one considers progesterone levels below 10 ng/ml as subnormal the probability of falsely diagnosing LPD is approximately 4%. A. CORPUS LUTEUM INSUFFICIENCY

In the life table analysis shown in Fig. 2 it is clear that subnormal progesterone levels result in reduced fecundity. In view of this lower pregnancy probability as a result of LPD several questions arise.

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1. Are LPD Inevitably Recurring Events? In a recent study (Hinney et al., 1996), of 905 infertile patients 733 were eumenorrhoic, of which 165 (22.5%) had plasma levels (single determination) below 8 ng/ml on days 7, 8, or 9 of the luteal phase. We concluded that on the basis of a single determination of progesterone level at one of the days when the CL is fully responsive to LH, these patients would be classified as having LPD. On the basis of data presented above for normal subjects with ovulatory cycles, however, the probability of measuring progesterone levels below 8 ng/ml in a single blood sample is 15%. Hence, it is likely that 15% of the 165 patients with progesterone levels <8 ng/ml might have had normal luteal phases. This would leave us with 140 patients with correctly diagnosed LPD, which is 15.5% of our total of 905 infertile patients. During the following cycle 109 of the 165 patients were reinvestigated and 2 withdrew during the following midluteal phase. Fifty-five (50.5%) of those with low progesterone levels in the previous cycle and (6.1%) of our total of 905 infertile patients again had progesterone levels <8 ng/ml in both blood samples and are thus suffering from chronic LPD. We have extended these studies and corroborated these earlier findings with those of earlier case reports (Aksel, 1980). Hence, we conclude that LPD may occur in infertile patients at irregular (unknown) intervals and it is rarely chronic (~6%).

2. What Do We Know about the Etiology of LPD? In an excellent review, McNeilly and Soules (1988) evaluated the postulated and proven causes of luteal phase deficiencies. Besides uterine factors (inadequate endometrial progesterone receptors and endometritis) and drug treatment (clomifen citrate, human menopausal gonadotrophins, and GnRH agonists or antagonists) they stress a number of neuroendocrine and ovarian factors which we elaborate on in the remainder of this chapter. Increased LH pulse frequency and abnormal follicular phase ratio (LH : FSH) were reported in women suffering from LPD (Jordan et al., 1994). An inadequate LH surge has also been postulated to be the reason for LPD (Ayabe et al., 1994). There are, however, numerous other reports that have not confirmed this view (Lenton et al., 1978; Lenton et al., 1982; Hinney et al., 1996). Inadequate perimenstrual and midfollicular FSH secretion was shown in LPD women (Sherman and Korenman, 1974; Cook et al., 1983) and in monkey (DiZerega and Hodges, 1981). Such follicular phase FSH deficiency may lead to abnormal follicular development and too-low preovulatory estradiol production (Sherman and Korenman, 1974; GeisthSvel

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and Skubsch, 1983; Ayabe et al., 1994). On the basis of 10 cases, however, McNeilly and Soules (1988) were unable to demonstrate any decreasing mean follicle diameter as determined by daily ultrasound scans and this is in line with our observation in which we scanned the preovulatory follicle diameters in 16 patients with chronic LPD (Hinney et al., 1996). In addition, we reported also no statistically significant differences in mean preovulatory estradiol levels when compared to those of a group of normal subjects (Hinney et al., 1996). Mild hyperprolactinemia may be the cause of LPD (Miihlenstedt et al., 1978; del Pozo et al., 1979) and it is our observation that subjects who react to stress with an exaggerated prolactin release often have LPD or a short luteal phase. In the course of our investigations of LPD we observed quite frequently that some patients reacted with exaggerated prolactin release in response to the stress ofvenepuncture. Although the number of patients that we could investigate thoroughly is too small to allow for a statistical treatment we give here one example of a latent hyperprolactinemic patient who obviously has chronic LPD. This patient was investigated on day 7 of her luteal phase. Serum progesterone levels taken during the luteal phase of the two preceding cycles, on the day prior to the thorough investigation, and early in the morning of the investigation day were well below the normal range, i.e., <8 ng/ml. Her serum prolactin levels at the beginning of the blood sampling period were exorbitantly high. In fact, they were at a level such that one could suspect prolactinoma (Fig. 5a). Within an hour levels decreased into the normal range, indicating that this patient reacted to the stress of the venepuncture procedure with exaggerated prolactin release. During the course of repeated blood sampling at 10-min intervals she had two LH episodes, with levels in the normal range, which were

