Evaluation of novel protease enzymes on growth performance and nutrient digestibility of poultry: enzyme dose response.

Two experiments were conducted to evaluate 3 novel proteases in broilers. In experiment 1, 600 male, Cobb 500 broilers were allocated to 1 of 12 experimental diets (5 birds/pen and 10 replicates/diet). A control (C) diet was formulated to be adequate in all nutrients. Proteases 1, 2, or 3 were added to this diet at 3 doses (1x, 3x, or 9x) in a 3 × 3 factorial arrangement of treatments. The factorial was augmented with 2 treatments of phytase at 500 or 1,500 FTU/kg added to the C diet. In experiment 2, 2,050 male Ross 308 broilers were allocated to 1 of 10 experimental diets (25 birds/pen and 9 replicates/diet). A C diet was formulated to be adequate in all nutrients. Protease 1, 2, or 3 was then added to the C diet at 3 doses (1x, 2x, or 4x) in a 3 × 3 factorial arrangement of treatments plus the C. In experiment 1, birds fed phytase gained more (P < 0.05) than birds fed protease, but neither were different than birds fed the C. Supplementation of 9x dose of any protease resulted in a reduction (P < 0.05) in BWG when compared with birds fed 1x dose of protease or phytase at 500 or 1,500 FTU/kg. Feed conversion ratio was improved (P < 0.05) in birds fed phytase compared with birds fed the C diet. Nitrogen digestibility was greater (P < 0.05) in birds fed protease 1 when compared with birds fed protease 2. Birds fed the 1x dose of protease or 500 FTU/kg of phytase had a greater (P < 0.05) N digestibility than birds fed 3x dose of protease. In experiment 2, protease supplementation significantly reduced (P < 0.05) BWG when compared with birds fed the C from hatch to 35 D post-hatch. Protease supplementation did not improve broiler growth performance or N digestibility above that of a nutrient adequate control diet or a diet supplemented with 500 FTU/kg of phytase.

Evaluation of novel protease enzymes on growth performance and apparent ileal digestibility of amino acids in poultry: enzyme screening.

Three experiments were conducted to evaluate eight neutral and six acid proteases on growth performance and apparent ileal amino acid digestibility (AID) of poults (Experiment 1) or chicks (Experiments 2 and 3). Two basal diets were formulated: a nutrient adequate positive control (PC), which met or exceeded the nutrient requirements for poults (Experiment 1) or chicks (Experiments 2 and 3) and a negative control (NC) formulated to achieve 85% (Experiments 1 and 2) or 80% (Experiments 3) of the requirement for protein and amino acids. Phytase was included in all diets to provide 500 phytase units (FTU)/kg and xylanase was included in all diets to provide 10,000 (Experiments 1 and 2) or 16,000 (Experiments 3) xylanase units (BXU)/kg. Proteases were supplemented in the NC diet at an equivalent amount of enzyme protein to create 16 experimental diets. There were five birds/pen and 10 replicate pens per treatment in each experiment. In experiment 1, birds fed the PC diet gained more (P < 0.05) than birds fed the NC. There were no differences in growth performance in birds fed the PC or NC in experiments 2 or 3. In all three experiments, birds fed the NC supplemented with neutral protease 1 had reduced (P < 0.05) feed intake (FI) or body weight gain (BWG) and increased (P < 0.05) feed conversion ratio (FCR) compared with birds fed the NC. Birds fed the NC diet supplemented with neutral protease 3, 7 (Experiment 1), or acid protease 4 (Experiment 3) had increased (P < 0.05) FCR and birds fed neutral protease 6 (Experiment 2) had reduced (P < 0.05) BWG compared with birds fed the NC. Apparent ileal amino acid digestibility was improved (P < 0.05) with protease supplementation to the NC diets (Experiment 1 or 3), but this was dependent on the protease and the amino acid. In conclusion, novel protease supplementation improved AID of amino acids but this was not reflected in improvements in growth performance of poults or chicks.

