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Study Question: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients?, Summary Answer: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment., What Is Known Already: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory., Study Design, Size, Duration: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018., Participants/materials, Setting, Methods: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed., Main Results and the Role of Chance: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy., Limitations, Reasons for Caution: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients., Wider Implication of the Findings: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients., Study Funding/competing Interest(s): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

In the past, explant tissue-culture methodologies have been used to grow gonads and study their development. Results from in vitro cultures of human gonads showed limited progress toward gonadal cell differentiation and were focused mainly on germ-cell differentiation. Thus, detailed studies focusing on human first-trimester gonadal tissue functionality in vitro are still missing. In this study we investigated the endocrine function of human first-trimester gonads in vitro. We included 27 female and 28 male gonadal samples, derived from a total of 55 cases, at postconceptional ages of 4.5 to 10.5 weeks. Tissues were cultured using an explant tissue-culture system for 14 days. Assays for testosterone (liquid chromatography-tandem mass spectrometry), anti-Müllerian hormone (AMH; ELISA), and inhibin B (ELISA) were performed using media collected after 7 and 14 days of culture. We demonstrated sex- and age-dependent secretion profiles of testosterone, AMH, and inhibin B in the culture media, which resemble the pattern of hormone production in human gonads in vivo, from the few available studies at the same age range. Our study shows that explant tissue-culture conditions are robust for culture of human first-trimester gonadal somatic cells. Thus, it can be used to study human gonadal development and related diseases as well as the effect of potentially hormone-disturbing substances in human gonads during development. However, detailed molecular studies are needed for better understanding of the mechanistic control of the endocrine function of human first-trimester gonads.

Context: Polycystic ovary syndrome (PCOS) is associated with increased risk of endometrial cancer. This is usually explained by chronic anovulation and deficient progesterone activity. However, the role of progesterone receptors (PRs) in endometrial proliferation is unclear., Objective: To evaluate PRs in relation to endometrial proliferation in women with PCOS., Design: Cross-sectional study and lifestyle intervention., Setting: Clinical and laboratory research unit at a university hospital., Participants: Twenty obese women with PCOS and 10 age- and body mass index-matched regularly menstruating controls., Intervention: Dietary management and physical exercise., Main Outcome Measures: Endometrial messenger RNA (mRNA) levels and immunostaining of the nuclear PRs A (PRA) and B (PRB), nongenomic progesterone receptor membrane component 1 (PGRMC1) and 2 (PGRMC2), and proliferation marker Ki67., Results: Before lifestyle intervention, mRNA expression of PRAB was lower while PRB was higher in proliferative endometrium of obese women with PCOS compared with controls (P < 0.05). After lifestyle intervention and weight loss, mRNA expression of PRAB was still low but PRB mRNA decreased and was not different to controls in proliferative endometrium (P < 0.01). The subgroup of PCOS women who remained anovulatory displayed higher protein levels of PRB, PGRMC1, PGRMC2 and of the proliferative marker Ki67 on cycle days 21 to 23 than controls (P < 0.05). In contrast, the subgroup of PCOS women with confirmed ovulation showed immunostaining, including Ki67, in secretory endometrium that was not different to controls, except for higher PRA (P < 0.05)., Conclusions: Lifestyle intervention improves, but not fully restores PR expression and decreases proliferation in secretory endometrium of obese PCOS women., (Copyright © 2017 Endocrine Society)

