Aspects of Mono- and Multiple Dominant Follicle Development in the Human Ovary

Chapter 1 The introduction of this thesis starts with general information on reproduction, sub­fertility and reproductive medicine. It continues with a brief overview of current knowledge regarding the function of the human ovary, describing ovarian development and early and advanced follicle development. The importance of the threshold/win-dow concept for the selection of a single dominant follicle is stressed in more detail. Furthermore, a description of disturbed follicle development and the classi.cation of anovulatory disorders is offered. After a discussion on management of subfertility and the differences between ovulation induction and ovarian (hyper)stimulation, the study objectives are provided. Chapter 2 Section 2.1 To test whether the administration of low-dose exogenous FSH initiated during the early, mid or late follicular phase can induce multiple dominant follicle development, a prospective, randomized trial was performed in normo-ovulatory women. Forty nor­mal weight women participated. Administration of a .xed dose (75 IU) of recombinant FSH was started on either cycle day 3, 5 or 7 until the induction of ovulation with human chorionic gonadotrophin. Frequent transvaginal ultrasound scans and blood sampling were performed. Multifollicular growth occurred in all groups (overall in 60%), although day 7 starters showed less multifollicular growth. Age, cycle length and initial FSH and inhibin B concentrations were similar between subjects with single or multiple follicle development. However, for all women, the lower the BMI, the more follicles emerged. If multifollicular growth occurred, the length of the luteal phase was reduced and midluteal serum concentrations of LH and FSH were decreased while oestradiol and inhibin A were increased. In conclusion, interference with decremental serum FSH concentrations by administration of low dose FSH starting on cycle day 3, 5 or as late as day 7, is capable of disrupting single dominant follicle selection. The role of BMI in determining ovarian response suggest that differences in pharmacokinetics of exogenous FSH are involved. Multifollicular growth per se has a distinct effect on luteal phase characteristics. Section 2.2 This study was performed to investigate the relationship between serum concentrations of inhibin A, B, and E2 and the number of developing follicles during the administration of FSH in various regimens in normo-ovulatory volunteers and to evaluate if inhibins act as suitable markers for the number of developing follicles during ovarian stimula­tion. To address this issue, serial hormone determinations and assessment of follicle numbers were carried out during unstimulated cycles and during various interventions with exogenous FSH. Subjects were randomized for FSH administration in a single high dose (375 IU: Group A) during the early follicular phase, 5 consecutive low doses starting in the mid follicular phase (75 IU: Group B) or daily low doses (75 IU) during the early to late follicular phase (starting on cycle days 3, 5 or 7: Groups C, D and E, respectively). This study showed that extending the FSH window increases the number of small antral follicles and hence inhibin B serum concentrations. If such an intervention re­sulted in multifollicular growth, mid follicular phase inhibin B (P = 0.001) as well as late follicular phase inhibin B and A levels were signi.cantly (P < 0.05 and P < 0.01, respectively) increased compared to monofollicular cycles or the natural cycle. Although mid follicular inhibin B levels correlated well with the number of small an­tral (P < 0.05) and pre-ovulatory (P < 0.001) follicles in the late follicular phase, mid follicular inhibin A and E2 serum concentrations only correlated with the number of pre-ovulatory follicles (P < 0.001 and P < 0.01, respectively). In conclusion, the present data extend our understanding of the relationship between follicle dynamics, serum inhibins and FSH during ovarian hyperstimulation. However, although mid follicular inhibin B does correlate with the number of developing follicles, it does not facilitate the identi.cation of women at risk for multiple follicle development. Chapter 3 Section 3.1 This section is meant as an introduction to section 3.2. It describes the enormous expansion IVF treatment has undergone since its .rst successful report in the natural cycle. In order to improve success rates of IVF treatment, GnRH agonists were intro­duced to prevent premature luteinisation and ovulation during ovarian hyperstimula­tion. The mechanism of action of GnRH agonists is discussed. Over the years IVF treatment has become extremely complex, expensive, time consuming and not without risks. Recently, serious concerns related to the management of assisted reproductive therapies, like IVF, have been expressed. The clinical availability of GnRH antagonists offers the opportunity to develop alternative approaches to ovarian stimulation, since these compounds are characterized by an immediate suppression of pituitary gonado­trophin release and a rapid recovery after cessation. Section 3.2 Extending the FSH window for multifollicular development by administering FSH from the midfollicular phase onward constitutes a novel, mild protocol for ovarian stimula­tion for IVF based on the physiology of single dominant follicle selection in normo­ovulatory women. To test the outcomes