Systematic molecular and clinical analysis of uterine leiomyomas from fertile-aged women undergoing myomectomy.

STUDY QUESTION: What are the distributions and associated clinical characteristics of mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2) and fumarate hydratase (FH) aberrations in uterine leiomyomas from fertile-aged myomectomy patients? SUMMARY ANSWER: These driver mutations account for the majority (83%) of tumours in fertile-aged patients. WHAT IS KNOWN ALREADY: Alterations affecting MED12, HMGA2 and FH account for 80-90% of uterine leiomyomas from middle-aged hysterectomy patients, while the molecular background of tumours from young myomectomy patients has not been systematically studied. STUDY DESIGN, SIZE, DURATION: A retrospective series of 361 archival uterine leiomyoma samples from 234 women aged ≤45 years undergoing myomectomy in 2009-2014 was examined. Associations between the molecular data and detailed clinical information of the patients and tumours were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA was extracted from formalin-fixed paraffin-embedded samples and MED12 exons 1 and 2 were sequenced to identify mutations. Level of HMGA2 expression was evaluated by immunohistochemistry. Biallelic FH inactivation was analysed with 2-succinylcysteine staining, which is an indirect method of assessing FH deficiency. All patients' medical histories were reviewed, and clinical information of patients and tumours was combined with molecular data. MAIN RESULTS AND THE ROLE OF CHANCE: The median age at operation was 34 years. The majority (58%) of patients were operated on for a single leiomyoma. Known driver mutations were identified in 83% of tumours (71% MED12; 9% HMGA2; 3% FH). In solitary leiomyomas, the MED12 mutation frequency was only 43%, and 29% were wild-type for all driver alterations. MED12 mutations were associated with multiple tumours, smaller tumour size and subserosal location. LIMITATIONS, REASONS FOR CAUTION: Although comprehensive, the study is retrospective in nature and all samples have been collected for routine diagnostic purposes. The use of paraffin-embedded samples and immunohistochemistry may have led to an underestimation of mutations. Due to the limited sample size and rarity of especially FH-deficient leiomyomas, the data are partly descriptive. WIDER IMPLICATIONS OF THE FINDINGS: The contribution of driver mutations in leiomyomas from young myomectomy patients is comparable to tumours obtained from hysterectomies of mostly middle-aged women. Our results support the earlier findings that MED12 mutations are associated with multiple tumours, smaller tumour size and subserosal location. The study emphasizes the distinct molecular background of solitary leiomyomas, and more research is needed to clarify the underlying causes of the notable proportion of wild-type leiomyomas. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the Academy of Finland (307773), the Sigrid Jusélius Foundation, the Cancer Foundation Finland and the iCAN Digital Precision Cancer Medicine Flagship. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Thyrocyte-specific Dicer1 deficiency alters thyroid follicular organization and prevents goiter development.

MicroRNAs (miRNAs) are important regulators of posttranscriptional gene expression and involved in embryonic development, regulation of cell differentiation, and growth. Dicer1 is a key enzyme in the maturation process of functional miRNAs. However, miRNA-mediated regulation of normal thyroid function and growth is largely unknown. To understand the role of miRNAs in the thyroid, we generated constitutive and tamoxifen-inducible, thyrocyte-specific Dicer1 knockout mice. The mice with perinatal Dicer1 deletion (cTgDcrKO) showed impaired follicular organization, increased fibrosis, and accumulation of adipocytes in the thyroid. Similar histological changes were observed in tamoxifen-induced adult Dicer1-deficient mice (iTgDcrKO). The thyroid phenotype in both knockout (KO) lines was associated with significantly down-regulated mRNA expression of thyroid transcription factor 1 (Ttf-1/Nkx2-1), thyroid peroxidase, and thyroglobulin (Tg) and up-regulated expression of genes involved in Tgf-ß signaling. Furthermore, in cTgDcrKO mice, which developed mild hypothyroidism, the protein expression of Nkx2-1, thyroglobulin, Paired box 8, and TSH receptor were clearly down-regulated compared with controls. Despite similar down-regulation of Dicer1 in cTgDcrKO and iTgDcrKO compared with controls, Dicer1 deletion in adult mice thyrocytes did not lead to acute hypothyroidism. No significant differences in thyroid weights between cTgDcrKO, iTgDcrKO, and controls were observed. However, a goitrogenic diet induced a 4-fold increase in thyroid weight in control animals, whereas it had no effect on iTgDcrKO thyroids. In summary, Dicer1 deficiency in thyrocytes is associated with intrathyroid fibrosis, adipogenesis, and enhanced expression of Tgf-ß signaling genes. Furthermore, our data indicate that Dicer1 is required for thyroid follicular organization, thyrocyte differentiation, and goiter development.

