【目的】从整体上把握多囊卵巢综合征的发生、发展及其规律, 为临床进一步诊断治疗提供整体思路和文献学基础。【方法】收集整理近10 年与多囊卵巢综合征研究相关的中、英文献，归纳和总结该病的病因病机、诊断、治疗，找出存在问题，提出发展方向。【结果】①多囊卵巢综合征主要病因为：肾虚、脾虚、肝郁、痰湿、痕血，并与饮食、环境、精神、遗传具有相尖性；②多囊卵巢综合征基本病机：本虚标实，肾虚、脾虚为本’肝郁、气滞、血痕、痰浊、痰湿为标；③PCOS 的分型大致有：肾虚血脐型、肾虚血痕痰浊型；脾肾阳虚型；痕血内阻型；肝气郁结型；脾肾阳虚型；痰湿阻滞型；④PCOS 的治疗以辨证分型论治为主，参考名老中医经验、专方专药、针灸、周期疗法、辨体质进行诊治，重视中西医结合在治疗中的应用。⑤PCOS 仍存在病因、病机认识不一，诊断存在争议’辨证分型众说纷纭等问题。⑥PCOS 发展方向：找出与PCOS 相尖性最强的病因，明确中西医发病机制，规范PCOS 诊断’研发有效药物，加强临床及实验研究，注重基础治疗和健康宣敦。【结论】多囊卵巢综合征的病因、病机、诊断及治疗研究已取得可喜的成绩，形成了完整的体系。但仍存在具体病因不明’发病机制不清，缺乏有效治疗方法等问题。今后应加强高级别基础和临床研究’尽快明确病因病机’找出有效治疗方案。关键词：多囊卵巢综合征；中医；病因病机；证治规律；药物

目的： PCOS 是女性最常见的内分泌疾病, 其症状与并发症严重危害了女性的身心健康。本研究通过收集整体近10 年关于PCOS 的中医临床研究类文献，对本病的证型、治法，尤其是用药处方进行统计、归纳、分析’从而总结出该病的病因病机分布及治疗规律，为PCOS 下一步的临床治疗及用药提供思路和依据。方法：以“多囊卵巢综合征”、“PCOS”、“多囊卵巢”、“不孕症”、“闭经”“中医” 、“中医治疗”、“中医药”等为关键词’搜索2006 年1 月至2016 年12 月刊登在中文学术期刊上有关多囊卵巢综合征中医临床研究类文献，通过建立数据库，对其中医证型及使用方药进行统计分析’归纳总结出多囊卵巢综合征的主要病因病机及治疗用药规律。结果：经过筛选最终得到120 篇文献，共记录病例5670 例，明确治法4616 例，明确证型3778 例。肾虚证型2598 例（ 68. 8% ）；痰湿阻滞型613 例(16. 2%)占84.8% 侨肾3172 例（ 68. 7% ）补肾法中辅以活血化痰之法者也497 例（ 78. 7% ）。药物使用频共中’补虚药（ 43. 7% ）、活血化痕药（ 16.1% ），占59.8% 。归纳的两种疗效评判标准中，总有效率分别为87.9% 、88.9% 。结论： 1. 肾、肝、脾功能失调为PCOS 的发病之源。2. 肾虚、痰湿、血痕是PCOS 的基本病机。3. 补肾健脾，活血化痰是治疗PCOS 的根本大法。4. 补虚药、活血化寐药为治疗PCOS 的主导药物类别。5 中医药治疗PCOS 有副作用小基本黛痛苦璧幢调筒H p O 轴的功能。6. 不同年龄段治疗目的不同导致疗效评判标准尚不统一。关键词：多囊卵巢综合征，中医，用药规律，文献研究

Orientador: Carminda da Cruz Landim / Banca: Márcia Maria Gentile Bitondi / Banca: Flávio Henrique Caetano / Banca: Hélio Conte / Banca: Maria Izabel Souza Camargo / Resumo: Nas abelhas eussociais há duas castas femininas, as rainhas que são responsáveis pela produção dos indivíduos que mantêm a população da colônia e fazem sua multiplicação e as operárias que compartilham entre si todas as tarefas de manutenção da colônia e das próprias atividades da rainha. Na espécie estudada, Melipona quadrifasciata anthidioides, uma abelha sem ferrão, as operárias normalmente produzem ovos em seus ovários na fase em que se ocupam do aprovisionamento das células de cria (operárias nutridoras), diferentemente de Apis mellifera em que feromônios da rainha são capazes de inibir a vitelogênese nos ovários das operárias. As diferenças funcionais entre rainhas e operárias em ambas espécies são controladas hormonalmente. O corpo gorduroso (CG) é um tecido constituído basicamente por um único tipo de célula designado trofócito, cuja função se assemelha em vários aspectos à dos hepatócitos. Os trofócitos ao mesmo tempo em que retiram substâncias da hemolinfa e as armazenam ou metabolizam, sintetizam outras que nela descarregam. Entre estas últimas está a vitelogenina, uma proteína precursora do vitelo acumulado pelos ovócitos durante a vitelogênese, como reserva para o futuro desenvolvimento do embrião. Neste trabalho a citoquímica dos trofócitos de rainhas virgens, fisogástricas e de operárias nutridoras foi estudada com microscopia eletrônica de transmissão (MET) e comparadas com a morfologia do desenvolvimento dos ovócitos no ovário com a finalidade de comparar a atividade dos trofócitos com a absorção de substâncias da hemolinfa pelo ovário. Em outra vertente a presença da vitelogenina foi pesquisada no CG e no ovário de ambas as castas, na tentativa de verificar se havia concordância quanto à produção da proteína nos trofócitos e sua presença nos ovários... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Eussocial bees have two female castes, the queens that are responsible for producing individuals that will keep the colony population and workers that share among them the tasks of colony maintenance and of the queen care. In the studied species Melipona quadrifasciata anthidioides, a stingless bee, workers normally produce eggs in their ovaries when they are provisioning the brood cells (nurse workers), differently from Apis mellifera, where pheromones from the queen are able of prevent the vitellogenesis in the workers ovaries. The functional differences between queens and workers are, in both castes controlled by hormones. The fat body (FB) tissue is constituted basically by a single kind of cell designated throphocyte whose function is similar in several aspects