from this protocol, 142 IVF patients were randomized to either a GnRH agonist long protocol or one of two GnRH antagonist protocols commencing recombinant FSH on cycle day 2 or cycle day 5. A .xed dose (150 IU/day) of exogenous FSH was used for ovarian stimulation, and GnRH antago­nist co-treatment was initiated on the day when the leading follicle had reached 14 mm diameter. Frequent transvaginal ultrasound scans and blood sampling were performed. This study showed that application of the described mild ovarian stimulation protocol Summary resulted in pregnancy rates per started IVF cycle similar to those observed after pro­found stimulation with GnRH agonist co-treatment despite a shorter stimulation and a 27% reduction in exogenous FSH. A higher cancellation rate before oocyte retrieval was compensated by improved embryo quality concomitant with a higher chance of undergoing embryo transfer. A relatively low number of oocytes retrieved after mild ovarian stimulation distinctly differs from the pathological reduction in the number of oocytes retrieved after profound ovarian stimulation (poor response) associated with poor IVF outcome. The relatively small number of oocytes obtained after mild ovarian stimulation may represent the best of the cohort in a given cycle. Chapter 4 Section 4.1 This section provides an introduction to alternative approaches to the management of anovulatory patients and ovulation induction. WHO 2 group patients represent a very heterogeneous group of patients, which shows a marked variation in ovarian response during ovulation induction. The importance of a more patient tailored approach to this category of patients is stressed. Section 4.2 Elevated LH concentration is a common feature in PCOS. This study was designed to investigate whether elevated LH levels in PCOS might be suppressed to normal range values by the administration of low doses of GnRH antagonist, which subsequently might reverse the anovulatory status of these patients. In order to address this issue, 24 PCOS patients with elevated endogenous LH concentrations were randomized into three different dose groups, receiving either 0.125 mg (group A), 0.250 mg (Group B) or 0.500 mg (Group C) ganirelix sc daily for 7 subsequent days. During the .rst day of treatment, LH and FSH levels were assessed at 20 minute intervals, during 8 hours. Thereafter LH, FSH, androgens, E2 and inhibins were assessed daily and frequent ul­trasound scans were performed for 7 days to record follicle development. The study showed that repeated GnRH antagonist administration induced a sig­ni.cant suppression of LH (and to a lesser extent of FSH) serum levels, which was comparable between the different doses. Six hours after ganirelix administration, en­dogenous LH was suppressed by 49%, 69% and 75%, and endogenous FSH was suppressed by 23%, 19% and 25%, respectively. The decrease in serum LH and FSH levels was transient and lasted for 12 hours, whereafter serum levels returned to base­line levels at 24 hours after drug administration. Only in the highest dose group a suppression of androgen levels after prolonged treatment was observed. E2 levels de­creased signi.cantly (P < 0.001) and suppression was most pronounced in group C. Inhibin B levels did not change during the treatment period. Spontaneous follicle de­velopment or ovulations were not recorded during the course of treatment. In conclu­sion, the present study demonstrated that the GnRH antagonist ganirelix is capable of normalising elevated LH in PCOS patients, in doses similar to the ones previously shown to prevent a premature LH rise during ovarian hyperstimulation for IVF. In addition, the transient suppression of elevated endogenous LH levels per se does not re-establish normal follicle development in PCOS. However, follicle development may be insuf.ciently supported by the accompanied subtle suppression of endogenous FSH. Similarly, a transient decline in E2 levels is not effectively restoring normal pituitary ovarian feedback. Moreover, these results support the contention of a limited role of LH in the pathogenesis of PCOS. Section 4.3 A placebo controlled double blind assessment was performed to assess whether the addition of metformin to gonadotrophin ovulation induction in insulin-resistant, nor­mogonadotrophic, anovulatory women alters ovarian responsiveness to exogenous FSH. After a progestagen withdrawal bleeding patients were randomized for either metformin (n = 11) or placebo (n = 9). In case of absent ovulation, exogenous FSH was subsequently administered to induce ovulation. Only during metformin treatment body mass index and androgen (androstenedione and testosterone) levels decreased, whereas FSH and luteinizing hormone levels increased signi.cantly. In the metformin group a single patient ovulated before the initiation of exogenous FSH. Signi.cantly more monofollicular cycles and lower pre-ovulatory oestradiol concentrations were observed in women receiving metformin next to FSH, compared to FSH alone. In con­clusion, metformin co-treatment in a group of insulin-resistant, normogonadotrophic, anovulatory patients resulted in normalisation of the endocrine pro.le and facilitated monofollicular development during FSH induction of ovulation. Chapter 5 In this chapter the reader is provided an overview of results and conclusions from the preceding studies. These .ndings are discussed in view of existing knowledge and future perspectives