Phylogenetic Variation of Chelonid Alphaherpesvirus 5 (ChHV5) in Populations of Green Turtles Chelonia mydas along the Queensland Coast, Australia.

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north-south trend. Received October 22, 2016; accepted May 7, 2017.

Nuclear ordering in lithium and an upper limit on its ambient pressure superconducting transition temperature.

We have discovered spontaneous ordering of nuclear spins in lithium metal by NMR measurements at very low temperatures. In low magnetic fields, B<0.2 mT, the NMR spectra show a pronounced low-frequency anomaly. Also, nonadiabatic response to a slowly varying magnetic field was observed. A rich phase diagram with three different nonparamagnetic regions is proposed. We estimate a critical spin temperature T(c) approximately 350 nK at B=0. We also report the absence of superconductivity in lithium at normal pressure down to T(e) approximately 100 microK (B<10 nT).

Effect of epilepsy and antiepileptic drugs on male reproductive health.

BACKGROUND: Men with epilepsy have reduced fertility, and antiepileptic drugs may affect semen quality. Moreover, animal studies suggest that valproate (VPA) may be associated with testicular atrophy. OBJECTIVE: To evaluate reproductive function in men with epilepsy. METHODS: Sixty men with epilepsy and 41 control men were evaluated for their reproductive health. Fifteen men were taking carbamazepine (CBZ) and 18 men oxcarbazepine (OXC) for partial epilepsy, and 27 men were taking VPA for generalized epilepsy. Reproductive hormones were assayed from serum samples, semen analysis and ultrasonography of the testicles were performed, and testicular volume was calculated. RESULTS: Men on CBZ had low serum dehydroepiandrosterone sulfate concentrations (p < 0.001), and men on VPA had high concentrations of serum androstenedione (p < 0.001). The frequency of morphologically abnormal sperm was higher among CBZ-treated (p < 0.01), OXC-treated (p < 0.05), and VPA-treated men (p < 0.01) than among the control men. Moreover, both CBZ and VPA were associated with poor motility of sperm (p < 0.05). In addition, the frequency of abnormally low sperm concentration was high in men on CBZ (p < 0.001), and the frequency of any sperm abnormality was high in men on VPA (p < 0.01). The VPA-treated men with abnormal sperm had smaller testicular volumes than the control men (p = 0.003). CONCLUSIONS: CBZ, OXC, and VPA are associated with sperm abnormalities in men with epilepsy. In addition, VPA-treated men with generalized epilepsy who have abnormal sperm may have reduced testicular volume.

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor.

Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.

Rye bread improves bowel function and decreases the concentrations of some compounds that are putative colon cancer risk markers in middle-aged women and men.

Cereal fiber may reduce the risk of colorectal cancer by diluting colonic contents due to increased fecal output, by accelerating intestinal transit, by increasing fecal frequency and by altering bacterial metabolism. The effects of whole-meal rye bread on some putative colon cancer risk markers were investigated in 17 healthy Finnish subjects using a randomized crossover trial with two 4-wk bread consumption periods and a 4-wk washout period between the bread periods. White wheat bread was used as a control. Test breads covered a minimum of 20% of the daily energy intake (range, 4330-14, 033 kJ/d). Intestinal transit time, stool weight, fecal bacterial enzyme activities and short-chain fatty acid, ammonia, diacylglycerol (DAG) and bile acid concentrations in feces (expressed per gram wet feces) were measured. Whole-meal rye bread significantly increased fecal output and fecal frequency and shortened mean intestinal transit time compared with wheat bread in both women and men. Activities of beta-glucuronidase and beta-glucosidase (expressed per gram wet feces) were significantly lower in men and urease activity significantly higher in women during the rye bread period (RBP). Fecal butyrate concentration was higher during the RBP in men. Fecal ammonia and DAG concentrations did not differ between bread periods. Fecal total and secondary bile acid concentrations were significantly lower during RBP in both women and men. This study shows that whole-meal rye bread significantly improves bowel function in healthy adults and may decrease the concentration of some compounds that are putative colon cancer risk markers.

