In-vitro synthesis of steroid hormones in corpora lutea from normal women and women treated with 300 fig norethisterone

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1 Human Reproduction vol.6 no 8 pp , 1991 In-vitro synthesis of steroid hormones in corpora lutea from normal women and women treated with 3 fig norethisterone T.Kim-Bjdrklund 1, B.-M.Landgren and L.Hamberger 2 Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm and department of Obstetrics and Gynaecology, Sahlgrenska Hospital, Goteborg, Sweden 'To whom correspondence should be addressed The in-vitro oestradiol (Ey and progesterone (P) production by corpora lutea (CL) obtained at sterilization from 3 untreated women and 43 women treated with norethisterone (NET) 3 /tg daily was measured. The CL were obtained at different stages of the luteal phase in the untreated women [luteinizing hormone (LH) to +3, n = 7; LH +4 to +7, n = 7; LH +8 to +11, n = 9; LH +12 to menses, n = 7] and on days LH +8 to +11 or cycle days 22 to 26 in the NETtreated women. In the treated women, four types of ovarian reaction were identified. Four women showed ovarian reaction Type A (completely inhibited ovarian activity), 14 women Type B (marked follicular activity, but no luteal function), 12 women Type C (normal follicular activity, followed by insufficient luteal function) and 13 women Type D (apparently normal follicular and luteal activity). The CL were incubated in Eagle's medium with and without stimulation by human chorionic gonadotrophin (HCG) for 2 and 4 h. In the untreated women, P and E 2 production increased significantly with both incubation time and stimulation by HCG throughout the luteal phase, except in the late luteal phase (LH +12 to menses) where P increased (P <.1) only after 4 h stimulation by HCG. The maximal production of P was found after 4 h incubation with HCG stimulation of CL tissue in the early-mid luteal phase (LH +4 to +7). In the treated women with luteal activity (Types C and D), the production of P increased significantly with incubation time and HCG stimulation, although the amount produced under all incubation conditions was significantly lower than in the corresponding control phase (LH +8 to + 11) in the untreated women. In addition, the P production in Type C was significantly lower than in Type D. Oestradiol production in Types C and D was similar to that of the corresponding control group of the untreated women. Based on the results of the present study, NET seems to have a direct effect on corpus luteum function, specifically by exerting an inhibitory effect on P production. Key words: corpus luteum/progesterone/oestradiol/in vitro incubation/low dose gestagen Introduction Since the first studies on the effects of low dose gestagens on pituitary-ovarian function were performed in the mid-sixties by Martinez-Manautou et al. (1966), a great number of papers have been published concerning the subject (Fotherby et al., 1968; Moghissi et al., 1971; Larsson-Cohn et al., 1971). It is now well known that the minipill does not always inhibit ovulation. However, the exact mechanism of its action on pituitary ovarian function is not yet fully understood. For instance, it has been shown by Mukherjee et al. (1972) that corpora lutea (CL) obtained from women treated with norgestrel (75 /ig daily) had a decreased capacity to synthesize progesterone during in-vitro incubation. On the other hand, Landgren and Diczfalusy (198) have reported that 4% of women treated with 3 /tg of norethisterone daily showed apparently normal ovulatory cycles, indirectly indicating that CL had been formed. This study was based on repetitive hormone determinations in peripheral blood. The aim of the present study was to assess further the effect of 3 fig of norethisterone, when given daily over 3 months, on the in-vitro steroid-producing capacity of the corpora lutea. Materials and methods Clinical material Seventy-three apparently healthy women, aged years with a history of regular menstrual cycles (25 35 days) and proven fertility, volunteered for the study. None of the women had used steroidal contraceptives or an intra-uterine device for a minimum of 3 months, or had had an abortion within the last 6 months or a delivery within 1 year before the start of the study. All 73 women underwent a legal sterilization ad modwn Pomeroy. They were divided into two groups; one group consisting of 3 untreated women while the remaining 43 women were treated orally with 3 fig of norethisterone (NET) daily over 3 months until the day of operation. The clinical data for the subjects are presented in Tables I and II. Estimation of the day of ovulation and collection of material During the menstrual cycle in which the laparotomy was performed, follicular growth, both in the untreated and treated women, was followed by ultrasonography and luteinizing hormone (LH) determinations; the latter was done using urine and plasma every day or every second day from cycle day 8 and until ovulation was detected. The day of the LH peak in urine was taken as day. In the untreated women, the operation was performed on different days of the luteal phase; LH to Oxford University Press