Memories of S S Hakozaki-Maru

textabstractOBJECTIVE: To compare the effects of different protocols of controlled ovarian hyperstimulation on thyroid function with those of the natural menstrual cycle. STUDY DESIGN: Prospective controlled study. SETTING: University Medical Center. PATIENTS: A total of 97 women without a history of endocrine disease undergoing intrauterine insemination either in a natural cycle, or with mild ovarian hyperstimulation, or in vitro fertilization (IVF). MAIN OUTCOME MEASURES: estradiol (E2), thyroxine binding globulin (TBG), free thyroxine (FT4), total thyroxine (TT4) and thyroid stimulating hormone (TSH) during the midluteal phase. RESULTS: In the IVF group midluteal E2, TBG, and TT4 were significantly higher; midluteal FT4 was significantly lower (mean difference: -1.46 pmol/L; P < 0.001) and midluteal TSH was significantly higher (mean difference: 0.52 mU/L; P = 0.015). CONCLUSIONS: Ovarian hyperstimulation in IVF is associated with lower midluteal FT4. and higher midluteal TSH levels compared to the natural cycle

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (including scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome

Study Question What is the recommended assessment and management of women with polycystic ovary syndrome (PCOS), based on the best available evidence, clinical expertise, and consumer preference? Summary Answer International evidence-based guidelines including 166 recommendations and practice points, addressed prioritized questions to promote consistent, evidence-based care and improve the experience and health outcomes of women with PCOS. What Is Known Already Previous guidelines either lacked rigorous evidence-based processes, did not engage consumer and international multidisciplinary perspectives, or were outdated. Diagnosis of PCOS remains controversial and assessment and management are inconsistent. The needs of women with PCOS are not being adequately met and evidence practice gaps persist. Study Design, Size, Duration International evidence-based guideline development engaged professional societies and consumer organizations with multidisciplinary experts and women with PCOS directly involved at all stages. Appraisal of Guidelines for Research and Evaluation (AGREE) II-compliant processes were followed, with extensive evidence synthesis. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework was applied across evidence quality, feasibility, acceptability, cost, implementation and ultimately recommendation strength. Participants/Materials, Setting, Methods Governance included a six continent international advisory and a project board, five guideline development groups, and consumer and translation committees. Extensive health professional and consumer engagement informed guideline scope and priorities. Engaged international society-nominated panels included pediatrics, endocrinology, gynecology, primary care, reproductive endocrinology, obstetrics, psychiatry, psychology, dietetics, exercise physiology, public health and other experts, alongside consumers, project management, evidence synthesis, and translation experts. Thirty-seven societies and organizations covering 71 countries engaged in the process. Twenty face-to-face meetings over 15 months addressed 60 prioritized clinical questions involving 40 systematic and 20 narrative reviews. Evidence-based recommendations were developed and approved via consensus voting within the five guideline panels, modified based on international feedback and peer review, with final recommendations approved across all panels. Main Results and the Role of Chance The evidence in the assessment and management of PCOS is generally of low to moderate quality. The guideline provides 31 evidence based recommendations, 59 clinical consensus recommendations and 76 clinical practice points all related to assessment and management of PCOS. Key changes in this guideline include: i) considerable refinement of individual diagnostic criteria with a focus on improving accuracy of diagnosis; ii) reducing unnecessary testing; iii) increasing focus on education, lifestyle modification, emotional wellbeing and quality of life; and iv) emphasizing evidence based medical therapy and cheaper and safer fertility management. Limitations, Reasons for Caution Overall evidence is generally low to moderate quality, requiring significantly greater research in this neglected, yet common condition, especially around refining specific diagnostic features in PCOS. Regional health system variation is acknowledged and a process for guideline and translation resource adaptation is provided. Wider Implications of the Findings The international guideline for the assessment and management of PCOS provides clinicians with clear advice on best practice based on the best available evidence, expert multidisciplinary input and consumer preferences. Research recommendations have been generated and a comprehensive multifaceted dissemination and translation program supports the guideline with an integrated evaluation program. Study Funding/Competing Interest(S) The guideline was primarily funded by the Australian National Health and Medical Research Council of Australia (NHMRC) supported by a partnership with ESHRE and the American Society for Reproductive Medicine. Guideline development group members did not receive payment. Travel expenses were covered by the sponsoring organizations. Disclosures of conflicts of interest were declared at the outset and updated throughout the guideline process, aligned with NHMRC guideline processes. Full details of conflicts declared across the guideline development groups are available at https://www.monash.edu/medicine/sphpm/mchri/pcos/guideline in the Register of disclosures of interest. Of named authors, Dr Costello has declared shares in Virtus Health and past sponsorship from Merck Serono for conference presentations. Prof. Laven declared grants from Ferring, Euroscreen and personal fees from Ferring, Euroscreen, Danone and Titus Healthcare. Prof. Norman has declared a minor shareholder interest in an IVF unit. The remaining authors have no conflicts of interest to declare. The guideline was peer reviewed by special interest groups across our partner and collaborating societies and consumer organizations, was independently assessed against AGREEII criteria and underwent methodological review. This guideline was approved by all members of the guideline development groups and was submitted for final approval by the NHMRC