Context: Polycystic ovary syndrome (PCOS) is a common cause of anovulation. It may also negatively affect the endometrium, which could lead to implantation failure and proliferative aberrations., Objective: Our objective was to study sex hormone receptors in the endometrium of women with PCOS., Design: This is a cross-sectional study and lifestyle intervention., Setting: Clinical and laboratory research unit was undertaken at a university hospital., Participants: Twenty overweight/obese women fulfilling all three PCOS criteria (anovulation, hyperandrogenism, and polycystic ovaries), 10 body mass index-matched regularly menstruating controls, 11 normal-weight women with PCOS, and 11 normal-weight controls., Intervention: Intervention for this study included dietary management and physical exercise., Main Outcome Measures: mRNA levels and immunostaining of estrogen receptor α (ERα) and β (ERβ), nongenomic estrogen receptor α36 (ERα36), and G-protein-coupled estrogen receptor-1 (GPER), and the androgen receptor (AR) on cycle days 6-8 and cycle days 21-23., Results: Before intervention, mRNA levels of ERα, ERα36, and the ERα/ERβ mRNA ratio were lower in proliferative endometrium of overweight/obese PCOS women compared with controls (P < .05). After intervention, ERα protein and the ERα/ERβ protein ratio in proliferative endometrium increased and were higher in PCOS women with improved menstrual function than in those without improvement (P < .05). In the subgroup of PCOS women with restored ovulation, only higher protein levels of GPER were found in secretory endometrium (P < .01). However, PCOS women who remained anovulatory had higher protein levels of ERα, GPER, and AR on cycle days 21-23 than controls (P < .05)., Conclusions: Lifestyle intervention alters, but does not fully restore, ER and AR expression in proliferative and secretory endometrium of obese women with PCOS.

Context: Available data concerning effects of testosterone on endometrium of postmenopausal women are seriously limited., Objective: Our aim was to compare the influence of treatment with testosterone and/or estrogen on endometrial proliferation in healthy postmenopausal women., Design: This was an open, randomized clinical study with parallel comparison of the groups., Setting: The study was conducted at a women's health clinical research unit and a research laboratory at a university hospital., Participants: Sixty-three women who had experienced natural menopause participated in this study., Interventions: After random assignment, the participants were administered orally testosterone undecanoate (40 mg every second day), estradiol valerate (2 mg daily), or both for 3 months., Main Outcome Measures: Endometrial thickness was measured, and endometrial proliferation evaluated on the basis of histopathology and expression of Ki-67, a proliferation marker., Results: Endometrial thickness was significantly increased by treatment with estrogen alone or in combination with testosterone but was unaltered by testosterone alone. Among the women receiving estrogen alone, the proportion exhibiting histopathology indicative of proliferation increased significantly to 50% (P < 0.05), there was a nonsignificant increase to 28% with the combined treatment, whereas testosterone alone had no effect at all. Expression of Ki-67 was up-regulated significantly in both glands and stroma (P < 0.05, respectively) in both estrogen treatment groups. However, the expression was significantly higher in stroma by estrogen treatment alone than after combined treatment (P < 0.05)., Conclusions: The short-term treatment with testosterone of postmenopausal women does not stimulate endometrial proliferation. In addition, testosterone appears to counteract endometrial proliferation induced by estrogen to a certain extent.

The development and functions of female reproductive tissues are regulated by the actions of two major sex steroid hormones, estrogen and progesterone. To investigate estrogen-dependent gene expression in the rat uterus, we studied the effect of ovariectomy with or without estrogen treatment on the uterine expression of 3000 genes using cDNA microarrays. Many genes were regulated by either treatment, but only few were reciprocally regulated by these contrasting treatments. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include follistatin-related protein, Thy-1 glycoprotein, alpha-fodrin, CD24, immediate early response 5, insulin-like growth factor-binding protein 2, growth response protein CL-6 (INSIG-1), ladinin1, class I major histocompatibility complex heavy chain, lactadherin, ezrin, and Fas-activated serine/threonine kinase. Because of their function as regulators of proliferation and apoptosis, CD24, insulin-like growth factor-binding protein 2, and Fas/Fas ligand were examined further by immunohistochemical expression and tissue localization analysis. Our analysis confirms a contrasting regulation of these gene products by ovariectomy and estrogen treatment. The present study identifies novel mediators of estrogen actions in the uterus and provides genome-wide expression data from which novel hypotheses regarding uterine function can be generated.