FIG. 5. At the beginning of the exam period serum prolactin levels in this patient were very high, which probably reflects the stress of venepuncture. In addition, the two LH pulses are accompanied by two large prolactin pulses which are above the normal range (see Fig. 3). This patient suffered from severe premenstrual mastodynia and her progesterone levels measured over three previous cycles were always in the subnormal range, which was probably due to the fact that the LH pulses did not elicit increased luteal progesterone release (left panel). Hence, this patient had malfunctioning small luteal cells (see text). After treatment with mild dopamine-containing extracts from the herb Vitex agnus castus, her prolactin response to stress and the accompanying LH prolactin pulses had normalized and the corpus luteum reacted normally to the regularly occurring LH pulses (right panel). Hence, it appears that the latent hyperprolactinemia induced a luteal phase deficiency.

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accompanied by two large prolactin pulses, of which the magnitude was again well above the normal range (see Fig. 3). Unlike in normal women, the corpus luteum did not react to the LH pulses (Fig. 3). Hence, the small luteal cells which normally respond to LH pulses with increased progesterone secretion did not release progesterone in amounts sufficient for a fertile luteal phase (see. Fig. 2). In addition, the three patients with a latent hyperprolactinemia all suffered from severe premenstrual mastodynia, which can probably be explained by the latent hyperprolactinemia. Increased stress-induced prolactin release and increased episodic prolactin release are typical features for latent hyperprolactinemia (Miihlenstedt et al., 1978), which raises the question whether latent hyperprolactinemia and LPD are causally linked events. Therefore, for the three patients whom we could study thoroughly we prescribed a prolactin-suppressive therapy utilizing an herbal preparation which contained an extract of Vitex a g n u s castus. In both cell culture and animal experimental work this extract has been shown to contain dopaminergic compounds producing mild prolactinsuppressive effects. Patients were treated over a period of 3 months and reexamined again on day 7 or 8 of the luteal phase. As shown in Fig. 5b, stress-induced and episodic prolactin release were still present but now clearly in the normal range. The two LH pulses that occurred during the investigation period stimulated progesterone release from the corpus luteum and the basal and LH pulse-induced progesterone levels were in the normal range. Hence, treatment with this mild dopaminergic plant extract normalized the latent hyperprolactinemia and the LPD. LPD may originate in the ovaries, McNeilly and Soules (1988) and attribute it to a reduced number of primordial follicles or accelerated luteolysis. Such accelerated luteolysis results in a luteal phase that is too short, thus enabling the trophoblast to nidate (Lenton et al., 1984). The reason(s) for such early luteal regression are largely unknown; however, cell and animal experiments point to the possibility that immune-competent cells which invade the corpus luteum at the time of luteolysis may be actively involved in luteolysis (Adashi, 1990; Wuttke et al., 1998, 1999). Cells of the white blood cell line produce a number of cytokines including monocyte chemoattractive protein (MCP-1) (Tsai et al., 1197; Haworth et al., 1998) and tumor necrosis factor-~ (TNF), both of which were shown to be luteolytic proteins (Pitzel et al., 1993; Wuttke et al., 1998). Hence, if these immune-competent cells invade too early, this in turn may cause the corpus luteum to regress too early and therefore result in infertility. At present there are a number of other possibilities that might explain improper luteal function. There is overwhelming evidence that the

