The role of aromatase inhibitors in ameliorating deleterious effects of ovarian stimulation on outcome of infertility treatment

Clinical utilization of ovulation stimulation to facilitate the ability of a couple to conceive has not only provided a valuable therapeutic approach, but has also yielded extensive information on the physiology of ovarian follicular recruitment, endometrial receptivity and early embryo competency. One of the consequences of the use of fertility enhancing agents for ovarian stimulation has been the creation of a hyperestrogenic state, which may influence each of these parameters. Use of aromatase inhibitors reduces hyperestrogenism inevitably attained during ovarian stimulation. In addition, the adjunct use of aromatase inhibitors during ovarian stimulation reduces amount of gonadotropins required for optimum stimulation. The unique approach of reducing hyperestrogenism, as well as lowering amount of gonadotropins without affecting the number of mature ovarian follicles is an exciting strategy that could result in improvement in the treatment outcome by ameliorating the deleterious effects of the ovarian stimulation on follicular development, endometrial receptivity, as well as oocyte and embryo quality

Development of a non-steroidal aromatase inhibitor-based protocol for the control of ovarian function using a bovine model

Five studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The general hypothesis was that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function for the purposes of fixed-time artificial insemination and embryo production. The specific objectives were to determine the effect of route and vehicle, type of aromatase inhibitor, and duration of aromatase inhibitor treatment (short vs prolonged) on ovarian follicles in cattle, and to test the efficacy of an aromatase inhibitor-based protocol to synchronize ovulation in cattle. In the first experiment, heifers were treated with letrozole intravenously (n=10) or intramuscularly (n=10) or allocated in iv and im control groups (n=5/group). During the second experiment, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The third experiment was designed with the goal of formulating and testing an intravaginal device that provides biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days. The biological significance of the pharmacokinetic differences between the letrozole intravaginal devices resulting from the third study was evaluated during the fourth study. A final study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based ovulation-synchronization protocol and to determine subsequent pregnancy outcomes. In all the studies, the effects of aromatase inhibitor on ovarian function were assessed by transrectal ultrasound examination of the ovaries, and blood samples were collected for hormone concentration determination. Results demonstrated that route of administration, or more precisely, the nature of iii the vehicle used for the administration of letrozole (intravenous, intramuscular depot, short release intravaginal or prolonged release intravaginal) has an impact on the effects of letrozole on hormonal profiles and ovarian dynamics. The intramuscular route appeared to provide a prolonged release of letrozole from the injection site which had a marked effect on estradiol production, dominant follicle lifespan, and CL form and function. Letrozole treatment during the ovulatory follicle wave by means of a gel-based intravaginal releasing device during the second study resulted in more rapidly growing dominant follicles and larger ovulatory follicles, delayed ovulation (by 24 h) of a single follicle and formation of a CL that secreted higher levels of progesterone. A wax-based vehicle allowed for a steady and continuous delivery of the active compound over the treatment period. During the third study, the addition of a letrozole-containing gel coating increased the rate of initial absorption and hastened the increase on plasma concentrations of the active ingredient, while the letrozole-containing wax-based vehicle prolonged drug-delivery from the intravaginal device. When tested in vivo during the fourth study, we confirmed that letrozole-impregnated intravaginal devices formulated with a wax base plus a gel coat vehicle was most suitable for the application of a letrozole-based protocol for the synchronization of ovulation in cattle, since it effectively delivered elevated concentrations of letrozole, and reduced estradiol production resulting in increased follicular growth and lifespan, without adversely affecting progesterone production. The application of a letrozole-impregnated intravaginal device for 4 days, combined with PGF treatment at device removal and GnRH 24 h post-device removal increased the percentage of ovulations and synchrony of ovulation in cattle, regardless the stage of the estrous cycle at initiation of treatment. As observed in previous studies, the effects observed could be associated with an increase in circulating LH iv concentrations. However, the effects of treatment on gonadotropin concentrations are inconclusive, possibly due to inadequate sampling frequency. The impact of letrozole treatment of oocyte fertility remains unknown. The results of the five experiments support our general hypothesis that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function in cattle. However, further research is needed in order to elucidate the effects of letrozole treatment during the proestrous on oocyte competence and fertility of the resulting ovulations in cattle