Prolactin (PRL) is one of several polypeptide factors known to exert trophic effects on the prostate. We have previously reported a dramatic prostate enlargement with concurrent chronic hyperprolactinemia and elevated serum androgen levels in a PRL transgenic mouse (Mt-PRL) with ubiquitous expression of the transgene. To address the role of local PRL action in the prostate, a new transgenic mouse model (Pb-PRL) was generated using the prostate-specific rat probasin (Pb) minimal promoter to drive expression of the rat PRL gene. Pb-PRL transgenic males developed a significant enlargement of both the dorsolateral and ventral prostate lobes evident from 10 wk of age and increasing with age. Expression of the transgene was restricted to the prostate and detected from 4 wk of age. Low levels of transgenic rat PRL were detectable in the serum of adult Pb-PRL animals. Serum androgen levels were normal. The Pb-PRL prostate displayed significant stromal hyperplasia, ductal dilation, and focal areas of epithelial dysplasia. Quantitative analysis of prostatic tissue cellularity demonstrated a marked increase in the stromal to epithelial ratio in all lobes of Mt-PRL and Pb-PRL transgenic prostates compared with controls. Microdissections demonstrated an increased ductal morphogenesis in dorsolateral and ventral prostate lobes of Mt-PRL prostate vs. Pb-PRL and controls. In conclusion, this study indicates the ability of PRL to promote, directly or indirectly, ductal morphogenesis in the developing prostate and further to induce abnormal growth primarily of the stroma in the adult gland in a setting of normal androgen levels.

The human uterine cervix is a fibrous organ with a high connective tissue content. An extensive remodeling of the connective tissue prior to parturition, i.e., cervical ripening, requires the presence of proteolytic enzymes. The exact mechanism of cervical ripening has not been clarified. We evaluated in vivo distribution and expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in the human cervix at term pregnancy and immediately after parturition compared with the nonpregnant state. Cervical biopsies were obtained from term pregnant, postpartum, and nonpregnant women. MMP-2 and MMP-9 proteins were localized by immunohistochemistry. Messenger RNA levels of MMP-2 and MMP-9 were evaluated by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR) using an invariable internal standard. The mRNA levels of MMP-2 and MMP-9 were increased in the cervix at term pregnancy and postpartum compared with the nonpregnant state. Cervical stromal fibroblasts and smooth muscle cells were identified as main sources of MMP-2, whereas the MMP-9 protein was observed exclusively in invading leukocytes. These data indicate the involvement of MMP-2 and MMP-9 in the cervical ripening process.

The action of steroid hormones is primarily mediated via a process that involves hormone binding to specific receptors in target cells, which leads to transcriptional activation of steroid-responsive genes and, subsequently, to a modification of cellular responses. The aim of the present study was to obtain information about the dynamics of the two types of estrogen receptors (ERs), alpha and beta, by comparing their concentration and distribution in the reproductive tract of the rat during the estrous cycle. Twenty-four 55- to 60-day-old female Sprague-Dawley rats were used. The stage of estrous cycle was determined by vaginal smear. ERalpha was the dominating subtype in uterus, oviduct, and cervix/vagina, with the distribution varying in stroma and epithelium during the estrous cycle. A low level of ERalpha mRNA was observed in ovarian stromal cells, with some scattered positive cells found among granulosa cells. ERbeta expression was observed in the different compartments of uterus and cervix/vagina, but cyclic variation during the estrous cycle was less evident than that of ERalpha. Only a few scattered cells that contained ERbeta mRNA were observed in oviduct. ERbeta mRNA was highly expressed in granulosa cells of developing follicles, with a weaker hybridization signal in new corpora lutea. Immunohistochemistry showed that protein levels of ERalpha and ERbeta have distinct specificity for tissues and cell types, similar to their respective levels of mRNA, as assessed by in situ hybridization. The precise physiological function and importance of ERbeta is still unclear. The relative physiological and pathological function of each ER subtype in the female reproductive tract remains to be further evaluated.

The uterus is an important target organ for steroid hormones. The effects of these hormones are mediated via specific receptors. The aim of this study was to compare the expression, distribution, and regulation of estrogen receptor (ER) alpha and beta in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E(2)), progesterone (P(4)), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ERalpha is the dominating subtype in the rat uterus. E(2) seemed to increase the ERalpha mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P(4) reduced ERalpha expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E(2) or P(4) did not alter the expression of ERbeta, on either the mRNA or protein level. In conclusion, the distribution and regulation of ERalpha and ERbeta differ in the different compartments of the rat uterus. The complex uterine responses to E(2) and P(4) are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERbeta probably plays a minor role in the regulation of uterine physiology.