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LH-induced process of ovulation induces arrays of events which include production and liberation of mitogenic and angiogenic factors. Among the mitogenic factors insulinlike growth factor I and tumor necrosis factor-a appear to be of utmost importance (for reviews see Adashi et al., 1985, 1989; Jones and Clemmons, 1995). They stimulate the postovulatory mitotic division of granulosa and theca cells (Hammond et al., 1982, 1989). Wherever tissues grow, angiogenic factors are necessary to guarantee proper blood supply (Koos, 1993; Smith et al., 1994). In the past a number of angiogenic factors have been identified within the ovary, which include vascular endothelial growth factor (VEGF), which was shown to be highly expressed in developing corpora lutea (Philips et al., 1990; Ravindranath et al., 1992). It is also likely that the basic fibroblast growth factor is involved in proper development of the corpus luteum (Gospodarowicz et al., 1987). It is currently discussed, though not proven, that only proper expression of these peptides leads to a fully functional CL and that any disturbed production may cause LPD. A corpus luteum consists of a variety of steroidogenic and nonsteroidogenic cell types. The most abundant cell types are the endothelial cells and the pericytes (O'Shea et al., 1979). Resident cells stemming from the white blood cell line are also present (Adashi, 1990) and fibroblasts are constituents of the corpus luteum. All cell types, the endothelial cells, the fibroblasts, as well as the cells stemming from the white blood cell line, appear to be involved in proper formation, function, and regression of the corpus luteum (Meidan and Girsh, 1997; Pitzel et al., 1998). This indicates that paracrine actions of growth factors, cytokines, and steroids are very important for proper function of the corpus luteum. The minority of cells are the steroidogenic cells which are divided into two cell types. First, the large luteal cells (LLC) stem from the follicular granulosa cells (Mori et al., 1983; Hansel and Dowd, 1986; Schwall et al., 1986; Ohara et al., 1987). They are not LH-receptive but produce a number of autocrine- and paracrine-acting peptides and eicosanoids. Also their steroidal products, progesterone and estradiol, have profound intraluteal functions (Maas et al., 1992). They guarantee the basal progesterone (and estradiol) production of the CL. Second, the small luteal cells (SLC) are derived from the follicular theca cells (Hansel and Dowd, 1986). During the course of CL ripening they acquire LH receptivity and respond to the regularly occurring LH pulses (see Fig. 3) with increased progesterone and estradiol secretion. In the course of studying pulsatile LH secretion and the response of the CL in LPD patients we observed some interesting new insights, which are discussed throughout the remainder of the chapter. In a total of 52 patients suffering from repetitively occurring LPD we observed in

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23 cases (44%) that no LH pulse occurred during the 8-h investigation period. This resulted in subnormal progesterone and estradiol levels. The hypothalamic GnRH pulse generator fails to elicit LH pulses at normal intervals in three patients and Fig. 6 shows an example. As a result serum progesterone and E2 levels are subnormal during the entire investigation period. As discussed earlier, progesterone feeds back into the hypothalamus to reduce the activity of the GnRH pulse generator (Soules et al., 1984; Nippoldt et al., 1989) and this effect involves activation of endogenous opioid peptide systems, most likely the hypothalamic ~-endorphin neurons (Quigley and Yen, 1980; Ropert et al., 1981; Steele and Judd, 1986). In these 23 patients the hypothalamic GnRH pulse generator seems to be oversuppressed even though serum progesterone levels were lower than normal. Therefore we name this subtype of LPD hypothalamic LPD. Interestingly, the hypothalamic pulse generator in these patients functions normally during the follicular phase (which is also shown in Fig. 6). Abnormal luteal function induced by too-infrequent GnRH-induced LH pulses was also reported in rhesus monkeys (Hutchinson et al., 1986) and clinically treated women (Sheehan et al., 1982). In a second subgroup of luteal-insufficient patients, LH pulses occurred regularly during the mid-/late luteal phase. The corpus luteum, however, responded poorly to these LH pulses, resulting in basal progesterone and estradiol levels in the subnormal range (Fig. 7). We interpret this as a failure of the SLC to respond to the regularly occurring LH pulses and consequently we call this condition small luteal cell defect (SLCD). In SLCD, for unknown reasons, the theca-derived SLC in the CL either did not differentiate well or did not acquire LH receptivity. Such cases of SLCD are almost as common as hypothalamic LPD. We observed 18 such cases (35%) in the 52 patients we have studied so far. In some of these patients we were also able to study pulsatile hormone release during the follicular phase and again found normal LH pulsatility (Fig. 7) and normal follicular development, including normal serum estradiol levels. The third and smallest subgroup of LPD is composed of those whose condition is also of ovarian origin. In 11 of the 52 patients (21%) with