Pituitary gonadotrophic hormone synthesis, secretion, subunit gene expression and cell structure in normal and follicle-stimulating hormone ß knockout, follicle-stimulating hormone receptor knockout, luteinising hormone receptor knockout, hypogonadal and ovariectomised female mice.

To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic-pituitary-gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle-stimulating hormone ß knockout (FSHßKO), follicle-stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild-type or heterozygote female mice. Serum levels of LH were elevated in FSHßKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHß subunit gene in FSHßKO mice. However, there was a significant increase in expression of the FSHß and LHß subunit genes in FSHRKO female mice. The morphology of FSHßKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHß and FSHß mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin-releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were associated with changes in expression of the gonadotrophin subunit genes and were reflected in the cellular structure and secretory granule appearance within the gonadotroph cells.

Laparoscopic vs robotic-assisted surgery for endometrial carcinoma in a centre with long laparoscopic experience.

Surgical outcomes and costs of laparoscopic and robotic hysterectomy for the treatment of endometrial carcinoma were compared in a centre with lengthy experience with laparoscopic surgery. The robotic cohort (n = 67) had a longer operative time than the laparoscopic cohort (n = 150) (p < 0.0001). Lymph node yields were similar for both surgical modalities, but the median of estimated blood loss was lower in the robotic group (50 ml vs 100 ml; p < 0.0001). The proportion of patients with hospital stay > 2 days and rate of overall complications were similar in both groups. Operative costs were (Euros) €1,680 and €3,860 for the laparoscopic and robotic procedure, respectively. We conclude that robotic technology is feasible but does not provide short-term benefits for the treatment of endometrial carcinoma in a centre where laparoscopy has been established as the standardised minimally invasive surgical method.

An investigation into pituitary gonadotrophic hormone synthesis, secretion, subunit gene expression and cell structure in normal and mutant male mice.

To investigate brain-pituitary-gonadal inter-relationships, we have compared the effects of mutations that perturb the hypothalamic-pituitary-gonadal axis in male mice. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, and gonadotroph structure and number were measured. Follicle-stimulating hormone (FSH)ß knockout (FSHßKO), FSH receptor knockout (FSHRKO), luteinising hormone (LH) receptor knockout (LuRKO), hypogonadal (hpg), testicular feminised (tfm) and gonadectomised mice were compared with control wild-type mice or heterozygotes. Serum levels of LH were similar in FSHßKO, FSHRKO and heterozygote males despite decreased androgen production in KO males. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHß subunit gene in FSHßKO mice. However, there was a significant increase in expression of the common α and LHß subunit genes in FSHRKO males. The morphology of FSHßKO and FSHRKO gonadotrophs was not significantly different from controls, except that the subpopulation of granules consisting of an electron-dense core and electron-lucent 'halo' was not observed in FSHßKO gonadotrophs and the granules were smaller in diameter. In the gonadotrophin-releasing hormone deficient hpg mouse, gonadotrophin mRNA and hormone levels were significantly lower compared to control mice and gonadotrophs were correspondingly smaller, with less abundant endoplasmic reticulum and reduced secretory granules. In LuRKO, tfm and gonadectomised mice, hyperstimulation of LHß and FSHß mRNA and serum protein concentrations was reflected by subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticulum and more secretory granules distributed adjacent to the plasma membrane. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH have been found between normal and mutant male mice. These changes are associated with changes in transcriptional activity of the gonadotrophin subunit genes and are reflected by changes in the cellular structure and secretory granule architecture within the gonadotroph cells.