The control and manipulation of angiogenesis in the primate ovarian follicle

The ovary is one of the most plastic tissues in the body undergoing constant serial remodelling throughout its reproductive lifetime, during both folliculogenesis and the formation and regression of the corpus luteum. The process of follicle growth and selection is intimately associated with the de novo establishment of vasculature supporting the developing follicles. Blood vessels are recruited from the ovarian stroma to form vascular sheaths surrounding each developing follicle supplying steroid hormone precursors, oxygen and nutrients to the expanding follicle. During folliculogenesis only the theca of the developing follicle becomes vascularised, the granulosa cells remaining avascular until ovulation at which point the basement membrane that has been separating the granulosa and theca breaks down. After ovulation the granulosa cells become heavily vascularised during the process of luteinisation and the formation of the corpus luteum. Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, requires the degradation of the established vessels followed by endothelial cell proliferation and finally stabilisation of the new vessels. Recently, techniques to quantify angiogenesis, identify putative molecular regulators, and inhibit them in vivo have become available. The work in this thesis applies these advances to the following questions:1) What is the effect of the inhibition of the gonadotrophins, using a gonadotrophin releasing hormone (GnRH) antagonist, on follicular angiogenesis? The hypothesis being tested was that follicular angiogenesis would be dependent on follicle stimulating hormone (FSH) / luteinising hormone (LH) and be severely inhibited by GnRH antagonist treatment. In vivo follicular angiogenesis was assessed by quantitative immunocytochemistry of bromodeoxyuridine and the endothelial cell marker CD31. The effect of treatment on follicular development and angiogenesis at the molecular level was assessed by in situ hybridisation of mRNA for vascular endothelial growth factor (VEGF) and aromatase. The results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control, it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle. The second chapter extends the findings by determining granulosa cell response to FSH stimulation with respect to induction of the VEGF and aromatase genes.2) What is the effect of inhibition of vascular endothelial growth factor, using the antagonist, VEGF trap R1R2, on follicular angiogenesis, follicular development, ovulation and the establishment of the corpus luteum (CL)? The hypothesis being tested was that VEGF is essential for increasing permeability and the growth of the selected follicles. The immunocytochemical techniques used in the first study were again employed. The effect of treatment on the molecular regulation of angiogenesis was assessed by in situ hybridisation of mRNA for VEGF and its two receptors. In vivo inhibition of VEGF caused dramatic reductions in angiogenesis and in VEGF receptor expression but did not reliably prevent dominant follicle growth or ovulation once dominant follicle selection had occurred.3) Is a novel factor, endocrine gland vascular endothelial growth factor (EGVEGF) expressed in our animal model? The hypothesis being tested was that EGVEGF is an additional angiogenic factor that is expressed in the marmoset and the human ovary. This was assessed by in situ hybridisation in various marmoset tissues as well as in the human corpus luteum. Findings demonstrated that EG-VEGF is expressed in the granulosa lutein cells in the human corpus luteum while the marmoset ovary does not appear to express EG-VEGF.This thesis has improved our understanding of the gonadotrophic control of follicular angiogenesis and the role VEGF plays in the latter stages of folliculogenesis

Stress-resilience differences related to emergence time in rainbow trout

In wild salmonid fish, individual behavioural traits have been suggested to be coupled with the timing of fry emergence form gravel spawning nests, in such a way that early emerging fish have shown to be more aggressive and to have a higher probability to become socially dominant than those fish emerging at a later stage. Besides aggression and dominance, other behavioural and metabolic traits such as boldness, metabolic rate or growth had also been coupled to emergence time. Altogether, early- and late-emerging fish have traits resembling those of proactive and reactive stress coping styles, respectively. Proactive fish are considered to be more resilient to stress. However, it is currently unclear if that coupling is maintained in farmed fish populations, which showed no consistent evidence of a clear relation between emergence time and stress coping style. In this study, fish were hatched and larvae were fractionated according their emergence time (Early fraction: first 20 % of fish to emerge; Intermediate fraction: mid 20 %; Late fraction: last 20 %). Several months later, the resilience against a mild stressor (30 min of high stocking density), along with the stress habituation ability was investigated in 5 g fish from the different fractions. Results showed that fish from different fractions di played a similar neuroendocrine response to a novel stressor. Interestingly, the capacity of habituation to stress was however better in the fish from the early emergence fraction, which showed no cortisol response to the stressor after being exposed daily for 15 days to another mild acute stressor (1.5 min of air exposure). These results demonstrate that at least some behavioural differences related to emergence time exist in a domesticated trout population. The aquaculture related implications of these stress resilience differences are currently under study