2 Steroidog nesl3 by corpora lutea Table I. Age, weight, height and index of obesity in 3 untreated control women and 43 women treated with 3 jig NET daily (mean and range) Index Untreated women (n = 3) Treated women Ovarian reaction 1 A (n =4) B (i = 14) C (n = 12) D (n = 13) Age (years) 36 (29-42) 39 (36-42) 36 (29-42) 38 (33-43) 38 (32-42) Weight (kg) 61 (48-84) 56 (46-59) 57 (5-78) 62 (47-78) 62 (48-9) Height (cm) 165 ( ) 166 ( ) 165 ( ) 164 ( ) 167 ( ) Index of obesity b.227 ( ).23 ( ).21 ( ).228 ( ).217 ( ) Type A: completely inhibited ovarian activity; Type B: marked follicular, but no lutea] function; Type C normal follicular activity, followed by insufficient lutea] function; Type D; apparently normal ovarian activity. b Index of obesity calculated according to Khosla and Lowe (1967) as [weight/(height) 2 ] x 1. (n = 7); LH +4 to +7 (n = 7); LH +8 to +11 (n = 9) and LH +12 to menstruation (n = 7). In the treated women, the operation was carried out on days LH + 8 to +11. In the women where no LH peak was detected, the operation was performed on days after the last menstrual-like bleeding. In these women, no CL were found at operation. They were thus excluded from the study. At operation, the CL was removed in toto by careful excision, immediately divided into two pieces, one piece being transferred into a vial containing chilled, oxygenated Eagle's medium (EM; Flow Laboratories, UK) and transported to the laboratory for further incubation, the other piece being fixed in Bouin's solution for dating and morphological analysis (the morphological results will be reported elsewhere). Peripheral blood was also collected every time the women were subjected to ultrasound examination and on the days before, during and after operation. Blood was drawn using a vacutainer, immediately centrifuged and the plasma frozen and stored at 2 C until analysed. Incubation procedure The CL was freed from the capsule, the large blood vessels were removed and the clotted blood and debris were separated from the tissue. The remaining tissue was then cut into small pieces of -3-5 mg (wet weight) using microscissors. The small pieces were placed in a 2 ml vial and preincubated immediately at +37 C in a gyratory waterbath for 1 h in 15 ml of fresh oxygenated (95% O2, 5% CO2) EM. From the preincubated tissue, 28 small, equally sized pieces were prepared, each about 1 mg wet weight. This procedure was carried out at +4 C. Each piece of tissue was quickly blotted on filter paper and placed into a 5 ml Erlenmayer flask. To 14 control pieces, 1 ml EM alone was added, while 1 IU of HCG (Gonadex 3 IU, Leo Ltd, Helsingborg, Sweden) was included in the 1 ml EM of 14 experimental tissue pieces. This concentration had been shown in earlier studies to produce a just maximal stimulation in Eagle's medium. Seven pieces from each group were incubated for 2 h and the other seven for 4 h. The incubation was performed at +37 C in a gyratory waterbath with an atmosphere of 95% O2 and 5% COj and a constant humidity of 9%. After incuba- Table n. Previous reproductive events in 3 untreated control women and in 43 women treated with 3 ^g NET daily Reproductive events Live births Still births Abortions Total pregnancy Untreated women (n = 'See Table I for description of groups. 3) Treated women (n = A B C )' D tion the tissue specimens were gently dried on filter paper, transferred to vials and stored at -2 C until assayed for protein content. The recovered incubation medium from each Erlenmayer flask was stored at -2 C until assayed for steroid content (within 3 months). Hormone and protein assays Oestradiol (E2) and progesterone (P) levels in the incubation medium and in the plasma samples were measured by radioimmunoassay, according to the method described by Aso et al. (1975). The intra-assay variations were 7.1 and 6.9 for the E? and P assay respectively, and the inter-assay variations were 13.2 and 15.8 respectively. LH in the plasma samples was determined by radioimmunoassay using the WHO method and reagents (Sufi etai, (1986), the results being expressed in terms of the International Reference Preparation of Human Pituitary LH for Immunoassay (JRP 68/4). The intra- and inter-assay variations were 6.4 and 12.2, respectively. LH in urine samples was assessed using an Ovukit (Nobel Medicare, Sundbyberg, Sweden). Total protein content was determined according to the method described by Lowry et al. (1951). Classification of ovarian reaction The ovarian reaction in the women treated with 3 /tg NET daily was classified according to Landgren and Diczfalusy (198). This