to the hepatocytes. The throphocytes at the same time that take substances from the hemolymph and storage or metabolize them also synthesize others that are discharged in the body fluid. Among these is the vitellogenin, a precursor protein of the yolk accumulated by oocytes during the vitellogenesis as a reserve for further development of the embryo. In this work the cytochemistry of trophocytes of virgin and physogastric queens and nurse workers was studied with transmission electron microscopy (TEM) and compared to the morphology of oocytes development in the ovary with the purpose of correlate morphologically the activity of trophocytes to the absorption of substances from hemolymph by the ovaries. The presence of vitellogenin was searched in the FB and ovaries extracts of both castes by electrophoresis, as an attempt to see if there was an agreement between production of the protein in the trophocytes and their presence in the ovaries. Finally, taking into account the action of the morphogenetics hormones over the trophocytes, these cells of both castes were incubated into medium containing juvenile hormones III... (Complete abstract, click electronic address below) / Doutor

L'ovaire représente à la fois un organe cible et le principal organe producteur d'estrogènes et de progestérone qui maintiennent le développement des caractères sexuels féminins et une fonction de reproduction normale. Cette production hormonale est contrôlée par les gonadotropines FSH et LH produites dans l'hypophyse, responsables dans l'ovaire de la croissance folliculaire et de l'ovulation, respectivement. Mon travail de thèse a identifié la signalisation prostaglandine D2 (PGD2), comme un nouvel élément-clé dans la signalisation des gonadotropines, contribuant à l'activation de l'expression des récepteurs FshR et LhR et des enzymes de la stéroïdogenèse SCC et StAR. La PGD2, produite dans plusieurs tissus par deux enzymes de synthèse, les prostaglandines synthases H et L-PGDS, est impliquée dans de nombreuses fonctions physiologiques et pathologiques. Comme dans l'ovaire pathologique, nous avons montré que la PGD2 avait aussi un rôle anti-prolifératif dans la cellule de granulosa de l'ovaire normal. Le cancer de l'ovaire représente la 4ème cause de mortalité par cancer chez la femme. Les mécanismes moléculaires impliqués dans le développement de ces tumeurs sont encore peu connus, bien que l'implication des estrogènes et de la Prostaglandine E2 (PGE2) dans la progression des tumeurs ovariennes épithéliales soit bien établie. D'autre part, les ovaires des souris invalidées pour les gènes codant les récepteurs aux estrogènes ou l'aromatase, possèdent des structures tubulaires contenant des cellules de Leydig et des cellules de Sertoli re-différenciées exprimant le facteur de détermination sexuelle mâle SOX9, alors qu'il n'est pas exprimé dans l'ovaire sain. Mon travail a montré que les estrogènes inhibent la transcription des gènes Sox9 et L-Pgds dans les lignées ovariennes tumorales BG1 et COV434 et que cette régulation est la résultante d'une inhibition, via le récepteur ERa et d'une activation via le récepteur ERß. Ces résultats sont en accord avec les études sur les effets prolifératifs d'ERa et le rôle anti-prolifératif d'ERß et suggèrent donc un rôle anti-prolifératif de la PGD2 dans l'ovaire tumoral et une régulation négative directe ou indirecte de l'expression de Sox9 et des Pgds par les estrogènes. / The prostaglandin D2 (PGD2) pathway is involved in numerous biological processes and while it has been identified as a partner of the embryonic sex determining male cascade, the roles it plays in ovarian function remain largely unknown. PGD2 is secreted by two prostaglandin D synthases (Pgds); the male-specific lipocalin (L)-Pgds and the hematopoietic (H)-Pgds. Here, we report the localization of H-Pgds mRNA in the granulosa cells from the primary to pre-ovulatory follicles. We used adult female mice treated with HQL-79, a specific inhibitor of H-Pgds enzymatic activity, to provide evidence of an interaction between H-Pgds-produced PGD2 signaling and FSH