Endometrial histomorphometry of trimegestone-based sequential hormone replacement therapy: a weighted comparison with the endometrium of the natural cycle.

Histomorphometric changes in the endometrium were evaluated under the effect of a trimegestone-based sequential hormone replacement therapy (HRT) regimen, and the findings were compared to those in endometrium of the natural cycle. Endometrial samples were taken from postmenopausal women who completed a randomized, double blind, dose-ranging study of oral trimegestone (0.05, 0.1, 0.25 and 0.5 mg per day) from day 15 to day 28 with continuous micronized oestradiol 2 mg daily for six treatment cycles. The HRT-treated endometrium, irrespective of the dose, had a smaller mean total glandular area, smaller average glandular diameter, smaller mean total vascular space area and diameter than the luteal phase. Stromal cellularity was similar in the four dose groups. There were reduced glandular secretions in the endometrium from the high dose group. The relative weighting of these histological parameters was evaluated by linear discriminant analysis. The weighted values were dose independent, and may therefore represent either a specific effect of trimegestone, number of days administered, or both. We have constructed an equation to assign a value for a histological parameter which determines the position on linear discriminant functions. These assigned values can be used in other sequential HRT regimens to determine the relative influence of a given progestogen on endometrial morphology in relation to different phases of the natural cycle.     

The distribution of endometrial leukocytes and their proliferation markers in trimegestone-treated postmenopausal women compared to the endometrium of the natural cycle: a dose-ranging study.

The effect of trimegestone-based sequential hormone replacement therapy (HRT) on the distribution of endometrial leukocytes and Ki-67 expression was investigated and the findings compared with the endometrium of the natural cycle. Endometrial cells positive for CD45(+), CD56(+), CD3(+), Ki-67(+) and CD45(+)/Ki-67(+) antigens were immunohistochemically evaluated in samples from postmenopausal women who completed a randomized, double-blind, dose-ranging study of oral trimegestone (0.05, 0.1, 0.25 and 0.5 mg per day) from days 15 to 28 with continuous micronized oestradiol 2 mg daily for six treatment cycles. The control samples were luteinizing hormone (LH)-dated endometrial biopsies. Cell counts were interpreted using linear discriminant analysis and unpaired t-test. The dose of trimegestone did not significantly affect the mean count of CD45(+), CD56(+), CD3(+), Ki-67(+) and CD45(+)/Ki-67(+) cells in the endometrial biopsies obtained from treated women. Endometrial sections from women who bled on the day of the biopsy contained higher numbers of CD45(+) and CD56 cells. In the trimegestone-treated endometrium, CD45(+), CD56(+) and CD3(+) cell expression was similar to the proliferative and early secretory phases of the natural cycle. However the expression Ki-67 and CD45(+)/Ki-67(+) cells was similar to the menstrual phase of the natural cycle endometrium. Women treated with four doses of trimegestone exhibited four different bleeding patterns. Therefore the endometrial infiltration with these cells did not explain the pattern of bleeding in women on this HRT regimen.     

Endometrial effects of three doses of trimegestone,a new orally active progestogen,on the postmenopausal endometrium

Objective: To study the effects of oral trimegestone on endometrial histology and vaginal bleeding when given in combination with oral 17-β-oestradiol. Methods: This was a prospective, randomised, double-blind, parallel groups, pilot comparative study. Thirty-eight healthy postmenopausal women with normal endometrial histology were given oral 17-β-oestradiol, 2 mg/day for three continuous cycles of 28 days, plus oral trimegestone, 0.10, 0.25 or 0.50 mg/day from day 15 to day 28 of each cycle. A Vabra biopsy was performed late in the oestradiol/trimegestone phase of cycle 3 and examined for histological evidence of secretory transformation of the endometrium. Characteristics of vaginal bleeding were recorded on a daily basis. Results: Thirty-seven women completed the study, of whom 31 yielded adequate tissue for histological assessment. All showed secretory transformation of the endometrium. Bleeding was of earlier onset and longer duration with the lowest dose of trimegestone. Conclusions: Trimegestone is a highly effective oral progestogen for endometrial protection, all doses inducing secretory endometrial transformation. Although bleeding patterns suggest a weaker effect of the lowest dose used, the minimum effective dose for endometrial protection has still to be determined and may be lower than those used in this study.     

