Tomasz Milewicz

Effects of levetiracetam and valproate on reproductive endocrine function studied in human ovarian follicular cells

Purpose:  Recent animal studies have indicated possible reproductive endocrine effects of levetiracetam (LEV). The aim of the present study was to investigate possible reproductive endocrine effects of LEV compared to valproate (VPA) using human ovarian follicular cells.

Progesterone-induced secretion of growth hormone ,insulin-like growth factor I and prolactin by human breast cancer explants

The aim of the study was to evaluate the potential of human breast cancer tissue to secrete growth hormone (GH) ,insulin-like growth factor I (IGF-I) and prolactin in response to 10−7 M progesterone stimulation. Explants were divided according to estrogen receptor (ER)/progesterone receptor (PR) phenotype (ER(−)PR(−); ER(+)PR(−); ER(+)PR(+); ER(−)PR(+)). Our results show distinct differences in cultured breast cancer tissue responses to progesterone stimulation with regard to secretion of proliferative agents such as GH ,IGF-I and prolactin. All but ER(−)PR(−) breast cancer cell types responded in vitro to progesterone stimulation with an increase in local GH secretion ,while in non-malignant tissue progesterone induced local GH secretion only in PR(+) cells. Moreover ,only in PR(+) cells did progesterone stimulate local IGF-I and prolactin secretion ,in both malignant and non-malignant tissue. This study provides evidence for the first time that in PR(+) breast cancer tissue ,progesterone may increase GH ,prolactin and IGF-I secretion in both malignant and surrounding non-malignant tissue. These hormones may act as local growth factors that stimulate the proliferation of mammary tumors.

Transrectal Ultrasound-Guided Hysteroscopic Myomectomy of Submucosal Myomas With a Varying Degree of Myometrial Penetration

STUDY OBJECTIVE To predict the 1-step complete resection rate after transrectal ultrasound-guided hysteroscopic myomectomy and to determine the usefulness of intraoperative transrectal ultrasonography (TRUS) in monitoring hysteroscopic electroresection of submucosal myomas. DESIGN Prospective cohort study (Canadian Task Force classification II-1). SETTING University hospital. PATIENTS One hundred twenty women with symptomatic (abnormal uterine bleeding or reproductive disorder), single, submucosal myomas underwent hysteroscopic electroresection. Groups 1 and 2 were monitored, respectively, with or without TRUS. Anatomical inclusion criteria were myoma ≤5 cm and myometrial free margin ≥3 mm above the myoma. INTERVENTIONS Myomas were evaluated preoperatively via sonohysterograpy and were graded according to the guidelines outlined by the European Society of Hysteroscopy (ESH), including size and myometrial free margin, and according to the STEPW (size, topography, extension, penetration, and lateral wall) classification. On the basis of sonographic findings, patients with myomas >3 cm received gonadotropin-releasing hormone therapy for 1 to 3 months. Hysteroscopic myomectomy was performed with or without TRUS guidance. At 4 to 8 weeks after the initial procedure, postoperative transvaginal ultrasonography, sonohysterography, or second-look hysteroscopy was performed. MEASUREMENTS AND MAIN RESULTS In the TRUS group, a significantly higher percentage of 1-step complete resections was observed than in the group without TRUS (91% vs 73%) (p = .02). This was associated with a statistically significant difference in the subgroups of myomas that were deeply penetrating into the myometrium (89% vs 55%) (p < .01). One-way logistic analysis of data for all treated patients indicated the use of TRUS, as well as the ESH and STEPW classifications, as significant factors influencing the 1-step complete resection. At multivariable logistic regression analysis, use of TRUS (odds ratio [OR], 2.74; p < .001), myomas graded 0 or 1 according to ESH (OR, 3.55; p < .001), and size <3 cm (OR, 2.35; p < .05) were significantly associated with 1-step complete resection (area under the curve, 0.80; p < .001). In the TRUS group there were two significant predictors: size <3 cm (OR = 5.21; p < .05) and myometrial free margin <5 mm (OR, 0.18; p < .05). CONCLUSION Intraoperative use of TRUS during hysteroscopic myomectomy increases the chance of complete 1-step removal of submucosal myomas that deeply penetrate the myometrium.