FIG. 6. Serum LH, estradiol, and progesterone levels in a woman suffering from a hypothalamic corpus luteum insufficiency (hypoth. LPD). Note the regularly occurring LH pulses with high frequency (nine pulses during 8 h) and low amplitude during the follicular phase (FP, left panel). Although this patient had ovulated, no high-amplitude LH episode could be identified during the luteal phase (LP, right panel).

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repetitive LPD normal LH pulsatility was observed during the follicular phase, which is associated with normal follicular development. During the mid-/late luteal phase these patients also had normally occurring LH pulses and the CL response to these LH pulses was clearly detectable. In between LH pulses, however, the basal progesterone release decreased to extremely low levels (Fig. 8). As the large luteal cells guarantee basal LH-unstimulated progesterone release, it appears that these granulosa cell-derived cells are not functioning adequately; therefore we call this subgroup of LPD the large luteal cell defect (LLCD). In all cases of hypothalamic LPD, SLCD, or LLCD, LH pulsatility, follicular development, and serum estradiol levels in the follicular phase were apparently normal. We conclude therefore that in SLCD and LLCD the postovulatory array of events leading to mitotic activity of the steroidogenic cells and to CL vascularization are disturbed. This, however, requires further investigation. 3. Is There a Way to Prove the Existence of Three Types of LPD? In many patients in whom LPD is suspected, it is routine to stimulate the CL with human chorionic gonadotrophin (hCG) to test whether the CL of SLCD patients reacts to this gonadotropic stimulus. Hence, we studied LH, estradiol, and progesterone pulsatility in patients with repetitive LPDs under unstimulated and hCG-stimulated conditions. The type of LPD was determined during a first cycle. During the next cycle, prior to hCG treatment, three blood samples were withdrawn to ascertain if an LPD had occurred again. Patients were then injected with 5000 IU hCG at days 5 or 6 and the CL response was studied at days 6, 8, or 9. Figure 9 shows a case of a normal subject under unstimulated and hCG-stimulated conditions. As one would expect, in the unstimulated cycle the CL responds to the LH episode properly with increased progesterone and estradiol secretion and basal levels were never abnormal. When the subject was treated with hCG during the next cycle, her CL increased progesterone and estradiol production approximately fourfold and no LH episode was observed during the investigational period. This points to a strong negative feedback of progesterone and/or E2 into the hypothalamus due to the supraphysiological concentrations of these steroids in the blood. In each of the four subjects studied so far, however, the CL reacted with increased progesterone and E2 secretion, Figure 10 shows LH and estradiol levels of an infertile woman suffering from repetitive LPD where LH levels at day 9 of the luteal phase of the first (control) cycle are very low; the very low amplitude fluctuations identify it further as hypothalamic LPD. As a result serum progesterone levels are continuously subnormal without significant