Acceptability and patterns of uterine bleeding in sequential trimegestone-based hormone replacement therapy: a dose-ranging study

Trimegestone is a norpregnane progestogen which is being developed in combination with oral oestradiol as postmenopausal hormone replacement therapy (HRT). In this multicentre dose-ranging study using randomized parallel groups, four doses of trimegestone were used to compare data on the patterns of uterine bleeding, the endometrial histology, and the control of menopausal symptoms in 203 women who completed treatment for 6 months. The treatment consisted of micronized oestradiol (2 mg/day) and one of four doses of trimegestone, which was administered sequentially for days 15-28 of the treatment cycle. Higher doses of trimegestone were associated with later onset of bleeding, which was lighter and of shorter duration than that observed with lower doses. The variability of the day of onset of bleeding in individual women was greater when bleeding occurred before the end of the progestogen phase (early bleeders) than when it occurred afterwards (late bleeders). All women enrolled in the study experienced good control of menopausal symptoms, with minimal progestogenic adverse effects, there being no statistically significant difference between the four dose groups.     

Effects of natural progesterone on the morphology of the endometrium in patients with primary ovarian failure

In 43 patients without ovaries, endometrial biopsies at day 21 of 75 substituted cycles were studied by light and electron microscopy. The morphology of the endometrium was compared after oral, vaginal or intramuscular administration of progesterone, and correlated with the serum levels of 17-beta oestradiol and progesterone and the pregnancies obtained after oocyte donation. After vaginal application of micronized progesterone, endometrial morphology closely matched that of a natural cycle. This therapy was able to support two ongoing pregnancies. No adequate endometrial response was noted after oral ingestion of progesterone. The maturation of the endometrium after intramuscular injections of progesterone in oil was heterogeneous. It was concluded that the vaginal route for administering micronized progesterone can be advised as the treatment of choice in patients without ovarian function.     

Comparison between 1 year oral and transdermal oestradiol and sequential norethisterone acetate on circulating concentrations of leptin in postmenopausal women

BACKGROUND: Oral and transdermal postmenopausal hormone replacement therapy (HRT) affects lipid and glucose metabolism differently, which is of significance in the release of leptin by adipocytes. Moreover, oestrogen and progesterone can stimulate leptin secretion in women of reproductive age. Therefore, we compared the effects of oral and transdermal oestrogen plus progestin regimen on plasma leptin in 38 healthy postmenopausal women with normal body mass index (BMI), who wished to use HRT to control incapacitating climacteric symptoms. METHODS: The women were randomized to treatment with oral HRT (2 mg oestradiol on days 1--12, 2 mg oestradiol plus 1 mg norethisterone acetate (NETA) on days 13--22, and 1 mg oestradiol on days 23--28, n = 19), or with transdermal HRT (50 microg/day of oestradiol on days 1--13, and 50 microg oestradiol plus 250 microg/day NETA on days 14--28, n = 19) for 1 year. Plasma samples were collected before and at oestradiol + NETA phase after 2, 6 and 12 months treatment and were assayed for leptin. RESULTS: The baseline leptin, ranging from 3.3 to 34.9 microg/l, was significantly associated with BMI (r = 0.78, P < 0.0001 ), but showed no difference between women in oral HRT (geometric mean 13.9 microg/l, 95% confidence interval (CI) 10.1--17.6 microg/l) or transdermal HRT group (geometric mean 12.0 microg/l, 95% CI 9.7--14.3 microg/l). Neither oral nor transdermal oestradiol + NETA caused any significant changes in plasma leptin (or BMI) after 2, 6, or 12 months of treatment. CONCLUSION: Leptin is an unsuitable factor to detect oestradiol + NETA-induced metabolic changes in postmenopausal women.     

