Hormones, herbal preparations and nutriceuticals for a better life after the menopause: part I.

In developed countries, women spend more than one-third of their life in the menopause and at least half of them experience vasomotor symptoms that impair their normal function and well-being. Long-term estrogen replacement therapy (HRT) with estrogen can suppress typical menopausal symptoms and prevents osteoporosis. When estrogen-only HRT is started within 10 years after the menopause, the prevalence of cardiovascular disease is reduced, mortality is lower, and the risk of breast cancer is not significantly increased. Postmenopausal genital and urinary problems with recurrent infections, incontinence, and dyspareunia can effectively be treated by vaginal application of estriol, which seems to be safe for women treated for breast cancer. HRT after the age of 60 years is associated with a lower number needed to treat than number needed to harm, implying that there would be one unfavorable side-effect for up to ten women experiencing a positive effect. However, further studies are needed regarding the risk-benefit ratio of HRT in women over 70 years. It is concluded that transdermal substitution therapy with estradiol may increase the number of quality-adjusted life years of postmenopausal women. The combination with nutriceutical food supplementation may add to this benefit, but complementary prospective trials are still needed.

Hormones, herbal preparations and nutriceuticals for a better life after the menopause: part II.

Long-term estrogen replacement therapy with estrogen has benefits for many postmenopausal women. However, some women prefer non-steroidal substitution with herbal preparations. The effectivity against vasomotor symptoms has been evidenced for the extracts of pine bark, of linseed and of Lepidium meyenii (Maca), whereas there is controversy about the effectiveness of genistein-rich soy extract. The extracts of cruciferous vegetables such as Broccoli and of linseed induce changes in the metabolism of estrogens, and antioxidants may reverse altered epigenetic DNA methylation, possibly reducing the risk of breast cancer or its recurrence. Indirect evidence from the literature and from clinical trials supports that a nutriceutical composed of plant extracts, low-dose vitamins and minerals may improve the quality of life by delaying certain age-related diseases. On the basis of epidemiologic studies, physiopathological considerations and controlled prospectieve trials, it is suggested that transdermal substitution therapy with estradiol together with nutriceutical food supplementation may increase the number of quality-adjusted life years of postmenopausal women, but complementary, large-scale, prospective trials are still needed.

The serum estradiol concentration is the main determinant of the estradiol concentration in normal breast tissue.

BACKGROUND: The estrogen concentration has been determined in breast tissue, focusing largely on samples obtained from breast cancers. In this study, estradiol concentration was determined in normal breast tissue obtained from women undergoing esthetic, and oncoplastic surgery. METHODS: Normal breast tissue was obtained during 68 operations for esthetic or reconstructive indications in women with and without a history of breast cancer. Our study included six different groups of women. The first three groups were normal cycling women, women taking oral contraceptives, and normal postmenopausal women, all undergoing a bilateral esthetic breast reduction. The second three groups were premenopausal and postmenopausal women, with a history of breast cancer and currently taking tamoxifen treatment, or postmenopausal women currently taking an aromatase inhibitor, needing contra-lateral corrective esthetic surgery. FINDINGS: In the group of women without history of breast cancer, normal cycling women (n=24) presented a strong correlation (r=0.853; P<0.0001) between 17ß-estradiol concentration in serum (median: 29.7 pg/mL; IQR: 10.8-82.3 pg/mL) and in breast tissue (30.6 pg/g; IQR: 18.6-183.8 pg/g). Postmenopausal women had low 17ß-estradiol concentrations both in serum and breast tissue (r=0.813; P<0.0001, n=16). Women taking oral contraceptives (n=12) had low serum and breast tissue levels of estradiol (r=0.376; P=n.s.). Premenopausal women (n=6, mean age: 44.2 years) with a history of breast cancer and currently taking tamoxifen, had very high concentrations of 17ß-estradiol both in serum (277.9 pg/mL; IQR: 96.2-544.7 pg/mL) and in epithelial cells (251.9 pg/g; IQR: 115.0-426.5 pg/g) (r=0.803; P<0.001). Postmenopausal women taking tamoxifen (n=4, mean age: 48.3) had low concentrations of 17ß-estradiol in serum (7.0 pg/mL; IQR: 5.7-16.3 pg/mL) and in epithelial cells (14.6 pg/g IQR: 13.3-16.3 pg/g) (r=0.10; P=n.s.). The estradiol concentration in the breast of premenopausal women taking tamoxifen was 8.2 times higher that observed in the breast of normal cycling women, and 17.3 times higher that observed in postmenopausal women taking tamoxifen. Women taking adjuvant aromatase inhibitors had extremely low concentrations of 17ß-estradiol both in serum and in breast tissue. INTERPRETATION: This study shows that serum estradiol levels largely determine estrogen levels in normal breast tissue.