Similar responses in hemostatic factors after consumption of wholemeal rye bread and low-fiber wheat bread.

OBJECTIVE: Both epidemiological and experimental evidence suggests a protective effect of dietary fiber against disease, such as cancer and cardiovascular disease. The effects of fiber on lipoproteins are modest, indicating that other mechanisms may be involved. As the hemostatic effects of different types of fiber are poorly known, we compared the effects of wholemeal rye bread and low-fiber wheat bread on factors related to coagulation, fibrinolysis and platelet function. DESIGN: Subjects consumed rye and wheat bread as part of their habitual diet in a cross-over manner for 4 weeks, with a 4 week washout period between the diet periods. SUBJECTS: Forty healthy subjects (18 men, 22 women), aged 43+/-2 y. RESULTS: Mean daily intake of fiber during the rye bread period was 31 g for men and 26 g for women, while the respective figures for the wheat bread period were 15 g and 12 g. However, no significant differences between the two periods were seen in factor VII coagulant activity (FVIIc), fibrinogen, prothrombin fragments 1+2 (F1+2), tissue plasminogen activator (tPA) plasminogen activator inhibitor 1 (PAI-1) or 2,3,-dinor-thromboxane B2. Fibrin degradation products, D-dimers, were slightly lower after the wheat period (P=0.046). CONCLUSIONS: The results indicate that wheat and rye bread do not differ in their effects on coagulation, fibrinolysis or platelet function. SPONSORSHIP: Fazer Bakeries Ltd, Lahti Finland; Vaasan & Vaasan Ltd, Helsinki, Finland.

Consumption of wholemeal rye bread increases serum concentrations and urinary excretion of enterolactone compared with consumption of white wheat bread in healthy Finnish men and women.

Rye is an important source of plant lignans in Finland. In the present crossover trial we wanted to study the effect of rye bread as part of the usual diet on serum and urine enterolactone (ENL) concentrations in healthy volunteers. Eighteen men aged 43 (sem 2.0) years and twenty-one women aged 43 (sem 1.6) years consumed wholemeal rye bread and white wheat bread in random order for 4 weeks. The bread periods were separated by a 4 week wash-out period. The breads provided at least 20% of the daily energy intake. The mean intakes of rye bread were 219 (sem 14.6) and 162 (sem 5.3) g/d and those of wheat bread were 200 (sem 9.6) and 153 (sem 5.8) g/d for men and women respectively. Blood samples were collected from all subjects and three 24 h urine samples were collected from ten men and twelve women at the end of both bread periods for the determination of serum concentration and urinary excretion of ENL. The mean serum ENL concentrations in both men and women at the beginning of baseline period and at the end of the rye-bread period remained constant and were significantly higher than those at the end of the wheat-bread period. Correspondingly, daily urinary ENL excretion increased significantly during the rye-bread period compared with the wheat-bread period and was 5- and 10-fold higher in men and women respectively in comparison with the amount of plant lignan precursors measured in the rye bread. These data indicate the presence of other precursors for ENL in rye which are not detected by the current method of measuring plant lignans in food. The possible role of fibre in enhancement of the formation of mammalian lignans from their plant precursors in the gut also remains to be determined.

Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies.

We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution. The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.

A shift from a male to a female majority in newborns with the increasing age of grand grand multiparous women.

In this longitudinal study, we investigated the relationship of birth order and the age of mother and father to the gender of 1795 newborns (mean +/- SD 12.5 +/- 1.6 per mother) of 143 grand grand multiparous (i.e women who have had >10 deliveries). The frequency of boys was 52.2% in the group of 1st to 9th paras and 46.2% in the group of 10th to 20th paras (P = 0.022). Mothers aged > or =35 years had 7.0% more female than male newborns (P = 0.024). The respective figure for fathers was 5.6% (P = 0.023). The interpregnancy interval evaluated for 96 mothers with 1091 deliveries had no correlation with the gender of the infants. In the stepwise logistic regression analysis, the age of the mothers remained the only significant independent factor for the shift from a male to a female majority in the newborns (P = 0.0389). The present data thus indicate that the age of the mother is the factor which explains why grand grand multiparous women deliver more girls than boys.