Time-dependent action of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and its metabolite DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) on human chorionic gonadotropin and progesterone secretion

Explants of human placenta were used to study the effects of two isomers of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) [p,p′-DDT and o,p′-DDT] and their DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) metabolites [p,p′-DDE and o,p′-DDE] on the secretion of progesterone and human chorionic gonadotropin (hCG). Explants were treated with 1, 10, 100 or 1000 ng/ml of each compound for 24 h or 72 h. We found opposite effects (stimulatory after short-term and inhibitory after long-term exposure) of all compounds on progesterone secretion. However, both short- and long-term exposure to all investigated compounds caused decreased hCG secretion. In conclusion, we suggest the existence of a local axis between steroid hormones and hCG in placenta. DDT (which has estrogenic properties) increases progesterone secretion and consequently decreases hCG secretion. After long-term exposure, the low level of hCG is insufficient to stimulate progesterone.

Regulation of Msx2 gene expression by steroid hormones in human nonmalignant and malignant breast cancer explants cultured in vitro

Muscle segment homeobox genes, which regulate developmental programs and are expressed in embryonic and adult tissue, play a role in development of some malignancies. There are no reports on the expression of these families of genes in breast cancer tissue. The aim of this study was to compare expression of Msx2 gene in breast cancer of different genotypes as well as in surrounding nonmalignant tissues. Explants obtained during surgery were divided according to their sex steroid receptor status determined by immunocytochemistry. Four explants obtained from malignant and nonmalignant tissue of each individual patient were incubated in a control medium or with the addition of progesterone (10− 7 M) alone, estradiol 17 β (10− 5 M) or both. The relative level of Msx2 transcripts was evaluated by a semiquantitative RT-PCR and cell proliferation by Alamar Blue test. Results of RT-PCR analysis showed that the relative expression of Msx2 gene depended on the presence of ER/PR receptors both in nonmalignant and malignant tissues Relative amount of Msx2 mRNA was the highest in surrounding nonmalignant ER + /PR − and ER − /PR + tissue, whereas in ER − /PR − and ER + /PR + tissue it was 1.4–1.6-fold lower. Tumorigenesis led to about a twofold decrease in the relative amount of Msx2 mRNA except for ER + /PR + immunophenotype, where no changes were observed. Addition of estradiol or progesterone to the culture of ER − /PR − type tissue explants did not change significantly the relative amount of Msx2 gene mRNA. An opposite effect was observed in ER + /PR − type of tissue. Addition of estradiol alone, or estradiol and progesterone together to tissue culture explants decreased two to three fold the relative amount of Msx2 gene mRNA in both, malignant and surrounding tissues. Progesterone alone had no effect on Msx2 gene expression in this type of tissue. The most complicated regulation was observed in ER + /PR + type of tissue. Culture of tissue explants supplemented with estradiol significantly increased the relative amount of Msx2 gene mRNA in the surrounding tissue. Progesterone enhanced the stimulatory effect of estradiol in surrounding tissues but not in the malignant tissue. Increased expression of Msx2 correlated with an increased proliferation in ER − /PR − and ER + /PR + types, but not in ER + /PR − type of tissues. In conclusion, obtained results provide evidence that estrogen affects Msx2 gene expression. Significant changes in the relative amount of Msx2 gene mRNA and lack of canonical ERE element in 5′-upstream sequence of this gene suggest that regulation takes place indirectly probably by protein-protein interaction. The decrease in the relative amount of Msx2 gene mRNA in ER + /PR − type tumor suggests that progesterone also affects Msx2 gene expression by an indirect mechanism(s).

Effect of hormone therapy on the enteroinsular axis.