Unravelling The Role Of Androgens In Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is a multifaceted hormonal disorder which affects 5-15% of reproductive-aged women worldwide. While classically recognised as an ovarian disorder, PCOS is associated with a variety of reproductive, endocrine and metabolic features including ovulatory dysfunction, infertility, hyperandrogenism, obesity, and an increased risk of type 2 diabetes mellitus and cardiovascular disease. The most consistently present of these is hyperandrogenism – supraphysiological levels of androgens such as testosterone (T) and dihydrotestosterone (DHT). Typically thought of as male steroid hormones, androgens have been shown to play important role in the maintenance of normal female reproductive function. Despite the high prevalence of hyperandrogenism amongst patients, the role of androgens in the etiology and pathogenesis of PCOS has yet to be determined. The aim of this work was to unravel the association between excess androgen exposure and the development and advancement of the PCOS phenotype in mouse model of androgen-induced PCOS. The first study contained within this work (Chapter Three) provides the first comprehensive characterization of a range of reproductive, endocrine and metabolic traits associated with PCOS in four distinct mouse models: a model of prenatal androgenisation utilising the potent non-aromatizable androgen DHT administered during days 16-18 of gestation, and three diverse models of postnatal androgen exposure employing a long-term treatment with either DHT, the proandrogen dehydroepiandrosterone (DHEA), or letrozole (an aromatase inhibitor) for 90 days beginning at 3 weeks of age. Prenatal androgenisation produced some reproductive and endocrine traits, but failed to induce the metabolic abnormalities seen in PCOS. DHEA treatment did not reproduce any features associated with PCOS while treatment with letrozole produced few PCOS-like characteristics and some aberrant changes not typical of the syndrome. Additionally, letrozole treatment did not reproduce any metabolic attributes of PCOS. On the other hand, postnatal exposure to excess androgen, by way of DHT treatment, produced a breadth of reproductive, endocrine and metabolic traits that mimic those seen in human PCOS. This study revealed that a treatment regime of long-term postnatal exposure to DHT reproduced the strongest PCOS-like phenotype in our mice and provides a robust animal model in which to study the pathogenesis of PCOS. The second study (Chapter Four) aimed to explore the involvement of genomic androgen receptor (AR)-mediated actions in the development of these PCOS traits. As a prenatally androgenised mouse model of PCOS had previously been reported to exhibit impaired neuroendocrine hypothalamic feedback of the hypothalamic-pituitary-gonadal (HPG) axis, we took this opportunity to utilise mice from our own prenatal model to investigate the neuroendocrine regulation of the HPG axis in PCOS in addition to the effects of AR inactivation on the PCOS phenotype. PCOS was induced in wild-type (WT) and androgen receptor knockout (ARKO) mice using DHT administered on days 16-18 of gestation. A subset of these mice were also exposed to 17β-estradiol for 7 days prior to collection, via a subdermal implant, to investigate the impaired estradiol negative feedback on the hypothalamus. WT mice with DHT-induced PCOS displayed several reproductive abnormalities including aberrant cycling and ovulatory dysfunction in addition to adipocyte hypertrophy and hepatic steatosis. However, diestrus serum luteinising hormone and follicle stimulating hormone, and estradiol-induced negative feedback as well as hypothalamic expression of several neuropeptides were unaffected by DHT treatment in WT mice. Mice both homozygous and heterozygous for the global inactivation of the AR (ARKO), did not display any PCOS traits when exposed to excess androgens during prenatal life. This study showed the importance of AR signalling in the development of PCOS and revealed that even AR haplosufficiency is adequate to prevent induction of PCOS by prenatal hyperandrogenism. Finally, the third study (Chapter Five) aimed to shed further light on the AR-mediated androgen actions in PCOS with a focus on identifying the tissue-specific targets of these actions. Employing our postnatal model of PCOS induction, this study investigates the effects of: 1) global loss of AR signalling (ARKO), 2) neuronal knockout (NeurARKO) and 3) granulosa cell-specific AR inactivation (GCARKO) on the development of the PCOS phenotype induced by exposure to exogenous DHT. As in our prenatal model, ARKO mice were fully protected from all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of a variety of reproductive and metabolic traits including classic polycystic ovaries, dysfunctional ovulation, obesity and dyslipidemia. In contrast, loss of AR signalling in granulosa cells did not impede the pathogenesis of PCOS-like features in GCARKO mice. To further examine the role of extra-ovarian AR signalling in PCOS, reciprocal ovary transplants were carried out in WT and ARKO mice. Results from ARKO hosts with transplanted WT ovaries revealed that excess androgen exposure requires functional extra-ovarian, and not intra-ovarian, AR signalling in order to produce features of PCOS. This study provides strong evidence that neuroendocrine genomic AR signaling is an important mediator in the development of PCOS. The studies contained within this thesis are the first to provide a comprehensive analysis of a mouse model of PCOS encompassing a breadth of reproductive, endocrine and metabolic features. This work has identified the optimal model in which to study this complex, multifactorial condition which affects a significant number of women worldwide. Additionally, our results have shown that the effects of androgens on the pathogenesis of PCOS are mediated via the androgen receptor in a dose-dependent manner such that two functional copies are required for DHT to reproduce features of PCOS in the mouse. Finally, in a crucial study to investigate the locus of androgen actions we have revealed the previously overlooked importance of extra-ovarian neuroendocrine androgen action in the origins and progression of PCOS, despite it being thought of primarily as an ovarian disorder. Overall, these studies have provided valuable insights into both the role of androgens in Polycystic Ovary Syndrome and potential new targets for the development of mechanism-based treatments of this disorder