Morphometric analysis of peri-implantation endometrium in patients having excessively high oestradiol concentrations after ovarian stimulation

The present study investigated whether high oestradiol concentrations after ovarian stimulation in infertile women affect endometrial development around the time of implantation. The glandular and stromal components of the endometrium were assessed by morphometric criteria. Endometrial biopsies were taken on day 7 (+/-1) after the ovulating dose of human chorionic gonadotrophin in stimulation cycles and on day 7 after the LH surge in natural cycles. Women (n = 38) undergoing assisted reproduction treatment were evaluated: 12 women in natural cycles, 11 women in stimulation cycles with oestradiol <20,000 pmol/l and failed fertilization after oocyte collection (moderate responders) and 15 women with an oestradiol concentration of > or =20,000 pmol/l in stimulation cycles (high responders). High responders showed delayed glandular maturation and advanced stromal morphology, whereas moderate responders demonstrated synchronous development of glandular and stromal features. In natural cycles, the glands were in phase. The effect of excessively high oestradiol concentrations could be explained by quantitative evaluation of the endometrial biopsies as gland--stromal dyssynchrony, which indicates a deficient secretory transformation of the endometrium that represents a suboptimal endometrial environment for implantation. This substantiates our previous clinical observation of significantly lower pregnancy rates in IVF cycles of women with high oestradiol concentrations (> or =20,000 pmol/l).     

Low dose transdermal estradiol/norethisterone acetate treatment over 2 years does not cause endometrial proliferation in postmenopausal women

OBJECTIVE: We investigated the effects of 2-year transdermal continuous combined estradiol (0.025 mg/day) and norethisterone acetate (0.125 mg/day) (Estragest TTS) on bleeding and on the endometrium. DESIGN: This double-blind, randomized, multicenter, parallel, 1-year trial enrolled 266 healthy women at least 2 years past menopause with intact uteri. Patients received a transdermal patch delivering either 0.025 mg estradiol and 0.125 mg norethisterone acetate daily or placebo. Of the 266 women initially included, 135 (96 Estragest TTS, 39 placebo) completed a second year open follow-up, where all women had the estradiol/norethisterone patch. Endometrial biopsies were performed at weeks 0, 48 (n = 171), and 96 (n =109). Effects on endometrial morphology and uterine bleeding were studied. RESULTS: The overall incidence of endometrial hyperplasia after treatment with the estradiol/norethisterone acetate patch for one year was 0.8% with only one case of atypical hyperplasia. There were no clinically significant changes in endometrial thickness in either treatment group. The proportion of bleed-free patients with the estradiol/norethisterone acetate transdermal system increased from 55% in cycles 1-3 to 83% in cycles 10-12. By the 12th cycle, 92% of patients receiving estradiol/norethisterone acetate patches were bleed-free. No additional hyperplasia was seen during the second year follow-up. CONCLUSIONS: A continuous combined transdermal patch delivering 0.025 mg estradiol/day and 0.125 mg norethisterone acetate/day provided good endometrial protection. The dose maintained a consistently high rate of amenorrhea in postmenopausal women.     

The effect of a change in the dose of trimegestone on the pattern of bleeding in estrogen-treated post-menopausal women: 6 month extension of a dose-ranging study

BACKGROUND: Abnormal bleeding pattern is one major reason for non-compliance with hormone replacement therapy (HRT) in post-menopausal women. We have previously documented that the dose of trimegestone is the main determinant of the pattern of bleeding in women treated with estradiol (E(2)) and sequential combined trimegestone administered in four doses. The objectives of this study were to test the effect of changing the dose of trimegestone and the duration of treatment on the pattern of bleeding in these women who then entered a 6 month extension phase where a single dose of trimegestone was given sequentially combined with E(2). METHODS: The menstrual diaries of 134 post-menopausal women who completed a dose-ranging study of trimegestone and then entered a 6 month extension phase were analysed. In the 6 month extension study, all women were given one dose of trimegestone (0.25 mg) in a sequential fashion (day 15-28) combined with continuous E(2) (2 mg/day). RESULTS: Women who had received trimegestone 0.25 mg/day during the first 6 months experienced no change in the bleeding pattern in the 6 month extension. Women who had been treated with 0.5 mg/day dose experienced earlier onset, and more prolonged bleeding (P < 0.0001) following the change to 0.25 mg/day. Women who previously received trimegestone doses of 0.05 and 0.1 mg experienced a later onset of bleeding, which was lighter and of shorter duration (P < 0.001) during the extension phase as compared with the first 6 months. CONCLUSION: The dose of trimegestone, and not the duration of treatment, appears to be the important determinant of the pattern of bleeding in post-menopausal women on this HRT regimen.     