Plant polyphenolics as anti-invasive cancer agents.

Because invasion is, either directly or via metastasis formation, the main cause of death in cancer patients, development of efficient anti-invasive agents is an important research challenge. We have established a screening program for potentially anti-invasive compounds. The assay is based on organotypic confronting cultures between human invasive cancer cells and a fragment of normal tissue in three dimensions. Anti-invasive agents appeared to be heterogeneous with regard to their chemical nature, but plant alkaloids, polyphenolics and some of their synthetic congeners were well represented. Even within this group, active compounds were quite diverse: (+)-catechin, tangeretin, xanthohumol and other prenylated chalcones, 3,7-dimethoxyflavone, a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin. The data gathered in this system are now applied in two projects. Firstly, structure-activity relationships are explored with computer models using an artificial neural network approach, based on quantitative structural descriptors. The aim of this study is the prediction and design of optimally efficient anti-invasive compounds. Secondly, the metabolism of orally ingested plant polyphenolics by colonic bacteria is studied in a simulator of the human intestinal microbial ecosystem (SHIME) and in human intervention trials. This method should provide information on the final bioavailability of the active compounds in the human body, with regard to microbial metabolism, and the feasibility of designing pre- or probiotics that increase the generation of active principles for absorption in the gastro-intestinal tract. The final and global aim of all these studies is to predict, synthesize and apply in vivo molecules with an optimal anti-invasive, and hence an anti-metastatic activity against cancer.

Number of women needed in a prospective trial to prove potential cardiovascular benefit of hormone replacement therapy.

INTRODUCTION: Hormonal replacement therapy (HRT) may be beneficial for the cardiovascular system if hormones are given shortly after the onset of menopause. So far, no randomized trial has provided conclusive results. MATERIALS AND METHODS: Based on Belgian population data, we calculated the number of women that should be included in a prospective double-blinded study to prove a potential cardiovascular benefit of HRT. Sample size calculations were based on the extrapolation of empirical observations made in three large databases from epidemiological studies carried out in Belgium during the past 20 years. RESULTS: The 10-year mortality varies with the age at which women are included in the observation. In the normal Belgian female population, the cardiovascular mortality risk is 0.85% and 1.58% for women aged 50-54 and 55-59 years, respectively. To prove that HRT induces a decrease of 10-year mortality of 30% in a normal population of 50-54-year-old women, 34 630 subjects would have to be included; for reductions of 20% and 10%, the numbers would be, respectively, 82 468 and 348 056. To prove a significant decrease in 10-year mortality starting with a normal population with an average age of 55-59 years, the numbers needed for hypothetical reductions of 30%, 20% and 10% would be, respectively, 18 514, 44 072 and 185 936. CONCLUSION: If cardiovascular mortality is the study end-point, it is obvious that such a study will be a gigantic task. Taking cardiovascular morbidity as the end-point, such a study would be feasible.

Tibolone and its metabolites inhibit invasion of human mammary carcinoma cells in vitro.

UNLABELLED: Tibolone is used in postmenopausal women to alleviate menopausal symptoms and to prevent osteoporosis, but it does not stimulate the endometrium and the breast. Up to date, little data are available on the effect of tibolone on breast cancer initiation and progression. OBJECTIVE: In the present in vitro study, we investigated the effect of tibolone and its metabolites (3alpha-OH tibolone, 3beta-OH tibolone, the Delta4 isomer and the sulphated isoform) on invasion of human breast cancer cells. METHODS: The effect on invasion was evaluated in the chick heart invasion assay using MCF-7/6 cells and in the collagen type I invasion assay using T47-D cells. Furthermore, the compounds were tested in aggregation and migration assays. RESULTS: We observed that, at a concentration of 100 microM, tibolone and its 3beta-OH metabolite possess anti-invasive activities in the two different invasion assays. However, this was neither due to effects on cell-cell adhesion nor on motility. In an attempt to probe the mechanism underlying the anti-invasive effect, we found that pro-MMP-9 release was markedly reduced in the supernatant of MCF-7/6 breast cancer cells treated with tibolone, 3alpha-OH tibolone and the Delta4 isomer but, interestingly, not with the sulphated metabolite. CONCLUSION: We conclude that tibolone and its 3beta-OH metabolite have an anti-invasive effect on the tested breast cancer cell lines in vitro. This effect on invasion is not correlated with an effect on cell-cell adhesion or motility but coincides with a decreased release of pro-MMP-9 in the medium.

Different responses to preoperative chemotherapy for invasive lobular and invasive ductal breast carcinoma.