Effects of levonorgestrel-releasing intra-uterine system on the expression of vascular endothelial growth factor and adrenomedullin in the endometrium in adenomyosis.

BACKGROUND: The levonorgestrel-releasing intra-uterine system (LNg-IUS) has been used to control menorrhagia, but irregular bleeding during the first 3 months of use was the most notable side effect. Endometrial angiogenesis is believed to be regulated by angiogenic factors. The study aim was to evaluate the effects of LNg-IUS on vascular endothelial growth factor (VEGF) and adrenomedullin (AM) expression in the endometrium. METHODS: VEGF and AM expression were analysed using the avidin-biotin immunoperoxidase method on endometrial curettage specimens from menorrhagic women associated with adenomyosis before and 3 months after LNg-IUS insertion. RESULTS: VEGF expression was abundant both in the endometrial glands and stroma before LNg-IUS insertion, but became scanty 3 months after insertion. No immunostaining for AM was noted in the endometrial glands and stroma before LNg-IUS insertion, whereas AM immunostaining became prominent in the endometrial glands and stroma 3 months after LNg-IUS use. CONCLUSIONS: This is the first study to demonstrate that LNg-IUS insertion results in decreased expression of VEGF and increased expression of AM in the endometrial glands and stroma after 3 months of use. The results obtained suggest that the increase in AM expression in the endometrium may be responsible for the frequent occurrence of irregular bleeding during the initial 3 months of LNg-IUS use.

Age-related uterine and ovarian hypertrophy in FSH receptor knockout and FSHbeta subunit knockout mice.

Female mice in which the gene encoding the follicle-stimulating hormone FSH receptor (FSHR) knockout (KO) or its ligand (FSHbetaKO) have been disrupted were infertile. Ovaries of these mice were significantly smaller than those of heterozygous littermates but significantly larger than those of hypogonadal mice of the same age. Uterine masses in all three mutants were <6 mg, significantly reduced compared with heterozygous mice. At 1 year of age uterine mass had increased to >12 mg in 63% of FSHRKO females and 88% of FSHbetaKO females. Despite the increase in uterine size there was no evidence of contractility: uteri were flaccid and unresponsive to electrical or pharmacological stimulation. In most females in which uterine growth had occurred there was evidence of ovarian growth with hypertrophy of the interstitial tissue, occurrence of ovarian cysts and epithelial and tubular inclusions. There was no evidence of uterine or ovarian hypertrophy in hypogonadal (hpg) mice at any age or in 1 year old females in which the FSH mutations were bred onto the hpg background. There was an inverse correlation of plasma LH concentrations and uterine mass in 1 year old mutant females with uterine hypertrophy. Ovariectomy of both FSHRKO and FSHbetaKO females with large uteri resulted in decreased uterine mass and increased plasma concentration of LH. The number of mice with ovarian pathology, reminiscent of the serous ovarian adenocarcinomas found in humans, was significantly greater in the FSHbetaKO mice, indicating that the presence of an intact FSH receptor on ovarian cells of FSHbetaKO females may allow constitutive basal stimulation of the ovary, which is absent in mice lacking FSH receptors.

How much LH do the Leydig cells see?