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs i. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (incl. scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

A randomized comparison of two ovarian stimulation protocols with gonadotropin-releasing hormone (GnRH) antagonist cotreatment for in vitro fertilization commencing recombinant follicle-stimulating hormone on cycle day 2 or 5 with the standard long GnRH agonist protocol

Extending the FSH window for multifollicular development by administering FSH from the midfollicular phase onward constitutes a novel mild protocol for ovarian stimulation for in vitro fertilization (IVF) based on the physiology of single dominant follicle selection in normo-ovulatory women. We compared outcomes from this protocol with two other stimulation protocols. One hundred and forty-two normo-ovulatory patients with an indication for IVF (or IVF/ICSI) were randomized to a GnRH agonist long protocol (group A; n = 45) or one of two GnRH antagonist protocols commencing recombinant FSH on cycle d 2 (group B; n = 48) or cycle d 5 (group C; n = 49). A fixed dose (150 IU/d) of rFSH was used for ovarian stimulation, and GnRH antagonist cotreatment was initiated on the day when the leading follicle reached 14 mm diameter. Group C showed a shorter duration of stimulation (median duration, 11, 9, and 8 d for groups A, B, and C, respectively; P &lt; 0.001), reflected in a significantly lower total dose of rFSH used (median amount of rFSH, 1650, 1350, and 1200 IU for groups A, B, and C, respectively; P &lt; 0.001). In group C more cycles were cancelled during the stimulation phase due to insufficient response, resulting in a lower percentage of oocyte retrievals (84%, 73%, and 63% for groups A, B, and C; P = 0.02). However, women in group C obtained better quality embryos (percentage of embryo score of 1 for best embryo, 29%, 37%, and 61% for groups A, B, and C, respectively; P = 0.008), resulting in more transfers per oocyte retrieval (68%, 71%, and 90% for groups A, B, and C, respectively; P = 0.04). After profound ovarian stimulation (groups A and B) only 7% of the patients who retrieved four oocytes or less conceived, whereas after mild stimulation (group C) 67% of these patients conceived (P &lt; 0.01). Overall, no differences were found among the three groups comparing pregnancy rate per started IVF cycle. In conclusion, application of the described mild ovarian stimulation protocol resulted in pregnancy rates per started IVF cycle similar to those observed after profound stimulation with GnRH agonist cotreatment despite shorter stimulation and a 27% reduction in exogenous FSH. A higher cancellation rate before oocyte retrieval was compensated by improved embryo quality concomitant with a higher chance of undergoing embryo transfer. A relatively low number of oocytes retrieved after mild ovarian stimulation distinctly differs from the pathological reduction in the number of oocytes retrieved after profound ovarian stimulation (poor response) associated with poor IVF outcome. The relatively small number of oocytes obtained after mild ovarian stimulation may represent the best of the cohort in a given cycle