Objective: Menopause is associated with a decline in insulin response to glucose and with insulin resistance. It has been proven that hormone therapy (HT) improves carbohydrate metabolism in postmenopausal women. However, it is known that gastrointestinal hormones play a key role in the coordination of digestion and absorption of ingested nutrients and in the regulation of pancreatic endocrine function. Therefore, the aim of this study was to investigate the effect of HT on gastrointestinal hormones and carbohydrate metabolism in postmenopausal women. Design: The prospective study was performed in 90 healthy postmenopausal women (mean age 54.5 years, standard deviation 3.34 years), of whom 49 completed the study. They were randomized and treated either with continuous transdermal HT (0.05 mg 17β-estradiol every 24 hours) combined with 5 mg oral dydrogesterone daily (group A, n = 25), or with oral HT (2 mg 17β-estradiol semihydrate every 24 hours) combined with 10 mg dydrogesterone as a continuous therapy (group B, n = 8). The control group (group C, n = 16) received no HT. Both basal and meal-stimulated plasma concentrations of glucose, insulin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and cholecystokinin (CCK), as well as basal estrogen levels, were measured before HT and after 6 and 12 months of treatment. At the same time intervals, all the studied parameters were measured for group C. Results: After 12 months of the transdermal HT, a decrease in both fasting (P < 0.002) and postprandial (P < 0.05) plasma glucose levels was observed. Oral HT reduced only the fasting plasma glucose level in the 12th month of treatment (P < 0.05). Regardless of the route of administration, HT reduced postprandial plasma levels of insulin (oral HT: P < 0.05; transdermal HT: P < 0.02). Fasting plasma levels of GIP were reduced after 6 and 12 months of transdermal HT (P < 0.002 and P < 0.001, respectively). Moreover, levels of postprandial GIP were reduced after 6 and 12 months of transdermal HT (P < 0.002 in both cases). Fasting and postprandial GLP-1 levels were reduced by transdermal HT after 12 months of supplementation. Oral HT also decreased these levels, but not significantly. The observed differences may, however, be related not only to the route of administration, but also to the difference in the dose of estradiol. Regardless of the route of administration, HT did not influence plasma levels of CCK. Conclusions: Hormone therapy significantly influences the enteroinsular axis in postmenopausal women and contributes to the normalization of plasma glucose levels.

Metformin increases pressure pain threshold in lean women with polycystic ovary syndrome.

Background Despite the strong preclinical rationale, there are only very few data considering the utility of metformin as a potential pain therapeutic in humans. The aim of this study was to determine the association between metformin therapy and pressure pain threshold (PPT) in lean women with polycystic ovary syndrome (PCOS). We hypothesized that metformin therapy in lean PCOS women increases PPT. Materials and methods Twenty-seven lean PCOS women with free androgen index phenotype >5 and 18 lean healthy controls were enrolled in the study. Fifteen of the PCOS women were randomly assigned to be treated with metformin 1,500 mg daily for 6 months. PPT and plasma β-endorphin levels were measured in all women at the beginning of the study and after 6 months of observation. Results We observed an increase in PPT values measured on deltoid and trapezius muscle in the PCOS with metformin group after 6 months of metformin administration (4.81±0.88 kg/cm2, P<0.001 on deltoid muscle, and 5.71±1.16 kg/cm2 on trapezius muscle). We did not observe any significant changes in PPT values in the PCOS without treatment group and in controls. We did not observe any significant changes in serum β-endorphin levels in any studied groups during the 6-month observation. Conclusion We conclude that metformin therapy increases PPT in lean PCOS women, without affecting plasma β-endorphin concentration. Our results may suggest the potential role of metformin in pain therapy. We propose that larger, randomized studies on metformin impact on pain perception should be performed.

Cyproterone, norethindrone, medroxyprogesterone and levonorgestrel are less potent local human growth hormone and insulin-like growth factor I secretion stimulators than progesterone in human breast cancer explants expressing the estrogen receptor.

The aim of the present study was to compare the ability of natural progesterone and synthetic progestins to stimulate local growth hormone (GH) and insulin-like growth factor I (IGF-I) secretion by breast cancer explants. Explants obtained during surgery were divided according to their estrogen/progesterone receptor phenotype – ER(+)PR(–); ER(+)PR(+); ER(–)PR(+) – as determined by immunocytochemistry. Natural progesterone (10-5 mol/l) and synthetic progestins (cyproterone acetate (5 × 10-7 mol/l), norethindrone (10-5 mol/l), medroxyprogesterone acetate (10-7 mol/l), and levonorgestrel (10-7 mol/l) were tested in vitro for their ability to induce secretion of proliferation-promoting agents such as human GH (hGH) and IGF-I. All hormone-dependent breast cancer cell types responded to progesterone stimulation with increased local hGH secretion, while in the non-malignant tissue this effect was observed only in PR(+) cells. Moreover, progesterone in only PR(+) cells in vitro stimulated local IGF-I secretion by both malignant and non-malignant tissue. Medroxyprogesterone and levonorgestrel increased GH secretion by both malignant and non-malignant ER(–)PR(+) breast cancer explants, while cyproterone stimulated it only in non-malignant tissue. None of the synthetic progestins tested in this experiment exerted an effect on GH secretion by both malignant and non-malignant tissue of ER(+) breast cancer explants. The present data additionally showed that, apart from cyproterone, which increased IGF-I secretion in the same manner as progesterone by both malignant and non-malignant ER(–)PR(+) breast explants, other progestins tested had either no effect on IGF-I local secretion or decreased it. Medroxyprogesterone and levonorgestrel induced a decrease in IGF-I secretion noted in ER(+) explants of non-malignant tissue and in malignant ER(–)PR(+) breast tissue. All progestins tested decreased IGF-I secretion by malignant ER(+)PR(+) explants. Taken together, the tested synthetic progestins widely used as oral contraceptives and in hormone replacement therapy were less potent than progesterone in inducing secretion of proliferation-promoting agents such as hGH and IGF-I in ER-containing breast tissue. Despite the lack of confirmation of the link between the use of progestins and breast cancer risk, patients should be informed that the use of certain estrogen/progestin preparations is of no influence on breast cancer risk while others may increase it.