The clinical outcome in pregnancies of grand grand multiparous women.

OBJECTIVE: To longitudinally evaluate maternal and neonatal complications with relation to birth order, with specific emphasis on grand grand multiparity (at least 10th para). METHODS: The maternal and neonatal outcome of 1200 pregnancies/deliveries in 96 grand grand multiparas was longitudinally investigated in 4 stages of the mothers' life: the primiparas, the multiparas (2nd-5th paras), the grand multiparas (6th-9th paras) and the grand grand multiparas stage. RESULTS: The frequency of hypertension, diabetes, placental complications, operative interventions at delivery, macrosomic infants, chromosomal abbreviations and fetal/neonatal anomalies increased with increasing birth order, being at a maximum in grand grand multiparas. The preterm delivery and perinatal mortality rate did not differ between the 3 groups of multiparas. Perinatal outcome was good in each group. CONCLUSIONS: Grand grand multiparity carries the risk of hypertensive and diabetic complications, which, in turn, often lead to induced or operative deliveries and placental complications. However, grand grand multiparity is not a major problem in societies with a good maternal health care system.

Grand grand multiparity and birth weight.

OBJECTIVE: To evaluate the association between birth weight and birth order in grand grand multiparous women (i.e., those who have had at least ten deliveries). METHODS: The longitudinal population consisted of 96 grand grand multiparous women with 1098 singleton deliveries. Two birth cohorts formed the cross-sectional reference populations: one from 1966 with 7564 deliveries and one from 1985-1986 with 5691 deliveries. In each population, birth weight was compared in four birth-order groups: first, second to fifth, sixth to ninth, and tenth to 12th born. RESULTS: The birth weight increased with birth order in each population, especially in the longitudinal one. The association remained even after adjusting for gestational age, sex of the newborn, maternal diabetes mellitus, and body mass index. Children born tenth to 12th were 83 g (95% confidence interval [CI] 29, 137) heavier than those born sixth to ninth; these in turn were 29 g (95% CI -27, 85) heavier than children born second to fifth, and those born second to fifth were 169 g (95% CI 54, 283) heavier than first born infants. Further indirect adjustment for the secular trend decreased these contrasts somewhat. CONCLUSION: Birth order is an independent determinant of birth weight even until the tenth delivery.

Conformational studies of human vitamin-D receptor by antipeptide antibodies, partial proteolytic digestion and ligand binding.

We have studied conformational changes of human vitamin-D receptor by using antipeptide antibodies, partial proteolytic digestion and binding of the natural ligand calcitriol or its synthetic analogs. Before exposing either [35S]methionine-labelled in vitro translated human vitamin-D receptor or a recombinant human vitamin-D receptor produced either in Escherichia coli or in Sf9 insect cells to limited proteolysis by trypsin or chymotrypsin, the proteins were treated with calcitriol or its synthetic analogs. The digestion products were analyzed by SDS/PAGE, immunoblotting with polyclonal antipeptide antibodies targeted against different domains of the receptor, and Edman N-terminal sequencing. After limited proteolysis with trypsin, two fragments of Mr 21,000 and Mr 34,000 could be localized into N-terminus and C-terminus of the receptor, respectively, by antipeptide antibodies. We found that treatment with calcitriol or its synthetic analogs leads to differential resistance of the ligand-binding domain of the recombinant receptor to partial proteolysis in vitro. We suggest that this is due to distinct conformational changes in the domain induced by the different ligands. The short N-terminal region and the Zn-finger domain form, however, a protease-resistant structure which is independent on the presence or absence of the ligand. When the C-terminal fragment of Mr 34,000 was further analyzed by Edman N-terminal sequencing, the major cleavage site in the receptor between amino acids Arg173 and His174 was revealed.