The purpose of this study was to assess the concentrations of LH that Leydig cells are exposed to upon in vivo stimulation of steroidogenesis. The concentrations of LH were measured in rats in testicular interstitial extracellular fluid, seminiferous tubular fluid and blood plasma from testicular veins from one testis before and from the other testis of the same rats after an intravenous injection of gonadotrophin-releasing hormone (GnRH) or saline, and compared with the concentrations in blood plasma from a peripheral vein. The concentrations of LH in interstitial fluid surrounding the Leydig cells before the injections were about 10% of the levels in blood plasma, and showed no significant rise at 15 min and a much smaller rise at later times in rats injected with GnRH than those seen in blood plasma from either of the two sources, which were similar. The concentrations of LH in tubular fluid were even lower and showed no change after GnRH. Testosterone concentrations in testicular cells, interstitial fluid and testicular venous blood plasma were significantly increased by 15 min after GnRH, when compared with saline-injected controls, with no change in the levels in tubular fluid. The rise in testosterone concentrations in testicular venous plasma after GnRH was smaller than those in the cells and interstitial fluid. In conclusion, the concentrations of LH reaching the testicular interstitial fluid were only about one-tenth of that measured in the circulation, presumably because the endothelial cells restrict access of the hormone to the interstitial fluid. This indicated that either the Leydig cells are extremely sensitive to LH stimulation or that testicular endothelial cells modulate the action of LH on the Leydig cells.

Lack of association of the common immunologically anomalous LH with endometriosis.

BACKGROUND: Subfertile women with endometriosis have been reported to demonstrate impaired follicular growth, ovulatory dysfunction and disturbed LH patterns. In addition, abnormal LH and/or LH receptors have been linked with endometriosis-associated infertility. Carriers of a variant of the beta-subunit of luteinizing hormone (V-LH) are largely healthy; however, differences in their gonadal function such as alterations in gonadal steroidogenesis, ovarian reserve, pubertal development and predisposition to diseases such as infertility and polycystic ovarian disease have been found. METHODS AND RESULTS: To explore the possible relationship between endometriosis and V-LH, we examined its frequency in 230 women undergoing laparoscopic surgery for the investigation of infertility. For the entire study population, 185 (80.4%) were wild type; 42 (18.3%) were heterozygous; and three (1.3%) were homozygous for V-LH. No difference was found between women with (n = 85) and without (n = 145) endometriosis concerning the frequency of the type of LH. CONCLUSION: Our results do not support the hypothesis that the variant form of LH is associated with an altered risk of endometriosis in the population tested.

Progestin-releasing intrauterine systems.

The levonorgestrel-releasing intrauterine system (LNG IUS) is an effective method for contraception. It has a strong antiproliferative action on the endometrium. The endometrium is transformed under the influence of local levonorgestrel and becomes unresponsive to ovarian estrogens. This process is associated with progressive reduction of menstrual blood loss and menstrual duration. Scanty and irregular bleeding and/or spotting is usual during the first 3 to 4 months. The reduction of menstrual blood loss continues and after the first 9 months many women have no bleeding at all. However, they have normal ovarian function. The absence of bleeding is a result of the local antiproliferative action of the LNG IUS on the endometrium, which is also responsible for many health benefits during the use of this method. As with oral contraceptives, the risk of pelvic inflammatory disease is reduced, because of reduced menstrual blood loss, endometrial suppression, and thickening of the cervical mucus. There are some steroidal side effects: mood changes, oily skin, and acne. Weight increase is similar to that associated with copper intrauterine devices: 500 g per year over 5 years. Users should be told that the LNG IUS does not prevent sexually transmitted infection, and therefore women at risk should also use condoms for their protection.

Therapeutic use of the LNG IUS, and counseling.

The intrauterine release of levonorgestrel (LNG) alters the function of the endometrium. This phenomenon offers special health benefits for users and a manner of local intrauterine therapy. In this review we discuss use of LNG-releasing intrauterine system (LNG IUS) in treatment and prevention of anemia and in the therapy of menorrhagia and dysmenorrhea. The antiproliferative effect of the LNG IUS on the endometrium offers targeted therapy against the proliferative action of estrogen on the endometrium during hormone replacement therapy. The LNG IUS as an alternative to sterilization is also discussed. There are also promising therapeutic fields such as use of the LNG IUS in the treatment of endometriosis and in the protection of endometrium exposed to tamoxifen during breast cancer treatment. Special attention is paid to counseling because successful use of the LNG IUS requires good training of the providers and extensive counseling of users.

