Matti Poutanen

To study further the role of gonadotropins in reproductive functions, we generated mice with LH receptor (LHR) knockout (LuRKO) by inactivating, through homologous recombination, exon 11 on the LHR gene. LuRKO males and females were born phenotypically normal, with testes, ovaries, and genital structures indistinguishable from their wild-type (WT) littermates. Postnatally, testicular growth and descent, and external genital and accessory sex organ maturation, were blocked in LuRKO males, and their spermatogenesis was arrested at the round spermatid stage. The number and size of Leydig cells were dramatically reduced. LuRKO females also displayed underdeveloped external genitalia and uteri postnatally, and their age of vaginal opening was delayed by 5-7 days. The (-/-) ovaries were smaller, and histological analysis revealed follicles up to the early antral stage, but no preovulatory follicles or corpora lutea. Reduced gonadal sex hormone production was found in each sex, as was also reflected by the suppressed accessory sex organ weights and elevated gonadotropin levels. Completion of meiosis of testicular germ cells in the LuRKO males differs from other hypogonadotropic/cryptorchid mouse models, suggesting a role for FSH in this process. In females, FSH appears to stimulate developing follicles from the preantral to early antral stage, and LH is the stimulus beyond this stage. Hence, in each sex, the intrauterine sex differentiation is independent of LH action, but it has a crucial role postnatally for attaining sexual maturity. The LuRKO mouse is a close phenocopy of recently characterized human patients with inactivating LHR mutations, although the lack of pseudohermaphroditism in LuRKO males suggests that the intrauterine sex differentiation in this species is not dependent on LH action.

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra-and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels. (Endocrinology 156: 2492-2502, 2015) S erum levels of sex steroid hormones are routinely mea- sured by immunoassay-based techniques in clinical and research settings. However, these assays have a questionable specificity, especially at the lower concentration range (1-4). In this respect, we have shown that immu-noassays for serum estradiol show interference by a C-reactive protein-related factor, which can influence the associations between serum estradiol and inflammationrelated sex steroid-dependent outcomes (5). Highly specific and sensitive assays for the quantification of sex

Human epididymal secretory protein E4 (HE4, also known as WAP four-disulphide core domain protein 2) is a new promising biomarker for ovarian cancer but its specificity against ovarian endometriotic cysts is only superficially known. We, thus, analysed serum HE4 concentrations together with a tumour marker CA125 in serum samples of women diagnosed with various types of endometriosis, endometrial cancer or ovarian cancer, and in samples from healthy controls. The mean serum concentration of HE4 was significantly higher in serum samples of patients with both endometrial (99.2 pM, P<0.001) and ovarian (1125.4 pM, P<0.001) cancer but not with ovarian endometriomas (46.0 pM) or other types of endometriosis (45.5 pM) as compared with healthy controls (40.5 pM). The serum CA125 concentrations were elevated in patients with ovarian cancer, advanced endometriosis with peritoneal or deep lesions, or ovarian endometriomas, but not in the patients with endometrial cancer. The microarray results revealed that the mRNA expression of the genes encoding HE4 and CA125 reflected the serum protein concentrations. Taken together, measuring both HE4 and CA125 serum concentrations increases the accuracy of ovarian cancer diagnosis and provides valuable information to discriminate ovarian tumours from ovarian endometriotic cysts.