Continuous intrauterine compared with cyclic oral progestin administration in perimenopausal HRT

OBJECTIVES: Two hormone replacement therapy (HRT) regimens of combined oral estradiol with either continuous intrauterine or cyclic oral progestin were compared for 2 years. METHODS: 200 perimenopausal women randomly received an intrauterine system with continuous levonorgestrel release (20 microg/24 h) combined with oral estradiol (2 mg daily), or a cyclic oral regimen of norethisterone acetate (1 mg on day 13-22) and estradiol (days 1-21; 2 mg, days 22-28; 1 mg). Efficacy on endometrial protection, vaginal bleeding patterns, blood loss and practical use were compared during 26 cycles. RESULTS: Endometrial protection was adequate in both regimens. The cyclic regimen induced a more regular bleeding pattern. The continuous local administration induced a reduction in bleeding (P=0.001) with an initial period of prolonged and frequent bleeding. 38% became amenorrhoeic. Women found both regimens acceptable. CONCLUSIONS: Continuous intrauterine Levonorgestrel administration by using an intrauterine system can well be recommended for use in combination with oestrogen replacement therapy in perimenopausal women.     

Efficacy and tolerability of fully transdermal hormone replacement in sequential or continuous therapy at two doses of progestogen in postmenopausal women

OBJECTIVES: Two randomized open-label studies were performed to evaluate fully transdermal hormone replacement therapy (HRT) with oestradiol (E2) and norethisterone acetate (NETA) in postmenopausal women. METHODS: Both hormones were delivered by transdermal matrix patches changed twice weekly. Subjects received E2, 50 microg/day and NETA, 170 microg/day or 350 microg/day, either continuously or sequentially. A one-year study (13 cycles of 28 days), including a reference regimen of transdermal E2 and sequential oral progestogen, was followed by a continuation study for a further year in 367 women. RESULTS: All regimens were highly and equally effective in the prevention of hot flushes. The fully transdermal regimens were associated with beneficial changes in the lipid profile. The sequential regimens provided effective scheduling of bleeding around the end of the progestogen phase. The continuous regimens were associated with irregular bleeding, which was rarely severe, and a gradually increasing incidence of amenorrhoea. With sequential or continuous therapy, bleeding was less severe at the lower dose of progestogen than at the higher dose. No endometrial hyperplasia was detected by biopsy in any treatment group. One serous endometrial carcinoma and one endometrial adenocarcinoma were detected. An endometrial thickness >5 mm did not predict the presence of hyperplasia at biopsy. Hormone-related adverse events were typical of those expected for HRT and dermal tolerability of the patches was good. CONCLUSIONS: Fully transdermal sequential or continuous HRT is effective and well tolerated in postmenopausal women. The lower dose of NETA may be preferable, because it confers adequate endometrial protection at a lower dose of progestogen.     