AIM: Preoperative chemotherapy (PCT) is used in primary breast cancer, to facilitate breast conservative surgery (BCS). Clinical and pathologic responses are important prognostic parameters. Biologic markers are needed to individualize treatment. PATIENTS AND METHODS: One hundred and thirty-five patients with breast carcinoma were treated with PCT, followed by surgery and adjuvant therapy. Clinical response and pathological complete response (pCR), biological markers and type of surgery were compared between invasive ductal (IDC) and invasive lobular carcinoma (ILC). RESULTS: Overall response (OR) for IDC was 75% compared to 50% for ILC (P=0.0151). Pathological CR was 15% for IDC and 0% for ILC (P=0.0066). Fifty-six percent of the responding patients had BCS, in contrast with 16% of the non-responders. BCS was performed in 50% of patients with IDC, in 38% of the patients with ILC. Salvage surgery was more necessary in ILC (19%) compared to IDC (4%) (P=0.0068). Patients with ILC were more frequently ER-positive and HER-2 negative than patients with IDC. CONCLUSIONS: Clinical and pathological responses are lower in ILC compared to IDC. After PCT, patients with large ILC should preferably be offered mastectomy with immediate breast reconstruction. However, PCT still remains valuable to evaluate tumor response and biologic factors in vivo.

Inhibition of tamoxifen's therapeutic benefit by tangeretin in mammary cancer.

Tangeretin, a molecule present in citrus fruits and in certain 'natural' menopausal medications, is an effective tumour growth and invasion inhibitor in vitro of human MCF 7/6 breast cancer cells. However, when added to the drinking water of MCF 7/6 tumour-bearing mice it neutralises the beneficial tumour-suppressing effect of tamoxifen. Tangeretin reduces the number of natural killer cells. This may explain why the beneficial suppressive effect of tangeretin on MCF 7/6 cell proliferation in vitro is completely counteracted in vivo.

Influence of tangeretin on tamoxifen's therapeutic benefit in mammary cancer.

BACKGROUND: Tamoxifen and the citrus flavonoid tangeretin exhibit similar inhibitory effects on the growth and invasive properties of human mammary cancer cells in vitro; furthermore, the two agents have displayed additive effects in vitro. In this study, we examined whether tangeretin would enhance tamoxifen's therapeutic benefit in vivo. METHODS: Female nude mice (n = 80) were inoculated subcutaneously with human MCF-7/6 mammary adenocarcinoma cells. Groups of 20 mice were treated orally by adding the following substances to their drinking water: tamoxifen (3 x 10(-5) M), tangeretin (1 x 10(-4) M), tamoxifen plus tangeretin (3 x 10(-5) M plus 1 x 10(-4) M), or solvent. RESULTS AND CONCLUSIONS: Oral treatment of mice with tamoxifen resulted in a statistically significant inhibition of tumor growth compared with solvent treatment (two-sided P = .001). Treatment with tangeretin did not inhibit tumor growth, and addition of this compound to drinking water with tamoxifen completely neutralized tamoxifen's inhibitory effect. The median survival time of tumor-bearing mice treated with tamoxifen plus tangeretin was reduced in comparison with that of mice treated with tamoxifen alone (14 versus 56 weeks; two-sided P = .002). Tangeretin (1 x 10(-6) M or higher) inhibited the cytolytic effect of murine natural killer cells on MCF-7/6 cells in vitro, which may explain why tamoxifen-induced inhibition of tumor growth in mice is abolished when tangeretin is present in drinking water. IMPLICATIONS: We describe an in vivo model to study potential interference of dietary compounds, such as flavonoids, with tamoxifen, which could lead to reduced efficacy of adjuvant therapy. In our study, the tumor growth-inhibiting effect of oral tamoxifen was reversed upon addition of tangeretin to the diet. Our data argue against excessive consumption of tangeretin-added products and supplements by patients with mammary cancer during tamoxifen treatment.

Changes of intracellular free calcium during intracytoplasmic sperm injection.

The present experiments were undertaken to investigate whether the procedure of intracytoplasmic sperm injection (ICSI) is associated with changes in the intracellular free calcium concentration ([Ca2+]i). [Ca2+]i was measured, using the calcium-sensitive dye fura-2, during and after impalement of mouse oocytes with an ICSI pipette and injection of a small amount of medium alone or of medium containing a normal human spermatozoon. Forty-five oocytes were injected with medium. Two different responses were observed: 20 of these cells showed a large increase of [Ca2+]i upon impalement; the other 25 cells did not show any change of [Ca2+]i, neither in the acute period nor in a late period 4 hr after impalement. All the cells that responded with an increase of [Ca2+]i subsequently lysed within the first 30 min following impalement, while all the cells with no [Ca2+]i change remained intact. This observation suggests that only traumatic impalement is associated with an increase of [Ca2+]i. Thirty-one oocytes were successfully, i.e., without subsequent cell lysis, injected with a normal mouse or human spermatozoon. In none of these cells could any acute or late change of [Ca2+]i be observed. The experiments illustrate that successful performance of the ICSI procedure, i.e., ICSI not followed by cell lysis, is not associated with changes of [Ca2+]i in mouse oocytes. This suggests that the ICSI technique, by itself, does not help in activating the oocyte via manipulation-induced changes of [Ca2+]i.