Long gonadotrophin releasing hormone agonist/human menopausal gonadotrophin protocol for ovarian stimulation in intrauterine insemination treatment.

OBJECTIVE: This prospective study was undertaken to examine the usefulness of a long gonadotrophin releasing hormone agonist (GnRH-a)/human menopausal gonadotrophin (hMG) protocol in intrauterine insemination (IUI) treatment. The results were compared to those of clomiphene citrate (CC)/hMG/IUI. STUDY DESIGN: Seventy-five patients were recruited to a GnRH-a/hMG group (group 1) while 88 patients underwent CC/hMG stimulation and served as controls (group 2). The study subjects were stimulated with a long GnRH-a/hMG regimen. IUI was performed 36 h after the administration of human chorionic gonadotrophin. RESULTS: The number of preovulatory follicles, the thickness of endometrium and sperm parameters were similar in both groups. The hMG requirements were significantly higher in group 1 than in group 2 (21.2 +/- 5.1 vs. 8.1 +/- 3.1 ampoules). The pregnancy rate was 20% in group 1 and 12.5% in group 2, the difference being not significant. CONCLUSION: The pregnancy rates were not significantly different between the GnRH-a/hMG/ IUI and CC/hMG/IUI groups. In addition, GnRH-a/hMG stimulation is notably more expensive than CC/hMG, and for these reasons, GnRH-a/hMG stimulation is not cost-effective in routine IUI therapy.

Comparison of fallopian tube sperm perfusion with intrauterine insemination in the treatment of infertility.

OBJECTIVE: To compare the efficacy of fallopian tube sperm perfusion utilizing a Foley catheter technique with standard IUI. DESIGN: Randomized controlled study. SETTING: The infertility units of the University Central Hospital and the Family Federation of Finland, Oulu, Finland. PATIENT(S): One hundred infertile women with unexplained factor, minimal to mild endometriosis, mild male factor, or ovarian dysfunction, undergoing 50 IUI and 50 fallopian tube sperm perfusion cycles stimulated by clomiphene citrate and hMG. INTERVENTION(S): Thirty-six hours after hCG administration, patients were randomized to either the IUI group (group 1, 50 patients and cycles) or the fallopian tube sperm perfusion group (group 2, 50 patients and cycles). Intrauterine insemination was performed using a standard method and fallopian tube sperm perfusion with a pediatric Foley catheter, which prevents the reflux of sperm suspension. MAIN OUTCOME MEASURE(S): Number of clinical pregnancies. RESULTS(S): The fallopian tube sperm perfusion method using a Foley catheter technique was easy to perform and convenient for the patients. The overall pregnancy rate per cycle was 8% for fallopian tube sperm perfusion and 20% for IUI, a difference that was not significant. CONCLUSION(S): The fallopian tube sperm perfusion method using a Foley catheter offers no advantage in comparison with the conventional IUI technique.

Is varicocele treatment useful?

The effect of the treatment of varicocele on symptoms and fertility was followed up in 66 patients for an average of 27 months (range 6-84 months). The patients had been treated either surgically (n = 45) or with embolization (n = 21). The mean duration of the inpatient periods and sick leaves were significantly shorter among the patients treated with embolization than among the surgical patients (2.4 vs 3.3 days and 4.8 vs 21.3 days, respectively). Scrotal problems disappeared either completely or almost completely in 73% of the patients treated for symptoms (n = 40). The number of spermatozoa increased significantly (from 6.7 mill/ml to 27.4 mill/ml) in the patients with infertility after the treatment of varicocele. Pregnancy started in 31% of the couples with infertility problems. A spontaneous abortion occurred in two cases, and six couples (23%) had a child of their own. It could be concluded that the treatment of varicocele is useful regardless of whether it is given for symptoms or infertility. It is also indicated for the patients with infertility problems who have a low number of spermatozoa and disturbed spermatozoan motility in sperm analysis.

Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene.

The recent finding that a mutation in the FSH receptor gene causes ovarian dysgenesis prompted the present study to determine the phenotype caused by this mutation. Twenty-two patients with ovarian dysgenesis and a 566C-->T mutation in the FSH receptor gene (designated FSH-resistant ovaries or FSHRO) were compared with 30 clinically similar patients with ovarian dysgenesis (designated ODG) who did not have this mutation. The genealogical studies suggested a founder effect of the FSH receptor gene mutation in Finland. Clinically, both groups of patients were characterized by primary or early secondary amenorrhea, variable development of secondary sex characteristics, and high serum levels of FSH and LH. Notable differences were observed in median adult height (FSHRO patients were shorter) and the occurrence of follicles judged by transvaginal sonography (observed in 6 of 8 FSHRO vs. 1 of 11 ODG) and ovarian histology (present in all 9 FSHRO vs. 1 of 4 ODG). These findings suggest that a subset of ovarian dysgenesis patients with the FSH receptor mutation 566C-->T is pathogenetically distinct, possibly due to residual receptor activity, and that these patients can be tentatively identified by demonstrating the presence of ovarian follicles and confirmed by mutation analysis.

Obesity and endocrine disorders in women taking valproate for epilepsy.

We recently reported the frequent occurrence of polycystic ovaries and hyperandrogenism in women taking valproate for epilepsy, especially when the medication was started before the age of 20 years. In the present study we evaluated the association of obesity and hyperinsulinemia with valproate-related polycystic ovaries and hyperandrogenism in women with epilepsy. Sixty-five women participated in the study. Twenty-two received valproate monotherapy and 43 received carbamazepine monotherapy. In addition to clinical examination, vaginal ultrasonography was performed to determine ovarian size, and the concentrations of serum sex hormones, insulin, insulin-like growth factor 1, and the insulin-like growth factor-binding proteins 1 and 3 (IGFBP-1 and IGFBP-3) were measured. Fifty-nine percent of the women on valproate were obese, and in a retrospective analysis an indisputable weight gain (mean, 21 kg; range, 8-49 kg) was found in 50% of the women taking valproate. Fourteen (64%) of the women on valproate had polycystic ovaries, hyperandrogenism, or both. These women were obese, and in addition to elevated serum androgen levels, they had high concentrations of fasting serum insulin and low levels of serum insulin-like growth factor-binding protein 1. Valproate therapy for epilepsy is associated with weight gain during treatment in approximately 50% of women patients. The weight gain can be progressive, and is associated with hyperinsulinemia and low serum levels of insulin-like growth factor-binding protein 1, which may lead to hyperandrogenism and polycystic ovaries.

Menstrual disorders in women with epilepsy receiving carbamazepine.

We measured concentrations of serum sex hormones and sex hormone binding globulin (SHBG) in relation to regularity of the menstrual cycles in 8 women before carbamazepine (CBZ) treatment was initiated and after 1 and 5 years of CBZ therapy. In addition, we evaluated menstrual cycle regularity and related endocrine changes in 56 women receiving CBZ treatment for > 5 years. Serum SHBG levels increased, and serum concentrations of 17 beta-estradiol (estradiol) and estradiol/SHBG ratio decreased during CBZ treatment. Two of the 8 patients (25%) in the prospective study group developed menstrual irregularities during the first 5 years of therapy. In the cross-sectional study group of patients treated with CBZ for > 5 years, the frequency of menstrual disturbances was also 25.0% (14 of 56 patients). Concentrations of serum sex hormones and SHBG were measured in 13 women with menstrual disorders and in 11 randomly selected women with regular cycles. In most cases, menstrual disorders were associated with increased serum SHBG and decreased serum estradiol levels and low estradiol/SHBG ratio. Long-term CBZ treatment results in increased serum SHBG levels and decreased estradiol effect, which correlate with the frequency of menstrual disorders in CBZ-treated women with epilepsy.