Low-dose exogenous FSH initiated during the early, mid or late follicular phase can induce multiple dominant follicle development

This prospective, randomized trial in normo-ovulatory women was designed to test whether administration of low-dose exogenous FSH initiated during the early, mid to late follicular phase can induce multiple dominant follicle development. Forty normal weight women (age 19-35 years, cycle length 25-32 days) participated. A fixed dose (75 IU/day) of recombinant FSH was started on either cycle day 3 (n = 13), 5 (n = 13) or 7 (n = 14) until the induction of ovulation with human chorionic gonadotrophin. Frequent transvaginal ultrasound scans and blood sampling were performed. Multifollicular growth occurred in all groups (overall in 60%), although day 7 starters showed less multifollicular growth. Age, cycle length and initial FSH and inhibin B concentrations were similar between subjects with single or multiple follicle development. However, for all women the lower the body mass index (BMI), the more follicles emerged (r = -0.44, P = 0.007). If multifollicular growth occurred, the length of the luteal phase was reduced (P = 0.002) and midluteal serum concentrations of LH (P = 0.03) and FSH (P = 0.004) were decreased and oestradiol (P = 0.002) and inhibin A (P = 0.01) were increased. In conclusion, interference with decremental serum FSH concentrations by administration of low dose FSH starting on cycle day 3, 5 or as late as day 7, is capable of disruptin

Development of a research agenda for general practice based on knowledge gaps identified in Dutch guidelines and input from 48 stakeholders

Background: Several funding organizations using different agendas support research in general practice. Topic selection and prioritization are often not coordinated, which may lead to duplication and research waste. Objectives: To develop systematically a national research agenda for general practice involving general practitioners, researchers, patients and other relevant stakeholders in healthcare. Methods: We reviewed knowledge gaps from 90 Dutch general practice guidelines and formulated research questions based on these gaps. In addition, we asked 96 healthcare stakeholders to add research questions relevant for general practice. All research questions were prioritized by practising general practitioners in an online survey (n = 232) and by participants of an invitational conference including general practitioners (n = 48) and representatives of other stakeholders in healthcare (n = 16), e.g. patient organizations and medical specialists. Results: We identified 787 research questions. These were categorized in two ways: according to the chapters of the International Classification for Primary Care (ICPC) and in 12 themes such as common conditions, person-centred care and patient education, collaboration and organization of care. The prioritizing procedure resulted in top 10 lists of research questions for each ICPC chapter and each theme. Conclusion: The process resulted in a widely supported National Research Agenda for General Practice. We encourage both researchers and funding organizations to use this agenda to focus their research on the most relevant issues in general practice and to generate new evidence for the next generation of guidelines and the future of general practice

Development of a research agenda for general practice based on knowledge gaps identified in Dutch guidelines and input from 48 stakeholders

Background: Several funding organizations using different agendas support research in general practice. Topic selection and prioritization are often not coordinated, which may lead to duplication and research waste. Objectives: To develop systematically a national research agenda for general practice involving general practitioners, researchers, patients and other relevant stakeholders in healthcare. Methods: We reviewed knowledge gaps from 90 Dutch general practice guidelines and formulated research questions based on these gaps. In addition, we asked 96 healthcare stakeholders to add research questions relevant for general practice. All research questions were prioritized by practising general practitioners in an online survey (n = 232) and by participants of an invitational conference including general practitioners (n = 48) and representatives of other stakeholders in healthcare (n = 16), e.g. patient organizations and medical specialists. Results: We identified 787 research questions. These were categorized in two ways: according to the chapters of the International Classification for Primary Care (ICPC) and in 12 themes such as common conditions, person-centred care and patient education, collaboration and organization of care. The prioritizing procedure resulted in top 10 lists of research questions for each ICPC chapter and each theme. Conclusion: The process resulted in a widely supported National Research Agenda for General Practice. We encourage both researchers and funding organizations to use this agenda to focus their research on the most relevant issues in general practice and to generate new evidence for the next generation of guidelines and the future of general practice