signaling. This leads to the activation of steroidogenic Scc and StAR gene expression through increased FshR and LhR receptor expression leading to progesterone secretion. We also identify a role whereby H-Pgds-produced PGD2 is involved in the regulation of follicular growth through inhibition of granulosa cell proliferation in the growing follicles. Indeed, we report an altered H-Pgds expression in human ovarian tumors alongside a partial or complete absence of H-Pgds protein in granulosa cell tumors, suggesting a potential association between decreased levels of H-Pgds expression and a tumoral phenotype. Together, these results show PGD2 signaling to be essential for FSH action within granulosa cells, thus identifying an important and unappreciated role for PGD2 signaling in controlling the balance of proliferation, differentiation and steroidogenic activity of these cells.

Class of 2008 Abstract / Objectives: To assess the effect of metformin usage and lifestyle modifications in women with polycystic ovary syndrome (PCOS) in achieving conception and live birth. Methods: A retrospective chart review of patients at a southwest reproductive health center was performed. Patients given a diagnosis of PCOS, treated with metformin alone, with at least 12 weeks of outcome data were enrolled. Diagnostic, reproductive history, and baseline endocrine and metabolic data were collected. All available metformin use, menstrual cyclycity, ovulation, pregnancy, pregnancy outcome, and alternative treatment data were captured. Results: A total of 1250 charts were reviewed and 103 patients were enrolled. Pre-treatment, a significant relationship between BMI and HDL, triglycerides/HDL, and fasting glucose (P <0.001, 0.018, 0.016) was noted with leaner patients having better metabolic profiles. The pregnancy, miscarriage, and live birth rates with metformin treatment were 55.3 %, 18.2 %, and 35.0 % respectively. Patients (40/103) that did not conceive with metformin attempted alternative fertility treatment; 55% became pregnant and 30% had a live birth. One third of all patients experienced minor adverse events, primarily gastrointestinal in nature. Logistic regression analyses comparing responders to nonresponders did not identify any baseline patient characteristics useful as significant predictors of success with metformin treatment. Conclusions: For the population under study, metformin use and lifestyle modifications resulted in conception and live birth for as many as 35 % of patients. Contrary to recent publications, it appears that this method of fertility treatment remains a viable option to treat infertility in patients with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome is the most common endocrine disorder affecting reproductive-aged women. It is diagnosed using a combination of menstrual irregularity, clinical and/or biochemical hyperandrogenism and polycystic ovary morphology upon ultrasound. Hyperandrogenism in females may clinically manifest as hirsutism, acne, alopecia, or other masculinization of features. Assessing total/free testosterone, dehydroepiandrosterone sulfate, and 17-hydroxyprogesterone provides biochemical evidence of hyperandrogenism. OBJECTIVE: To determine self-reported clinical signs of androgen excess using data from the Ovulation and Menstruation Health (OM) Study, a diverse, multi-ethnic cohort study being conducted at Boston University School of Medicine. METHODS: Data was collected from participants enrolled in the Ovulation and Menstruation Health Study pilot cohort. This epidemiologic survey captured demographics, menstrual cycle patterns, PCOS histories, reproductive histories and manifestations of androgen excess in a diverse patient population. Participants were women ages 18-45 who had the capacity to ovulate/menstruate at the time of the study, had no history of chemotherapy, radiation, or surgical menopause, and were not pregnant at the time of the study. To assess androgen excess, participants were asked to self-report hair growth in nine body areas, acne on the face and back and hair loss on the scalp. The