The SOCS (suppressors of cytokine signaling) proteins have been suggested to function as inhibitors of cytokine receptor signaling. We have analyzed SOCS-2, SOCS-3, and CIS expression in relation to GH actions in the rat. SOCS-2, SOCS-3, and CIS transcripts were detected in various GH responsive tissues, including liver, muscle, and fat. In addition to the finding that different tissues express different levels of SOCS-2, SOCS-3, and CIS messenger RNA (mRNA), the steady-state levels of these SOCS transcripts were dependent on the endocrine status of the animal. SOCS-3 expression was 5-fold higher in fat from old compared with younger rats. Hypophysectomy reduced the levels of SOCS-2 and CIS mRNA in liver, muscle, and fat, whereas SOCS-3 expression was unchanged. Using primary cultures of rat hepatocytes, GH was shown to increase SOCS-2, SOCS-3, and CIS mRNA levels with different kinetics. SOCS-3 was rapidly and transiently induced, whereas SOCS-2 and CIS were increased in a slower fashion. Glucocorticoids blocked GH-induced SOCS-3 expression in cultured hepatocytes, whereas SOCS-2 and CIS expression was potentiated. Our data fit well with a concept of SOCS proteins acting as modulators of GH signal transduction.

Thioredoxin is a major cellular dithiol reductant with a large number of functions in electron transport and thiol redox control of enzymes and transcription factors. To investigate the expression and regulation of thioredoxin in the uterus, 35 rats were ovariectomized (OVX) and treated 14 days after surgery with either growth hormone (GH), dexamethasone (DEX), or estradiol (E2), or combinations of these, for 24 h. Thioredoxin mRNA levels were determined by solution hybridization. The animals receiving E2 or a combination of E2 and DEX showed significantly increased thioredoxin mRNA levels, by 4-fold and 5-fold, respectively, as compared to the OVX control group. The GH or GH+DEX-treated groups did not display any difference in thioredoxin mRNA levels. Thioredoxin mRNA was also measured at different time points in uteri from OVX rats treated with daily E2 injections and was found to be transiently increased, with a maximum 48 h after the initiation of the E2 treatment. In contrast, the thioredoxin mRNA level in the liver of OVX rats was about 10-fold higher than in the uterus but remained unaffected by the different hormone treatments. We conclude that thioredoxin mRNA is expressed in the rat uterus, and up-regulated by E2 in a tissue-specific manner, with a maximum at 48 h after the initiation of hormone treatment.

Hepatic estrogen receptors (ER) mediate estrogenic effects on mammalian liver metabolism and are thereby involved in the regulation of important physiological/pathological processes, such as coagulation, atherosclerosis, and hypertension. The regulation of the formation of the ER in primary cultures of rat hepatocytes was studied by assaying ER and ER mRNA under different endocrine conditions. The ER concentration was measured using two different methods, a ligand-binding technique and an ER enzyme immunoassay. The results obtained by the two methods showed good correlation, and linear regression analysis gave a correlation coefficient of 0.95. ER concentrations fell to low steady state levels within 16 h after establishing the cell culture and remained low in the absence of hormonal substitution. Upon medium supplementation with pituitary GH and the glucocorticoid dexamethasone (DEX) in combination, the ER concentration increased 6-fold from 4.2 +/- 1.0 to 25.8 +/- 7.0 fmol/mg cytosolic protein. ER mRNA was measured by solution hybridization. Substitution with GH and DEX in combination increased ER mRNA to 210 +/- 14% of control levels. No effect on ER mRNA stability was seen after hormone treatment. It is concluded that the regulatory effects of GH and DEX on the hepatic ER in this in vitro system are very similar to the effects of these hormones under in vivo conditions. The inducible expression of the ER has never before, to our knowledge, been demonstrated in any mammalian liver cell culture system.