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fluctuations. Upon hCG treatment at day 6 of the luteal phase of the next cycle serum progesterone levels were significantly increased compared to those of the previous cycle and were above the normal range indicating that hCG continuously stimulated the small luteal cells of this CL. This is in striking contrast to the hormone profile of a woman suffering from repetitively occurring small luteal cell defects (Fig. 11). At day 9 of the luteal phase of the first cycle an LH pulse had occurred prior to the beginning of blood sampling and another occurred at the end. Both stimulated progesterone secretion only slightly and never into the normal range. Consequently, the hCG injection at day 6 of the ~following luteal phase failed to stimulate progesterone secretion significantly and most of the progesterone levels remained in a subnormal range. Interestingly, progesterone in the hCG-stimulated luteal phase exerted a weak feedback into the hypothalamus. Therefore LH pulses occurred at the typical luteal frequency. The woman suffering from a repetitively occurring large luteal cell defect (Fig. 12) had a small LH pulse at the beginning of the blood sampling period and another marked LH pulse at the end of the sampling period. Both pulses stimulated progesterone such that the values increased into a normal range for a short period of time. They then dropped, however, quickly into the subnormal range. In the following cycle the hCG treatment at day 6 stimulated progesterone secretion very markedly such that no endogenous LH pulse occurred. Luteal progesterone secretion in this woman and three others with an LLCD fluctuated markedly, albeit no LH pulses occurred. HCG has a long half-life in the blood and it can be assumed that the levels of the injected hCG did not fluctuate. Therefore it must be concluded that the marked progesterone pulses seen in this type of LPD are due to an intraluteal pulse generator. Such pulsatile release of luteal progesterone in the absence of exogenous LH pulses was also shown in women with normal luteal function (Filicori et al., 1984; Hinney et al., 1995) and in monkeys (Healy et al., 1984) and is believed to be due to auto-/paracrine-interacting peptides, steroids, and eicosanoids. In fact, when the local release of progesterone, oxytocin, and angiotensin II were studied in porcine CL, pulsatility could be demonstrated throughout the lifespan of the CL (Jarry et al., 1990; Einspanier et al., 1991). Thus for in this chapter much attention has been paid to LH pulsatility occurring in the midluteal phase and to the response of the corpus luteum with more emphasis on progesterone release than on that of estradiol. When estradiol levels were analyzed in response to LH pulses and in relation to the different subtypes of LPDs it was evident that LH pulses also elicited increased serum estradiol levels and

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in some cases of LPD estradiol levels were also subnormal. In cases of small luteal cell defects LH pulses or treatment with hCG often did not stimulate luteal estradiol production. The estradiol levels during the luteal phase of normal subjects and those with a LPD often overlapped and an attempt to demonstrate statistically significant differences between estradiol levels in women with normal ovulatory cycles and those with the three different subtypes of LPD failed. In the literature there is also some indication that luteal phase estradiol levels are unimportant and no good indicator for proper or improper function of the CL. Furthermore, luteal estrogen is not required for the establishment of pregnancy (Zegers-Hochschild and Altieri, 1995). V. TREATMENTOF LUTEAL PHASE DEFICIENCIES

The most common treatment of a LPD is the attempt to stimulate luteal progesterone secretion by hCG. In IVF patients this is the most effective treatment and superior to progesterone (administered intravaginally) substitution (Soliman et al., 1994). As demonstrated above, this is successful in the most common type of LPD, hypothalamic LPD, as well as in the least common type of LPD, i.e., LLCD. In case of SLCD, we demonstrated that treatment with hCG (or clomifen citrate) is ineffective because the CL is unresponsive to any gonadotrophic stimulus. This explains why in a number of patients with LPD gonadotrophic support of the corpus luteum is ineffective in establishing pregnancy (Jones et al., 1974). Since the large luteal cells in SLCD patients appear to function properly and since they are regulated by paracrine factors, attempts to treat these patients with such factors may prove to be effective tools. This, however, needs further detailed (and elaborative) studies. Currently it can be advised that intravaginal application of progesterone (Rosenberg, 1980) may be the optimal therapy for such cases. REFERENCES Adashi, E. Y. (1990). The potential relevance of cytokines to ovarian physiology: The emerging role of resident ovarian cells of the white blood cell series. Endocr. Rev. 11, 454-464. Adashi, E. Y., Resnick, C. E., D'Ercole, A. J., Svoboda, M. E., and Van Wyk, J. J. (1985). Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr. Rev. 6, 400-420. Adashi, E. Y., Resnick, C., Hernandez, E. R., Svoboda, M. E., Hoyt, E., Clemmons, D. R., Lund, P. K., and Van Wyk, J. J. (1989). Rodent studies on the potential relevance of insulin-like growth factor (IGF-I) to ovarian physiology. In "Growth Factors and the Ovary," (A. N. Hirshfield, Ed.), pp. 95-105. Serono Symposia. Plenum Press, New York.

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