nine body areas were scored using the modified Ferriman-Gallwey (mFG) scoring system. Reference images created by a medical illustrator were used for hirsutism and alopecia grading while clear descriptions were provided for grading acne severity. Clinical hirsutism was defined as total mFG score of ≥ 8, or ethnic specific cutoff for East Asian (≥ 2) and Southeast Asian (≥ 3) women. Alopecia was defined as scalp hair loss ≥ 2. For participants that consented to medical record validation total, free and bioavailable testosterone lab levels were assessed for biochemical hyperandrogenism evaluation. RESULTS: Beginning August 9, the day the study opened to the public, 249 participants completed the pilot survey questionnaire. These participants were 66.8% white (n=165), 6.5% Hispanic or Spanish origin (n=16), 10.5% Black or African-American (n=26), 1.6% East Asian (n=4), 2.0% Southeast Asian (n=5), 2.4% South Asian (n=6), and 10.9% were of mixed ethnic backgrounds (n=27). 22.5% (55/245) of these women had clinical hirsutism by total mFG score. Mean total mFG scores were highest in women who were South Asian at 13.8±9.1 (n=6) and Hispanic at 8.6±8.7 (n=16). Moderate-severe acne was reported in 23.6% (58/246) of respondents, 24.8% (41/165) of white women, 26.7% (4/15) of Hispanic women, 15.4% (4/26) of Black women, 0.0% (0/4) of East Asian women, 20.0% (1/5) of Southeast Asian women, 50% of South Asian women (3/6) and 20% (5/25) of women of mixed ethnicities. 9.4% (23/246) of all pilot women reported alopecia, highest in Black (26.9%, 7/26) and East Asian women (25%, 1/4). Among women that had a PCOS diagnosis there was a higher presence of clinical hirsutism, higher acne severity, and higher prevalence of alopecia when compared to non-PCOS women. In addition, 33%(4/12) of the 44 women that consented to medical record validation had total testosterone levels above the normal range. CONCLUSIONS: This pilot population demonstrated an ethnic dependent pattern of development for hirsutism, acne and alopecia. Additionally, women who had a PCOS diagnosis were more likely to report having the clinical signs of androgen excess than those without a diagnosis. / 2020-06-14T00:00:00Z

Blastocyst implantation is the process in which a competent blastocyst acquires the ability to tether into the mother endometrium. At the same time, the endometrial tissue undergoes the process of decidualization to support the anchoring of the blastocyst and provides the blastocyst with nutrition until the fully functional placenta is formed. Although the process of implantation and decidualization are under control of progesterone and estrogen, the precise mechanisms involved in this regulation are not fully understood. Here, we report the expression and function of a transcription factor, HAND2, in sensitizing mouse uterus for implantation and decidualization. In mouse, HAND2 expression was localized mainly in the endometrial stromal cells even before the blastocyst implantation. The expression of HAND2 increased after blastocyst implantation and correlated with the increase in decidual compartment. The expression of HAND2 depended on progesterone but not estrogen. Further investigation using conditional knockout mouse revealed that HAND2 was important for both implantation and decidualization. Hand2d/d mice were infertile and had defects in decidualization. It seemed that HAND2 was an important factor that mediates the anti-estrogenic effect of progesterone on luminal epithelial proliferation. The abnormal in expression of Mucin 1, Calcitonin and E-Cadherin in Hand KO uterus may be responsible for defects in the uterine receptivity. The expression of HAND2 was also critical in decidualization in vitro. Silencing and over-expression HAND2 disclosed the roles of HAND2 in regulating the expression of FOXO1A, IGFBP1, BMP2 as well as WNT4. It seemed that HAND2 promoter worked in tissue specific manner and although both HOXA10 and cAMP binding sites were found in proposed HAND2 promoters, its activity was stimulated by cAMP and steroid hormones rather than the expression of HOXA10.