3 T.Kim-Bjdrklund, B.-M.Landgren and L.Hamberger classification is based on the peripheral E? and P levels. The following ovarian reaction types were described: Type A, completely inhibited ovarian activity; Type B, marked follicular activity but no luteal function; Type C, normal follicular activity, followed by insufficient luteal function, and Type D, apparently normal ovarian activity. Statistical methods Ethical aspects Informed consent was given by all volunteers after the objectives of the study were carefully explained to them. It was particularly emphasized that they might withdraw from the study at any time. Permission to conduct the study was granted by the local Ethics Committee of the Karolinska Hospital. Incubation time Days from I X peak Fig. 1. Geometric mean levels and 95% confidence limits of E? produced by CL from normally menstruating women (untreated women) during different stages of the luteal phase. The incubation was performed for 2 and 4 h in the absence and presence of HCG (1 IU/ml). Number of subjects in each group is given in Materials and methods (Mens = menstrual bleeding). Results All untreated women showed well-formed CL at operation and their peripheral levels of E2 and P were in the range of the mean values for the corresponding day of the luteal phase. In the treated women four different types of ovarian reaction were identified (Landgren and Diczfalusy, 198). Four women showed Type A, 14 women showed Type B, 12 women showed Type C, and 13 women Type D. At operation, the women with ovarian reaction Type A had inactive ovaries (no sign of follicle growth), and all women with ovarian reaction Type B showed antral follicles in at least one ovary. Corpora lutea were present in all women exhibiting ovarian reaction Types C and D. The CL obtained from the untreated women and from the women with ovarian reaction Types C and D were incubated as described in the Materials and methods, and in-vitro synthesis of E2 and P was documented. Oestradiol production The E2 production by CL obtained from the untreated women is shown in Figure 1. Oestradiol increased significantly with the incubation time throughout the lutea] phase (P <.5). Addition of HCG significantly stimulated the E2 production only after 4 h incubation (P <.1: LH to + 3, LH +4 to +7, LH +12 to menses, P <.5: LH + 8 to +11). No difference in the magnitude of E2 production with the age of the CL could be observed before or after stimulation with HCG. Oestradiol production by CL in the treated women is shown in Figure 2. The production of E2 by the CL in ovarian reaction Types C and D exhibited a significant increase when the incubation period was prolonged from 2 to 4 h (P <.5). When HCG was added, a significant stimulation of E2 was elicited after both 2 h (P <.5) and 4 h (P <.5) incubation in each of the groups h 4h Incubation time Fig. 2. Geometric mean levels and 95% confidence limits of Ej produced by CL from 12 women who showed ovarian reaction Type C and 13 women who showed ovarian reaction Type D during treatment with 3 /tg NET daily for 3 months. Incubation was performed for 2 and 4 h in the absence and presence of HCG (1 lu/ml) in the incubation medium (Mens = menstrual bleeding). Progesterone production Progesterone production in the untreated women is presented in Figure 3. The highest basal level of P production was found in the mid-luteal phase (LH +8 to +11). Progesterone levels increased significantly with the incubation time (P <.1: LH to + 3, LH +4 to +7, P <.5: LH +8 to +11) and with the addition of HCG (P <.1) throughout the luteal phase, except in the late luteal phase (LH +12 to menses), where the P production increased only after 4 h incubation with HCG (P <.1). Progesterone production by the CL from treated women is shown in Figure 4. The P production in Types C and D increased significantly when the incubation period was prolonged from 2 h to 4 h (P <.5) and when HCG was added In all assays, a log normal distribution of individual values was assumed (Gaddum, 1945). All mean values are presented as geometrical means with 95% confidence limits. The significance of differences between levels was assessed by calculating F-values from appropriate contrasting values after an analysis of variance. When comparisons between the treated women and untreated women were performed, levels were always referred to the group of subjects in phase LH +8 to +11 in the untreated women.