Effects of the levonorgestrel-releasing intrauterine system on proliferation and apoptosis in the endometrium.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNg-IUS) has been shown to be effective in the management of menorrhagia. In order to evaluate the effects of LNg-IUS on endometrial proliferation and apoptosis, proliferating cell nuclear antigen (PCNA) expression, apoptosis, Fas and Bcl-2 protein expression in the endometrium were determined at the early proliferative phase of the menstrual cycle before and 3 months after LNg-IUS insertion. METHODS: PCNA, Fas and Bcl-2 protein expression were analysed using an avidin-biotin immunoperoxidase method. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labelling (TUNEL) method. RESULTS: PCNA, immunolocalized both in the nuclei of endometrial glands and stroma was less abundant 3 months after insertion (P < 0.05). Bcl-2 protein, immunolocalized in the cytoplasm of endometrial glands but not in the stroma, became scanty 3 months after insertion. Fas antigen, immunolocalized only in endometrial glands before insertion, became prominent in both endometrial glands and stroma 3 months after insertion. The apoptosis-positive rate of the nuclei in both endometrial glands and stroma was significantly higher 3 months after insertion relative to that before insertion (P < 0.05). CONCLUSIONS: LNg-IUS resulted in a decrease in endometrial proliferation and an increase in apoptosis in endometrial glands and stroma. The increase in apoptosis associated with increased Fas antigen expression and decreased Bcl-2 protein expression in the endometrium may be one of the underlying molecular mechanisms by which LNg-IUS insertion causes the atrophic change of the endometrium.

Assessment of mechanisms of thyroid hormone action in mouse Leydig cells: regulation of the steroidogenic acute regulatory protein, steroidogenesis, and luteinizing hormone receptor function.

Recently, we demonstrated that triiodothyronine (T(3)) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T(3) on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T(3) acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T(3) (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T(3) increased the levels of cytochrome P450scc mRNA by 83%, whereas those of cytochrome P450 17alpha-hydroxylase and 3ss-hydroxysteroid dehydrogenase decreased. The diminished response in steroidogenesis following chronic T(3) exposure was not a result of alterations in StAR mRNA stability, but rather was due to inhibition of transcription of the StAR gene. Similar acute stimulatory and chronic inhibitory responses to T(3) were found when LHR mRNA expression and LHR ligand binding were examined. Transfections with an LHR or StAR promoter/luciferase reporter construct demonstrated that a 173-bp fragment of the LHR promoter containing an SF-1 binding motif was involved in T(3) response, as was the SF-1 recognition site at -135 bp in the StAR promoter. Furthermore, the importance of SF-1 in T(3) function was also verified employing mutation in the bases of SF-1 sequences using electrophoretic mobility shift assays. The potential physiological relevance of these findings was demonstrated when similar responses were obtained in mice rendered hypo and hyperthyroid. Collectively, these observations further characterize the thyroid-gonadal connection and provide insights into the mechanisms for a dual regulatory role of thyroid hormone in Leydig cell functions.

A common polymorphic allele of the human luteinizing hormone beta-subunit gene: additional mutations and differential function of the promoter sequence.