In-vivo administration of progesterone inhibits the secretion of endometrial leukaemia inhibitory factor in vitro

Human interleukin for DA cells, also called leukaemia inhibitory factor (LIF), is of cardinal importance for successful murine embryo implantation. Recent studies suggest it may also play an important role in human embryo implantation. Our objective was to study the hormonal regulation of the production/secretion of LIF by the human endometrium. Endometrial LIF secretion in specimens obtained from women without ovarian function (n+/-14) at day 10 (4 mg of oestradiol regimen) or day 20 (oestradiol plus 300 mg of progesterone) of a simulated menstrual cycle was examined. LIF was detected in all cultured explants obtained both in the proliferative and secretory phase of the stimulated cycles. The levels of cytokine production by day 10 endometrial culture explants were 5-fold higher than by day 20 endometrial samples (mean+/- SEM, 24.3+/-8.6 versus 4.5+/-2.1 pg/mg, P < 0.01). This suggests that progesterone significantly down-regulates the endometrial LIF secretion. The effect of progesterone on LIF secretion by the endometrium in vitro was also examined. Explants of endometrium obtained from the same patients on day 10 of cycle were treated with 0.5 ng/ml of progesterone in vitro. This progesterone treatment significantly reduced LIF secretion by endometrial explants in vitro (mean+/-SEM, 20.3+/-4.8 versus 10.7+/-2.3 pg/mg, P < 0.05). These results suggest that LIF endometrial production is regulated by progesterone both in-vivo and in-vitro. The possible mechanisms of LIF regulation are discussed briefly.      

Charcoal treatment and risk of escape ovulation in oral contraceptive users

Ovulatory potential was studied during the use of two oral contraceptive pill preparations, after repeated mid-cycle administration of activated charcoal. Eleven women used monophasic pills containing gestodene, 75 microgram, plus ethinyl oestradiol, 30 microgram, or norethisterone acetate, 1 mg, plus ethinyl oestradiol, 30 microgram, for 4 months each, in randomized order. During both pill treatments the third cycle was a control cycle, and during the fourth cycle of both pill types, 5 g of activated charcoal was ingested four times a day, starting 3 h after pill intake, on cycle days 12, 13 and 14. Ovarian activity was monitored by intravaginal ultrasonography of follicles and by measurements of serum concentrations of LH, FSH, oestradiol and progesterone throughout the control and charcoal-treatment cycles of both pill treatments. None of the women ovulated. Follicular activity seen in two women did not correlate with charcoal administration. It is concluded that the possible enterohepatic recirculation of gestodene and norethisterone is not of clinical importance. Repeated charcoal treatment, when administered 3 h after but at least 12 h before pill intake, can be used to treat diarrhoea in women taking oral contraceptives.     

Clomiphene citrate does not affect the secretion of alpha3, alphaV and beta1 integrin molecules during the implantation window in patients with unexplained infertility.

BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of beta1, alpha3 and alphaV integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5-9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of alpha(v) but not beta1 and alpha3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 +/- 0.12 versus 2.21 +/- 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of alpha(v), beta1 and alpha3 integrin molecules, which might play a role in implantation, was not affected.     

Replacement of frozen-thawed embryos in artificial and natural cycles: a prospective semi-randomized study

This prospective partly-randomized study assessed the relative efficacy of two strategies of patient management for the replacement of frozen-thawed embryos. A luteinizing hormone-releasing hormone (LHRH) agonist was used to induce a temporary hypogonadism in a group of patients who were then prepared for implantation by endometrial priming with hormone replacement therapy (HRT): oral oestradiol valerate and then oestradiol valerate and injections of progesterone. A second group of patients had their frozen-thawed embryos replaced during their natural cycles. Of the 84 patients treated with the LHRH regimen, 80 had embryos replaced and 16 (20%) clinical pregnancies were established. Of the 78 patients treated with natural cycles, 70 had embryos replaced and 14 (20%) achieved clinical pregnancies. There were no statistical differences between the two groups in terms of age, obstetric history, duration of infertility, number of oocytes retrieved and fertilized or the number of embryos frozen following ovarian stimulation in the embryo 'generating' cycle. In terms of pregnancy rates, both protocols were equally effective. However, the LHRH-HRT protocol was of great value in the management of oligomenorrhoeic patients and in establishing standard conditions for implantation in cyclic patients.     