4 Steroidogenesis by corpora lutea 25 2h 4h +4 to +7 2h 4h h 4h Incubation time Days from + I2tomens LH peak Fig. 3. Geometric means and 95% confidence limits of progesterone (P) produced by the corpora lutea (CL) from normally menstruating women (untreated women) during different stages of the luteal phase. For further explanation see the legend to Figure 1. c '(I) O Q. O) "O Q_ 8 o Q_.3UU n Q Control thcg il n 111 I'fl in! M MI f i h I 2h 4h Incubation time D Ovarian reaction Fig. 4. Geometric means and 95% confidence limits of P produced by corpora lutea from women treated with NET. For further explanation see the legend to Figure 2. to the medium (P <.5). Ovarian reaction Type D produced higher amounts (f <.1) of P than Type C during all different incubation conditions. A comparison of the E 2 and P production by CL obtained during the period LH +8 to +11 in the untreated women with that from treated women showing ovarian reaction Types C and D is presented in Figures 5 and 6, respectively. Both before and after stimulation with HCG, the E2 production in Types C and D was not significantly different, whereas the P production was significantly lower in treated than in untreated women. Discussion The present study shows that the control E 2 production by the CL of untreated women did not vary significantly with the different stages of the luteal phase. Similar results have previously been reported by Patwardhan and Lanthier (198) and McNeilly 2 4 Hours of incubation Fig. 5. Geometric means and 95% confidence limits of F^ produced by CL from nine normally menstruating women during the period LH +8 to +11 and in 12 and 13 women that showed ovarian reaction Types C and D, respectively, during treatment with 3 /xg NET. Incubation was performed for 2 and 4 h in the absence and presence of HCG (1 IU/ml). c ) o Q_ O).E 5 Ea. LLJ o rr LLJ LLJ o orr 2t> n ± Control TypeC Type D -<5> + hcg A -o- +hcg / / o +hcg / \ t < ' " Hours of incubation \ \ /.. Fig. 6. Geometric means and 95% confidence limits of P produced by CL specimens. For further details see the legend to Figure 5. The statistical significances are denoted as follows: *P <.5, **P <.1, ***P <.1. et al. (198) who measured E2 concentration of CL. On the other hand, Hunter and Baker (1981) found that the E2 production by the CL was highest during the early luteal phase and t ' t 153

5 T.Kim-BJdrkhind, B.-M.Landgren and L.Hamberger Swanston et al. (1977) showed that, when E 2 was extracted from the CL, the content was highest in the mid-luteal phase. These discrepancies in results may be due to the different ways of classifying the luteal phase and/or to different numbers and ages of the subjects investigated. It is well known that HCG stimulates E2 production via activation of cyclic adenosine monophosphate (camp) when added to the incubation medium of minced and sliced human CL (Nakashima, 1979; Hunter and Baker, 1981) and to cultures of isolated luteal cells (Richardson and Masson, 1981; Dennefors etal., 1982; Polan etai, 1984; Devoto etal., 1989). In the present study, E2 production by CL from the untreated women increased significantly after 4 h stimulation with HCG. The magnitude of the stimulation did not vary with the age of the CL, indicating that the activation of the aromatization by HCG under the present incubation conditions occurs throughout the luteal phase. The ability of HCG to stimulate the E 2 production during the late luteal phase is of interest, since this is probably the time when the CL is rescued by HCG if pregnancy occurs. One might therefore expect CL of the late luteal phase to react by a significant rise in Ej production during HCG stimulation. This was also the case in our study, when incubation was prolonged to 4 h. Previous investigations have shown that incubations of isolated CL (Hunter and Baker, 1981; Dennefors et al., 1982) as well as cultures of human granulosa luteal cells (Veldhuis et al., 1983; Polan et al., 1984; Hillensjo et al., 1985; Jalkanen et al., 1986) produced the highest amounts of P during the mid-luteal phase. Similar results were obtained in this study. The highest amount of P in the control incubation in the untreated women was obtained in the mid-luteal phase (LH +8 to -+-11) and the amounts produced by the young and premenstrual CL were low. This may be explained by the fact that the CL becomes fully mature as an