A common genetic variant (V) of the human luteinizing hormone (LH) beta-subunit gene was recently discovered. The V-LH molecules have higher bioactivity in vitro, but shorter half-life in circulation, which apparently is related to the alterations of LH function observed in individuals homo- and heterozygous for the V-LHbeta allele. We have now studied whether additional mutations in the V-LHbeta promoter sequence could contribute to the altered physiology of the LH variant molecules. The 661 bp 5'-flanking region of the V-LHbeta gene, retrieved from human genomic DNA by PCR, contained eight single-nucleotide changes, as compared with the wild-type (wt) LHbeta promoter. The finding was consistent in DNA samples of different ethnic groups. Reporter constructs with various lengths of the wt- and V-LH promoter sequences, driving the firefly luciferase reporter gene, were transfected into an immortalized mouse pituitary cell line, LbetaT(2), known to express the endogenous LHbeta gene, and into a non-endocrine human embryonic kidney cell line, HEK 293. Basal expression levels of the V-LHbeta promoter constructs were on average 36% higher in LbetaT(2)cells ( P < 0.001; n = 29), and 40% higher in HEK 293 cells ( P < 0.001; n = 16), as compared with the respective wt sequences. Numerous qualitative and quantitative differences were found between the two cell lines in responses of the two promoter sequences to stimulation with 12- O -tetradecanoylphorbol-13-acetate, forskolin, 8-bromo-cAMP, progesterone and gonado- tropin-releasing hormone. In conclusion, the V-LHbeta promoter has higher basal activity, and differs in response to hormonal stimulation, as compared with the wt-LHbeta promoter. The altered promoter function of the V-LHbeta gene provides evidence for differences in regulation of the wt- and V-LHbeta genes, which may contribute to the differences observed in pituitary-gonadal function between carriers of the two LHbeta alleles. The findings also suggest a novel evolutionary mechanism whereby polymorphic changes resulting in altered bioactivity of a gene product may be compensated for by additional mutations in the cognate promoter sequence, changing transcription of the same gene.

The effect of intrauterine and oral levonorgestrel administration on serum concentrations of sex hormone-binding globulin, insulin and insulin-like growth factor binding protein-1.

BACKGROUND: The concentrations of sex hormone-binding globulin (SHBG) have been shown to decrease during the use of levonorgestrel (LNG)-containing contraception. This decrease has been thought to be due to the androgenic action of LNG. In endogenously hyperandrogenic women, particularly in those with increased body weight, serum SHBG correlates with circulating insulin-like growth factor binding protein-1 (IGFBP-1) concentration, and both are inversely related to insulin. LNG-containing combined contraceptives have also been reported to increase the pancreatic insulin secretion. OBJECTIVE: To examine whether serum insulin and IGFBP-1 levels are related to SHBG during the use of intrauterine or oral levonorgestrel contraception. METHODS: Thirty-one fertile women were divided into three study groups: A copper-releasing intrauterine device (IUD) was inserted in control group (n= 10), and the LNG-releasing intrauterine contraceptive system (LNG-IUS) in group II (n= 10), and 30 mirog LNG-containing contraceptive minipills were given in group III (n=11). Twenty-nine women completed the study and one woman was excluded because of a high body mass index. Fasting concentrations of blood glucose, insulin, SHBG, IGFBP-1, testosterone and LNG before and after three-months-use of contraception were measured. RESULTS: SHBG concentrations decreased slightly during oral LNG contraception, but not during the use of the LNG-IUS. No change was found in blood glucose, serum insulin, serum IGFBP-1 and serum total testosterone concentrations in either group. In our study group, including women with normal body weight, no correlation was detected between insulin and SHBG concentrations before or after LNG contraception, whereas an inverse correlation was found between insulin and IGFBP-1 levels at the baseline as well as after LNG-IUS use (R2= 0.578; p=0.001). Multiple regression analysis showed no significant association between the levels of SHBG and IGFBP-1 as dependent factors, and glucose, insulin, LNG, age, waist-hip ratio and body mass index as dependent factors. CONCLUSIONS: Our data imply that the effect of levonorgestrel on variables associated with endogenous hyperandrogenism remains borderline in women with normal body mass index.

Functional assessment of the calcium messenger system in cultured mouse Leydig tumor cells: regulation of human chorionic gonadotropin-induced expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence of Ca2+. The mechanism of the Ca2+ effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan receptor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 +/- 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca2+, whereas EGTA and verapamil completely abolished the increase caused by Ca2+. Cells expressing SF-1 marginally increased StAR expression, but coordinately elevated StAR mRNA levels in response to hCG and hCG plus Ca2+ compared with those in mock-transfected cells. On the other hand, overexpression of the nuclear receptor DAX-1 remarkably diminished (P < 0.0001) the endogenous SF-1 mRNA level as well as hCG-induced StAR mRNA expression. In summary, our results provide evidence that extracellular Ca2+ rapidly increases [Ca2+]i after hCG stimulation, presumably through opening of the transmembrane Ca2+ channel. Neither extracellular Ca2+ nor K+ alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly potentiate hCG induction. The Ca2+-mediated increase in hCG involved in StAR expression and P production is well correlated to the levels of SF-1 expression. The stimulatory effect of hCG that rapidly increases [Ca2+]i is responsible at least in part for the regulation of SF-1-mediated StAR expression that consequently regulates steroidogenesis in mouse Leydig tumor cells.