Methods to increase the success rate of artificial insemination with donor semen.

Donor insemination was performed in two fertility clinics using cryopreserved semen from the same sperm bank. Donors were selected in agreement with American Fertility Society recommendations. In the first clinic, cup insemination was combined with intra-uterine insemination in 321 cycles. Follicular development was closely monitored with vaginal echography and serum hormonal measurements. In the second clinic, 1287 insemination cycles were performed using intra-cervical insemination, timed only on basal body temperature. In the first clinic, a total of 53 pregnancies were obtained, of which seven were in 55 unstimulated cycles (12.5%), 21 in 159 cycles with clomiphene citrate stimulation (13.1%, P > 0.1), and 25 in 107 cycles stimulated with clomiphene plus human menopausal gonadotrophin (HMG) (23.9%, P < 0.03). No multiple pregnancies were observed in the latter group. In the second clinic, 159 pregnancies occurred during 1287 insemination cycles (12.4%). It is concluded that neither intra-uterine insemination, nor hormonal and echographic cycle monitoring, nor clomiphene citrate enhances the success rate of artificial insemination with donor semen, but HMG treatment probably exerts a favourable effect.

Intracellular pH changes during zona drilling.

OBJECTIVE: To investigate whether zona drilling of oocytes, a technique used to locally disrupt the zona pellucida, causes important changes in intracellular pH. DESIGN AND METHODS: In a first set of experiments, intracellular pH was measured during zona drilling of mouse oocytes, using pH-sensitive microelectrodes. In a second set of experiments human oocytes that failed to fertilize were used to measure intracellular pH during drilling. In these cells intracellular pH was measured using microfluorimetry with the pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein. RESULTS: In mouse oocytes intracellular pH dropped from 7.25 +/- 0.02 (mean +/- SEM) to 7.09 +/- 0.03 during zona drilling, followed by a recovery to pH 7.17 +/- 0.02 after 4 minutes (n = 20). In human oocytes, intracellular pH dropped over 0.36 +/- 0.10 pH units during drilling, followed by a recovery that was complete within 4 minutes (n = 14). CONCLUSION: Zona drilling is associated with a significant cytoplasmic acidification both in mouse and human oocytes. This effect is perhaps related to the high incidence of cytoplasmic degeneration after zona drilling of human oocytes.

Tubal hydatidiform mole.

We report a tubal hydatidiform mole, most likely resulting from dispermic fertilization. Early hatching of the embryo, because of a defective zona pellucida, may have favored tubal implantation.

Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro.

Zona drilling (ZD) and zona cutting (ZC) were used in an IVF programme to assist fertilization in semen defect patients. Twenty-seven patients consented to ZD where acidified Tyrode's was used to create a hole in the zona pellucida. In 19 patients, ZD increased the fertilization rate to 29% compared with 8% (P less than 0.001) in their routine IVF cycles, and in eight patients precluded from routine IVF, a fertilization rate of 14% was achieved. Twenty-two patients consented to ZC where a slit in the zona is made mechanically. In 12 patients ZC increased the fertilization rate to 31% compared with 14% (P less than 0.01) from previous routine IVF cycles, and in 10 patients precluded from routine IVF, a fertilization rate of 34% was achieved. In 13 cycles, 68 uncut control oocytes were inseminated. In five cycles both control and ZC oocytes were fertilized (n.s.d.). In eight cycles no control oocytes were fertilized compared with 27% of ZC oocytes. The polyspermy rate was 4.6%. Twenty-four per cent of ZD and 12% of ZC (P less than 0.01) oocytes and embryos were degenerate after 42 h. Both ZD and ZC can increase the fertilization rate of sub-optimal semen, however, in our hands neither technique produced a pregnancy.

Normal survival and in-vitro development after cryopreservation of zona-drilled embryos in mice.

The importance of the zona pellucida on survival after freezing and thawing was investigated. Zona-drilled and zona-intact mouse embryos were fertilized in vitro, cultured to the 2, 4 and 8-cell stages and frozen using conventional methods. Zona drilling did not affect the survival or development of frozen embryos to the blastocyst stage in vitro. We conclude that partial damage to the zona pellucida during micromanipulation procedures is compatible with rates of survival and development which are not different to those observed in zona-intact control embryos.

Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse.

Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3.8-90%) at low sperm concentrations (less than 200,000/ml) compared with zona-intact controls (0-45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (less than 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested. The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%). Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.