Endometrial and menstrual response to subcutaneous oestradiol and testosterone implants and continuous oral progestogen therapy in post-menopausal women

A regimen of subcutaneous implants of oestradiol and testosterone in combination with continuous oral norethisterone (0.35mg to 5mg daily) was used to treat 71 non-hysterectomised post-menopausal women for up to 30 mth in an attempt to avoid the withdrawal periods associated with conventional cyclical therapy, while at the same time protecting the endometrium from oestrogenic overstimulation. Amenorrhoea, defined as no vaginal bleeding for at least 3 mth, occurred immediately in 5.4–55.6% of women, the percentage depending on the daily dose of the progestogen. In those women who bled, the dose of norethisterone was adjusted at 3-mth intervals. Despite this protocol, only 51.0% of the patients were amenorrhonic after 6 mth, and 63.2% after 1 yr. Although eight women did develop amenorrhoea for 12–27 mth, there was a high drop-out rate by the others, mainly because of unacceptable irregular bleeding. Irrespective of the bleeding pattern, endometrial biopsies 6 mth after treatment revealed endometrial atrophy. It is concluded that this form of therapy is inferior to oral continuous combined hormone replacement where amenorrhoea can almost invariably be achieved.     

Endometrial oestrogen and progesterone receptors and their relationship to sonographic appearance of the endometrium

The rapid development of ultrasonographic equipment now permits instantaneous assessment of follicles and endometrium. The sonographic appearance of the endometrium has been discussed in relation to in-vitro fertilization (IVF) cycles. However, a generally agreed view of the relationship of the sonographic appearance to fecundity in IVF cycles has not emerged. We have studied the relationship between steroid receptors and the sonographic appearance of the preovulatory endometrium in natural cycles and ovulation induction cycles. Preovulatory endometrial thickness was not found to be indicative of fecundity, although a preovulatory endometrial thickness of <9 mm related to an elevated miscarriage rate. The preovulatory endometrial echo pattern did not predict fecundity. No relationships were found among endometrial appearance, endometrial steroid receptors and steroid hormone concentrations in serum. Oestrogen or progesterone receptor concentrations were not related to endometrial thickness or to concentrations of serum oestradiol, the only significant correlation being found between the endometrial concentrations of oestrogen and progesterone receptors. The ratio of progesterone:oestrogen receptor concentration was somewhat less in echo pattern B (not triple line) endometrium compared with pattern A (triple line) endometrium. Oestrogen and progesterone receptor concentrations appeared stable on gonadotrophin induction, though fewer numbers were found during clomiphene cycles than in natural cycles. With regard to the distribution of receptor concentration between clomiphene and natural cycles, most women using clomiphene had very low oestrogen receptor populations. Pregnancy rates were low, in spite of high ovulatory rates during clomiphene treatment and were mainly related to low oestrogen receptor concentrations in preovulatory endometrium.     

Outcome of treatment subsequent to the elective cryopreservation of all embryos from women at risk of the ovarian hyperstimulation syndrome.

From 1st June 1989 to 31st May 1991, 78 women with a serum oestradiol level greater than 3500 pg/ml on the day of the ovulatory trigger, following pituitary suppression with buserelin and ovarian stimulation with human menopausal gonadotrophins (HMG), had all their embryos electively cryopreserved at the pronucleate stage to minimize the risk of developing ovarian hyperstimulation syndrome (OHS). Treatment with buserelin was continued in the luteal phase. A median of 19 oocytes (range 7-43) was obtained and 12 embryos (range 1-37) frozen per cycle. Twenty-one (27%) women developed OHS (six severe). Women developing OHS had higher (P less than 0.05) serum oestradiol concentrations on the 7th day after oocyte retrieval, compared to those who did not. No differences were found for any of the following criteria: aetiology of infertility, age, total dose of HMG, number of oocytes, fertilization rate or freeze-thaw survival of embryos. Subsequently, 125 frozen-thawed embryo replacements have been undertaken, using buserelin and hormone replacement therapy (HRT) (n = 93) or natural cycles (n = 32). The overall freeze-thaw survival and implantation rates per embryo were 71.8 and 11.7%, respectively. The pregnancy rates in natural cycles (19%) and buserelin/HRT cycles (29%) were not significantly different.