Follicle-stimulating hormone ligand and receptor mutations, and gonadal dysfunction.

In contrast to the general contention, infertility can be an inherited condition. Some of the genetic causes of male and female infertility have turned out to be due to inactivating mutations in the gonadotropin and gonadotropin receptor genes. The topic of the present text is to review current knowledge on mutations affecting the function of follicle-stimulating hormone (FSH). This gonadotropin, by binding to its specific G protein-coupled cell membrane receptor (FSHR), is important for normal gonadal function. Mutations affecting gonadotropin genes are extremely rare, but recent genetic studies have revealed that the pathogenesis of subfertility or infertility can be due to mutations in the FSH receptor (FSHR) gene. While mutations affecting FSHR are sporadic, polymorphism of the FSHR gene seems to be a common phenomenon. To date, six inactivating and only one activating mutation have been detected in the FSHR gene. In contrast to LHR gene, the majority of these mutations affect the extracellular domain of the receptor. Together with animal models using the transgenic and knock-out approaches, systematic analysis of alterations in the FSHR gene increases our knowledge on the structure and function of the FSHR and demonstrates that the integrity of each FSHR segment is required for proper expression of the fully active protein and for normal gonadal function. Mutations in the FSHR gene have different consequences in the reproductive function depending on the sex of the patient: while normal ovarian function is critically dependent on FSH, male fertility is possible with minimal or absent FSH action.

The frequency of an inactivating point mutation (566C-->T) of the human follicle-stimulating hormone receptor gene in four populations using allele-specific hybridization and time-resolved fluorometry.

We have described previously in the Finnish population an inactivating point mutation (566C-->T) in the human FSH receptor (FSHR) gene. In women, this mutation causes hypergonadotropic ovarian failure with arrest of follicular maturation and infertility, whereas in men, there is variable suppression of spermatogenesis, but no absolute infertility. To determine whether the same FSHR mutation occurs in other populations, its frequency was determined in Finland, Switzerland, Denmark, and the Chinese population of Singapore. The mutation was screened for using genomic DNA extracted from whole blood or dried blood spots. Exon 7 of the FSHR gene was first amplified using a pair of biotinylated primers. The PCR products were then immobilized on streptavidin-coated microtitration wells and hybridized using short allele-specific oligonucleotide probes labeled with europium. Time-resolved fluorometry was used for europium signal detection. To test the reliability of this method, 40 isolated DNA samples and 35 dried blood spot samples were blindly tested for the 566C-->T FSHR mutation. The analyses yielded identical results with denaturing gradient gel electrophoresis and allele-specific restriction enzyme digestion of the same samples, thus demonstrating the reliability of the tested method. Automation of this procedure allows the screening of large numbers of samples, which was subsequently carried out to investigate the frequency of the 566C-->T mutation in the study populations. A total of 4981 samples from the above-mentioned 4 countries were analyzed. The frequency of the 566C-->T mutation was 0.96% for all Finnish samples (n=1976), with a strong enrichment of the mutant allele in the northeastern part of the country. Only 1 mutation carrier was identified in the samples from Switzerland (n=1162), whereas none was found in samples from Denmark (n=1094) and the Singapore Chinese (n=540). These results suggest that the 566C-->T mutation of the FSHR gene is enriched in Finland, but is uncommon in other populations.

The ultrastructure of human endometrium is altered by administration of intrauterine levonorgestrel.