Bisphenol (BP) A is a common manufacturing chemical in polycarbonate plastics and has been widely used in plastics, epoxy resin liners of canned foods, dental materials, and thermal receipts. Human exposure to BPA is associated with a negative impact on human health including the development and function of the reproductive system due to its action as an endocrine-disrupting chemical (EDC). Numerous experimental studies have demonstrated that BPA impairs both male and female reproductive function, despite the variation in study paradigms such as dose, exposure route, timing, and outcomes measured. Due to the toxicological effects of BPA, BPA analogues such as BPS have been used as alternatives for BPA. However, recent evidence has suggested these BPA analogues can induce similar or even more severe toxic effects as BPA, and health risks of exposure to replacement bisphenols need to be considered. Therefore, my study was designed to examine whether prenatal exposure to BPE and BPS negatively impacts on male and female reproductive function in mice. Pregnant females were orally administrated corn oil (control), BPA, BPE, and BPS (0.5, 20, or 50mg/kg/day) from gestational day 11 (the presence of vaginal plug=1) to birth, and reproductive tissues in F1 mice were collected and analyzed in both neonatal and adult mice. In males, I observed reduced sperm counts and quality, disrupted stages of spermatogenesis in adults and increased germ cell apoptosis in neonatal testis following prenatal BPA, BPE or BPS exposure. Particularly, I found the expression of methyltransferases for DNA methylation and histone modification was also affected by prenatal exposure to BPA, BPE, or BPS in neonatal testis, suggesting a potential of epigenetic alterations in F1 males. In females, prenatal exposure to BPE and BPS accelerated the onset of puberty, disrupted estrous cyclicity, and caused several fertility problems especially in aged mice. In the neonatal ovaries, I also observed that BPE and BPS inhibit germ cell nest breakdown comparable to BPA. These results suggest that prenatal exposure to BPE and BPS with physiologically relevant doses affects male and female reproductive function probably due to germ cell development defects in the developing gonads. Finally, to understand their complete impact on male and female fertility, a study of transgenerational effects of BPE and BPS is performed to examine the transgenerational effects of prenatal exposure to BPA, BPE and BPS on reproductive function in F3 offspring. To be called transgenerational, expression of the specific phenotype will be continued at least across three generations. As described in previous studies, the direct exposure of a pregnant female (F0 generation) results in the exposure of the embryos (F1 generation) and the germline that will generate the next generation (F2 generation). Thus, I orally exposed to control treatment (corn oil), BPA, BPE or BPS (0.5 or 50 μg/kg/day) from gestational day 7 to birth in pregnant females (F0). Mice from F1 and F2 offspring were used to generate the F3 generation. In F3 males, prenatal exposure to BPA, BPE, and BPS induces persistence and even more severe phenotypes in sperm counts and motility in the F3 generation than in the F1 offspring. The expression of DNA and histone methyltransferases were transgenerationally increased by BPA, BPE and BPS exposure in both neonatal and adult testis. In F3 females, prenatal exposure to BPA, BPE, and BPS accelerated the onset of puberty and exhibited abnormal estrous cyclicity, and those females exhibited similar fertility problems as those in the F1 generation. However, BPA, BPE and BPS exposure did not affect neonatal follicular development such as germ cell nest breakdown or follicle numbers in the ovary on postnatal day 4. Taken together, our results suggest that prenatal exposure to BPA analogues, BPE and BPS, have transgenerational effects on male and female reproductive function in mice. Our findings suggest the hypothesis that transgenerational epigenetic alterations in germ cells may lead to reproductive disorders/dysfunction in the F3 generation.