Plasma fibrin clot properties in postmenopausal women: effects of hormone therapy.

Objective:Postmenopausal women are at risk of thromboembolic events. It is unclear whether menopause alters fibrin clot properties. The aim of our study was to assess the effects of menopause and hormone therapy on clot characteristics. Methods:Ex vivo plasma clot permeability, turbidity, and susceptibility to lysis were determined in 70 premenopausal and 70 postmenopausal women (a case-control study). From the postmenopausal group, 30 women were randomly assigned (1:1) to a 24-week oral or transdermal treatment with 17&bgr;-estradiol, combined with norethisterone acetate (2 mg + 1 mg/d or 0.05 mg + 5 mg/d, respectively). Results:Compared with premenopausal women (aged 29.2 ± 2.60 y), postmenopausal women (aged 49.7 ± 3.4 y; P = 0.009) were characterized by higher fibrinogen levels (by 36.8%), lower C-reactive protein levels (by 36.9%), and lower clot permeability (by 10.5%); also after adjustment for fibrinogen (all P < 0.05), with no difference in turbidimetric or fibrinolytic variables. Estrogen plus progestogen therapy led to higher maximal absorbency of fibrin gels by 6.0% (P < 0.05), whereas all other fibrin variables remained unchanged. Compared with transdermal hormone therapy, 24-week oral therapy was associated with higher absorbency of plasma clots by 16% (P < 0.05), without any other changes in fibrin characteristics. Conclusions:Menopause age is associated with the formation of denser fibrin clots. Estrogen plus progestogen therapy has a minor effect on plasma fibrin properties, but leads to the formation of thicker and more branched fibrin fibers, particularly during oral administration.

Selenium status parameters in patients with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. To date, no systematic study of interactions between selenium status parameters (SSPs: serum selenium concentration, plasma glutathione peroxidase, GPX3, plasma selenoprotein P, SELENOP), sex hormones, thyroid function parameters, and other laboratory parameters in patients with PCOS has been undertaken. Therefore we aimed to compare such parameters in women with PCOS and in the control groups, and to investigate the multidimensional interactions between various parameters in PCOS patients and in controls. The subjects were diagnosed either with PCOS (n=28, 25.4±5.2 y) or with PCOS+Hashimoto disease (n=13, 27.3±5.6 y). Female patients having normal menses were recruited into the first control group (n=70, 26.8±7.3 y) or to the second control group comprising women only with Hashimoto disease (n=10, 26.2±6.9 y). No apparent differences in SSPs between control subjects and patients with PCOS, also complicated with Hashimoto disease, were identified, though such differences were noticeable for total testosterone (tT), sex hormone binding globulin, free androgen index, dehydroepiandrosterone sulfate (DHEAS), and insulin profile. The correlation between tT and DHEAS was found the strongest. The other group of mutually highly and positively correlated parameters consisted of GPX3, follicle stimulating hormone, free triiodothyronine and free thyroxine. All the latter parameters correlated negatively with vitamin D3. SSPs took part in interactions with thyroid hormones, sex hormones and some other parameters, but only for GPX3 such interactions were statistically significant. The significance of these findings remains open for further investigation, particularly in patients with PCOS and/or Hashimoto disease.