We studied the effect of intrauterine administration of levonorgestrel (LNG) on the ultrastructure of the endometrium. Twenty-one endometrial biopsy specimens, collected from nine fertile women during normal menstrual cycles and after 1, 3 or 6 months of use of a levonorgestrel-releasing intrauterine contraceptive system (LNG IUS), were studied using transmission and scanning electron microscopy. During the 6 month exposure to LNG IUS, changes took place in the endometrium. The glandular epithelial cells became lower. The junctional complexes between epithelial cells remained unchanged, whereas the lateral microvillar interdigitations became more prominent. The basal lamina under the epithelium became wavy but remained uniform and practically uninterrupted; only solitary epithelial cell protrusions through the basal lamina were seen. The stromal cells were largely decidualized. We conclude that in parallel with the generally known cellular effects, the use of the LNG IUS results in distinct changes in the basal lamina between the endometrial epithelial and stromal cells. The especially well-developed and uninterrupted basal lamina may be involved in the mechanism of the LNG IUS-induced endometrial suppression. Furthermore, the complex intercellular junctions between the epithelial cells, normally loosening around the time of implantation, persist during the local administration of levonorgestrel. This may have a pivotal role in the contraceptive effect of the LNG IUS.

PIP: The suppressive effect of intrauterine administration of levonorgestrel (LNG) on the ultrastructure of the endometrium was assessed through analysis of 21 endometrial biopsy specimens collected from 9 fertile women both during normal menstrual cycles and 1, 3, or 6 months after insertion of an LNG intrauterine system (LNG-IUS). After 6 months of LNG-IUS use, the morphology resembled that seen during the secretory phase of the normal menstrual cycle. Transmission and scanning electron microscopy revealed distinct changes in the basal lamina between the endometrial epithelial and stromal cells after LNG-IUS insertion. The glandular epithelial cells became thinner and the lateral microvillar interdigitations more prominent. The basal lamina under the epithelium became wavy but remained uniform and practically uninterrupted--a mechanism that may be salient to LNG-induced endometrial suppression. Only solitary epithelial cell protrusions through the basal lamina were seen. Stromal cells were largely decidualized. The junctional complexes between epithelial cells remained unchanged in contrast to the loosening normally seen around the time of implantation, which may have a key role in the contraceptive effect of the LNG-IUS.

Gonadotropin-independent regulation of steroidogenesis in the fetal rat testis.

UNLABELLED: The goal of the present study was to determine whether the onset of fetal Leydig cell steroidogenesis is dependent on gonadotropic stimulation. The relationships between the onset of pituitary LH synthesis and secretion, and the response of testicular steroidogenesis to LH and various putative paracrine factors were examined. We found by reverse transcription-polymerase chain reaction (RT-PCR) that the LHbeta-subunit gene expression in the fetal pituitary gland starts on embryonic day (E) 16.5. Plasma LH was very low (< 5.0 ng/L) until E19.5 and increased significantly thereafter. In contrast, the greatest increase in the testicular testosterone had already occurred between E18.5 and E19.5. Hence, fetal testicular steroidogenesis must start independent of LH stimulation. Basal testosterone production in incubations of fetal testis (E16.5-19.5) was high, 50-80% of the hCG-stimulated level. In contrast, in dispersed fetal Leydig cells, basal steroidogenesis was consistently low. This suggests the presence of paracrine factors in the intact testes that stimulate their steroidogenesis. Effects of various putative paracrine factors were thereafter tested on the fetal testis. We found for the first time that both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP-27) markedly stimulated fetal, but not adult, Leydig cells. IN CONCLUSION: 1) Pituitary LH cannot be the initial stimulus for fetal testicular steroidogenesis. 2) Some paracrine factor(s) probably turn on and maintain early fetal testicular steroidogenesis before the later onset of LH secretion, although a constitutive component in the onset of steroidogenesis is also possible. 3) VIP and PACAP-27 are likely candidates for a paracrine stimulus of the fetal testis.