IGF1 and its receptor IGF1R have been correlated with the proliferation of granulosa cells as well as steroid synthesis. Studies have shown that conditional ablation of Igf1r in granulosa cells leads to follicular arrest at a secondary stage, absence of ovulation and infertility. With a high homology between IGF1R and INSR, the full effects of insulin signaling could be masked by just a single receptor knockout. Therefore, utilizing Esr2-iCre we generated a granulosa specific double knockout mouse model. These mice have severely disrupted follicular development, with a block at a primary stage. Granulosa cells do not proliferate, while the oocytes appear activated resulting in reduction of ovarian size, absence of estrous cyclicity and infertility. Since an early granulosa cell knockout leads to block in follicular development, it masks the receptor function during ovulation, and CL formation. With the use of Pgr-Cre, the follicular development goes undisturbed until the periovulatory stages. Pgr-Cre knockout of Insr and Igf1r results in reduced ovulation, and progesterone synthesis. Few oocytes, that do escape, get fertilized but fail to thrive, and do not implant. Pgr-Cre is also active in the uterine endometrium. Ablation of Insr and Igf1r in the uterus results in reduced endometrial proliferation during the preimplantation period, complete absence of implantation and decidualization. Collectively, these results indicate the importance of INSR and IGF1R during follicular development, and ovulation, as well as in uterine proliferation, implantation, and decidualization.

The nonapeptide family of hormones is deeply conserved in evolution. In teleost fishes, as in all vertebrates, two nonapeptide families exist. These are vasotocin (avp) and oxytocin (oxt). While vasotocin has been shown to regulate individual aspects of reproductive physiology in several teleost species, an integrative assessment of its role on male and female reproduction is currently lacking even in widely used fish models, such as the zebrafish (Danio rerio). Taking advantage of the genetic tractability of the zebrafish, and its emerging status as model to study reproductive physiology, I generated avp -/- mutants using a CRISPR/Cas9 based approach to determine reproductive consequences in female and male zebrafish. Following the identification of a female-specific reproductive phenotype which manifests as a reduction in oocyte release and decreased quivering behaviour, I investigated the potential mechanistic basis at the level of the gonad. In avp -/- ovaries, significantly fewer eggs were present compared to WT fishes. When comparing the distribution of oocyte maturation stages, a significantly lower percentage of stage I and higher percentage of stage V oocytes was present in avp-/- ovaries. The altered distribution in oocyte maturation stages coincided with significant decreases in ovarian transcript abundance of nanos2, a germ-cell specific marker suggesting a possible role for Avp in germ-cell maintenance. Additionally, I observed a decrease in the ovarian concentration of the prostaglandin PGF2, which coincided with a reduction in ovarian transcript abundance of pla2g4ab, a paralogue of the phospholipase A2 involved in mobilizing arachidonic acid, a precursor of PGF2,. Together, these finding suggests a role for Avp in PGF2 -mediated ovulation. Because Avp has pleiotropic effects and may thus affect female reproductive physiology indirectly, we assessed somatic growth, a key regulator of sexual maturation in zebrafish, as well as aspects of the endocrine stress axis known to affect oocyte growth in avp -/- mutants. While avp -/- mutants did not exhibit differences in somatic growth up to sexual maturation or GSI, mutants exhibited hypercortisolism. While other zebrafish knock-out mutants exhibiting persistent hypercortisolism do not share the observed reproductive phenotype, future studies investigating potential contributions of pleiotropic Avp effects are nevertheless warranted. Overall, I demonstrate that avp, while not essential, affects female reproductive success, at least iii in part by regulating oocyte maturation. This finding is in line with the recent findings from other vertebrate and invertebrate species, suggesting an evolutionarily ancient role in these processes. It is anticipated that such novel insights into the regulation of female oocyte maturation have in addition to increasing our understanding of female reproduction, translational potential for captive breeding (aquaculture, species conservation) and ecotoxicology (insight into mode of action of specific EDCs).