endocrine organ to produce P during the mid-luteal phase (Corner, 1956), which is also reflected by circulating levels of this steroid. In addition, there is some evidence indicating that P stimulates its own secretion from the CL, thus acting as an intrinsic ovarian luteotropin (Rothchild, 1981). Addition of HCG to the medium did stimulate the production of P by CL during all stages of the luteal phase. However, the time required for stimulation varied between the young and the premenstrual CL. In the young CL, the stimulation was more marked after 2 h incubation, whereas in the old CL the stimulation was more pronounced after 4 h. The rapid response of the young CL to HCG may be explained by the fact that large fresh human CL showed the highest binding capacity for HCG (Wardlaw et al., 1975) and also that the number of receptors for LH in the luteal cells is highest during this period (Hansel and Dowd, 1986). The retarded response of the premenstrual CL to HCG can be explained by a decreased sensitivity of the luteal cells to this hormone. Our findings confirm those of Dennefors et al. (1982) who reported that complete loss of HCG sensitivity in the human CL does not occur until the onset of menstruation. Martinez-Manautou et al. (1967) had already established in the mid-sixties that mini-pill formulations containing progestogens do not always inhibit ovulation. In this study, four of the 43 women (1%) reacted to treatment with 3 /xg NET daily with complete suppression of ovarian reaction (Type A), 14 (31%) 154 showed follicular activity only (Type B), 12 (3%) presented decreased P profiles indicating CL insufficiency (Type C) and 13 (3%) reacted to treatment with apparently normal ovarian activity (Type D). These results are in good agreement with earlier findings (Landgren and Diczfalusy, 198). The women who reacted with complete suppression of ovarian activity had ovaries lacking follicles or CL. The women with high circulating E2 levels had antral follicles in one or both ovaries, and women with E2 and P rise had CL. The CL of these women produced P and E2, which increased significantly in the presence of HCG. The basal P production before as well as that after HCG stimulation was, however, significantly lower than in the corresponding control phase in the untreated women (LH +8 to +11), suggesting a direct effect of NET on P production. Moreover, a clear difference in the inhibitory effect of NET on P production could be observed, this being that inhibition was more pronounced in the CL of Type C, as indicated by the significantly lower levels of P in this group when compared to TypeD. The E2 production of the CL was not affected by NET treatment. The E2 levels between the NET treated and untreated women were not significantly different. Mukherjee et al. (1972) previously reported that, in women treated with norgestrel (75 fig daily) who formed CL, the peripheral levels of E2 were not different from the controls, suggesting that norgestrel did not affect the Ej production. On the other hand, the ability of the CL to produce P in vitro appeared to be reduced. These findings together with ours, suggest that in women who form CL during treatment with low dose progestogens, the effect of the progestin is probably more specific to the CL cells that produce P than those responsible for E2 production. Acknowledgements We wish to thank Dr Sten Cekan for statistical advice, Dr Ana-Rosa Aedo for fruitful discussions and Mrs Britt Masironi and Karin Regne"r for their assistance in the preparation of the figures and typing the manuscript. References Aso.T., Guerrero,R., Cekan,Z. and Diczfalusy,E. (1975) A rapid 5 hour radioimmunoassay of progesterone and oestradiol in human plasma. Clin. Endocrinoi, 4, Comer,G.W., Jr (1956) The histological dating of the human corpus luteum of menstruation. Am. J. Anat., 98, Dennefors,B.L., Sjogren.A. and Hamberger.L. (1982) Progesterone and adenosine 3',5'-monophosphate formation by isolated human corpora lutea of different ages: Influence of human chorionic gonadotropin and prostaglandins. J. Clin. Endocrinoi. Metab., 55, Devoto.L., Vega.M., Navarro.V., Sir,T., Alba.F. and Castro.O. (1989) Regulation of steroid hormone synthesis of human corpora lutea; failure of follicle-stimulating hormone to support steroidogenesis in vivo and in vitro. Fertil. 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