Oocyte morphology and estrogen concentrations following a reduction in progesterone in beef cattle

Low dosages of progestogens promote persistent follicles, high systemic estrogen and low fertility. The objectives of this study were to determine effects of a reduction in progesterone on (1) morphology of oocytes and intrafollicular concentrations of estradiol. Cows on low progesterone (n = 12) received used intravaginal progesterone inserts on d 4 after estrus and prostaglandin (PG) F2alpha (25 mg, i.m.) on d 6. Control animals (n = 12) received saline on d 6. The oocyte and follicular fluid were recovered from the largest follicle on d 8 or d 10.;Serum estradiol was lower during d 4--6 but greater (P \u3c .01) during d 7--10 in cows treated with progesterone inserts and PGF 2alpha while the largest follicle was larger in treated cows on day 10 only (14 vs. 12 mm; P \u3c .05). Intrafollicular concentrations of estrogen were greater in treated than in control cows (990 +/- 87 vs 191 +/- 106; P \u3c .01). Progesterone in follicular fluid (mean = 42 ng/ml) did not differ. Oocytes were observed in oocyte nuclear stage I in the control group on d 8. All other oocytes were in nuclear stage II. In addition, the degree of clumping of mitochondria, the percentage of intact cumulus cell processes and percentage of normally shaped mitochondria was greater in oocytes from d 8 control cows than in all other groups.;Changes in concentrations of estradiol and oocyte morphology typically associated with the preovulatory period had occurred within 2 d after a reduction in progesterone, even when low peripheral concentrations of progesterone were maintained. These earliest stages of oocyte maturation occurred in response to a reduction in progesterone. Similar changes in oocyte morphology were observed in control animals by d 10 of the estrous cycle, probably representing the onset of atresia

Hydroxysteroid (17beta) dehydrogenase 1 expression enhances estrogen action and serves as a potential drug target for reducing estrogen signaling in the uterus and mammary glands

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is a steroidmetabolizing enzyme with a preface for converting the low active estrogen estrone (E1) to highly active estradiol (E2). Accordingly, HSD17B1 is expressed particularly in E2-producing tissues such as the human placenta and ovaries. Moreover, HSD17B1 is expressed in peripheral estrogen target tissues such as the breast and uterus, where it controls local intratissue E2 concentration. Exposure to elevated concentrations of estrogens is associated with increased risk of several diseases, including breast cancer, which is the most common cancer in women worldwide. To investigate the significance of HSD17B1 in steroid production in vivo and in estrogen-dependent diseases, transgenic mice universally expressing human HSD17B1 enzyme (HSD17B1TG mice) were used. These mice showed increased peripheral conversion of E1 to E2 in a variety of tissues, including the uterus and mammary glands. Female HSD17B1TG mice developed endometrial hyperplasia, a precursor to endometrial carcinoma. The hyperplasia was reversed by ovulation induction, while initially the HSD17B1TG females failed to ovulate. Treatment with an HSD17B1 inhibitor also partly reversed the hyperplastic morphology. The HSD17B1TG females developed mammary cancer at older age. Mammary gland epithelial restricted HSD17B1 expression formed mammary lesions with a disrupted myoepithelial cell layer and inflammatory cell infiltration that was reduced by blocking estrogen receptor signaling with an antiestrogen, ICI 182, 780. The HSD17B1TG mice were also successfully used as a preclinical model for screening the efficacy of HSD17B1 inhibitors in vivo by crossing HSD17B1TG mice with estrogen reporter mice (ERELuc mice). In these bi-TG mice, both an immature uterus growth response and estrogen receptor activity were used as readouts, and both were reduced by HSD17B1 inhibitor treatment. These studies elucidate the potential of HSD17B1 enzyme to enhance the action of E1 in peripheral tissues, thus, indicating that inhibition of HSD17B1 is a plausible approach to treating estrogen-dependent diseases.Hydroksisteroidi (17beta) dehydrogenaasi 1 ilmentyminen lisää estrogeeniaktivaatiota ja toimii potentiaalisena lääkekohteena vähentämällä estrogeenisignalointia kohdussa ja maitorauhasissa Hydroksisteroidi (17beta) dehydrogenaasi tyyppi 1 (HSD17B1) on steroideja metaboloiva entsyymi, joka pääasiassa muuttaa vähemmän aktiivista estrogeeniä, estronia (E1), biologisesti aktiivisemmaksi estradioliksi (E2). Ihmisillä HSD17B1 ilmentyy erityisesti estrogeenejä tuottavissa kudoksissa kuten ihmisen istukassa ja munasarjoissa, joissa se osallistuu estradiolin tuotantoon. Lisäksi HSD17B1 ilmentyy perifeerisissä estrogeenin kohdekudoksissa, kuten rinnassa ja kohdussa, joissa se säätelee paikallista kudoksen sisäistä E2-konsentraatiota. Estrogeenien kohonnut altistus on liitetty useiden sairauksien kohonneeseen riskiin kuten rintasyöpään, joka on naisten yleisin syöpä maailmanlaajuisesti. HSD17B1-entsyymin merkityksen tutkimiseksi käytettiin muuntogeenisiä hiiriä, jotka tuottavat ihmisen HSD17B1-entsyymiä (HSD17B1TG-hiiret). Näillä hiirillä on lisääntynyt perifeerinen E1:n muuntuminen E2:ksi useissa kudoksissa, kuten kohdussa ja maitorauhasessa. HSD17B1TG-naarashiirille kehittyi kohdun limakalvon liikakasvua, joka on kohtusyövän esiaste. Lisäksi HSD17B1TG-hiiret eivät ovuloineet ja indusoimalla ovulaatio myös kohdun limakalvon liikakasvu korjautui. HSD17B1-inhibiittorihoito lievensi kohdun limakalvon liikakasvua. HSD17B1TG-naaraille kehittyi myös vanhemmalla iällä maitorauhassyöpiä. Maitorauhaseen rajattu HSD17B1:n ilmentyminen aiheutti maitorauhasleesioita, joissa epiteelisolujen proliferaatio oli lisääntynyt, tiehyiden myoepiteelikerros oli hajonnut ja niihin oli kertynyt inflammatorisia soluja. ICI 182,780, antiestrogeenihoito, vähensi leesioiden määrää. HSD17B1-inhibiittoreiden testaamiseksi in vivo kehitettiin uudenlainen hiirimalli, jossa HSD17B1TG-hiiret risteytettiin estrogeeni-reseptorin aktivoitumista raportoivaan hiireen (ERELuchiiret). Näissä kaksoissiirtogeenisissä hiirissä esipubertaalisen kohdun kasvua ja lusiferaasiaktiivisuutta käytettiin vasteena ja HSD17B1-inhibiittorihoitolla nämä vasteet vähenivät. Nämä tutkimukset vahvistavat, että HSD17B1-entsyymin ylituotto lisää E1:n vaikutusta perifeerisissä kudoksissa, joten HSD17B1:n esto voisi olla uudenlainen lähestymistapa kohtu- ja rintasyövän hoidossa