The role and regulation of ovarian preantral follicular development and atresia by GDF-9 and FSH
I. Background:
Ovarian folliculogenesis is an important process involving cell proliferation and differentiation within the developing follicles. The transition of the developing follicle from the preantral to early antral stage is primarily controlled by intraovarian regulators such as growth differentiation factor-9 (GDF-9), but is responsive also to FSH. While FSH promotes growth of cultured follicles, this response is markedly enhanced by the presence of GDF-9, or 3,5,3’-triiodothyronine (T3). In vivo studies have shown that the effect of thyroid hormone on FSH-induced follicular growth is specific to the preantral stage. It is well established that adequate levels of circulating T3 are important for normal female reproductive functions. Women suffering from thyroid disorders frequently experience impaired fertility, a condition readily improved with the restoration of euthyroid state. While hypo- and hyper-thyroidism are associated with suppressed ovarian follicular growth and infertility, the cellular and molecular mechanism(s) involved is not known. While GDF-9 is required for FSH receptor (FSHR) expression in preantral follicles, whether this latter response is required for the FSH-T3 interaction during preantral growth is unclear. In addition, whether T3 has an effect on the FSHR mRNA expression in granulosa cells and thus influence the responsiveness of the cells to gonadotropic stimulation remains to be elucidated.
In the present study, we investigated the interaction between T3 and FSH in the control of preantral follicular growth, and to determine whether T3 plays a role in the regulation of FSHR expression, and if this regulation is mediated through the synthesis and action of oocyte-derived GDF-9.
II. Overall Goal:
To better understand the cellular and molecular regulatory mechanism in the control of ovarian follicular development and atresia, especially in relation to the actions of and interactions between gonadotropin and thyroid hormone.
III. Overall Objective:
To assess the role of GDF-9 in the increased responsiveness of the preantral follicles to FSH and T3 stimulation.
IV. Specific Objectives:
1. To assess the effect of FSH and T3 on preantral follicular development in vitro.
2. To assess the time - dependent effect of FSH and T3 on GDF-9 mRNA expression in cultured preantral follicles in vitro.
3. To assess the effect of GDF-9 down-regulation on GDF-9 expression and follicular cell apoptosis in preantral follicles cultured in the presence of FSH and T3.
4. To assess the effect of GDF-9 down-regulation and FSH - T3 interaction in the regulation of preantral follicular growth in vitro.
5. To assess the time and concentration - dependent effect of FSH and T3 on FSHR expression in cultured preantral follicles in vitro.
6. To assess the effect of GDF-9 down-regulation on FSHR mRNA expression induced by co-treatment of preantral follicles with FSH and T3 in vitro.
V. Experimental Approaches:
My training program involves participation in seminars, journal club and work-in-progress sessions participated by clinicians, basic scientists and pre-doctoral and post-doctoral trainees. In addition, my research program involves biomedical research and clinical components which are co-supervised by a basic scientist (Dr. Ben Tsang) and clinical investigator (Dr. Arthur Leader). My research program is briefly described hereafter.
1) Biomedical Research
a) Objective:
To examine the action and interaction of FSH and T3 in the regulation of the follicular growth, especially in their transition from preantral to early antral stage and the involvement of GDF-9 in this process, using an established follicle culture system and a gene manipulation strategy in vitro.
b) Methodology:
Preantral follicles (diameter: 150-180 µm) isolated from ovaries of 14-day old SD rats were cultured in serum-free medium for various durations (0 - 60h) and concentrations of FSH (0 - 100 ng/ml) or T3 (0 - 10-8 M). FSHR mRNA, GDF-9 mRNA abundance and 18S rRNA levels were determined by quantitative real-time PCR.GDF-9 Morpholino antisense oligos (GDF-9 MO, 10 µM) or its control (CTL MO, 10 µM) were injected into the oocyte and the follicles were cultured for 4 – 6 days ± FSH and/or T3. Apoptosis (TUNEL) and GDF-9 expression (IHC) were performed at the end of the culture period.
c) Results:
i) While T3 alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth during 4-days of culture.
ii) Addition of T3 or FSH to the preantral follicles markedly increased GDF-9 mRNA abundance in 12-18h (P<0.05).
iii) GDF-9 in the cultured follicles was predominantly immunolocalized in the oocyte. Intra-oocyte injection of GDF-9 MO decreased oocyte GDF-9 expression and induced follicular apoptosis, as well as suppressed growth of preantral follicles cultured with FSH and T3 (P<0.001). Addition of GDF-9 (100 ng/ml) to the MO-treated follicles significantly prevented the decrease in preantral follicular growth (P<0.01).
iv) T3 significantly increased FSHR mRNA abundance in the presence but not absence of FSH at 36h of culture period (P<0.001). Maximal responses were observed at 10 ng/ml FSH and 10-9 M T3.
v) The level of FSHR mRNA was significantly decreased in GDF-9 MO-injected follicles cultured for 36h with both FSH and T3 (P<0.001).
d) Conclusion:
Our findings suggest that thyroid hormone plays an important role in the regulation of ovarian follicular growth. It interacts with FSH in promoting preantral follicular growth and this action is mediated through increased FSHR expression which in turn is dependent on adequate oocyte-derived GDF-9 expression and action. This study offers new insight into the molecular and cellular mechanism of impaired follicular growth during hypothyroid state.
2) Clinical Investigations
a) Objective:
To confirm the information gained from the basic science research described above is important to our understanding of clinical conditions associated with infertile women with hypothyroidism.
b) Methodology:
Based on the in vitro findings from my basic science project, I have learned that thyroid hormone promotes FSH-induced preantral follicular growth through up-regulation of FSHR and its mechanism depends on the expression of oocyte-derived GDF-9. To determine if any of these observations are relevant to the clinical setting and to learn how I can translate this basic research information into a clinical research and treatment, I visited the Ottawa Fertility Centre to learn how infertile hypothyroidism patients are managed. I have also reviewed charts of the infertile patients with hypothyroidism who visited the Ottawa Fertility Centre and assessed their serum hormone levels (gonadotropin, sex hormone, thyroid hormone, prolactin, etc.), follicle growth response to hormonal stimulation (by ultrasonography) and clinical outcome (fertility). What I have learned is that thyroid dysfunction is an important cause of infertility and most of the patients with hypothyroidism could become pregnant after thyroid function has been restored by the thyroid hormone replacement therapy. Presented hereafter is a typical case of infertile women with hypothyroidism who has undergone infertility treatment at the Ottawa Fertility Centre which I have followed during the course of my study.
Case Study:
35 years old, Primary infertility,
Oligomenorrhea of 6 month’s duration with periods every 65 days,
Primary hypothyroidism, L-thyroxine 0.88 mg daily
TSH = 13.0, Free T4 = 8; Free T3 = 2.6; LH = 4; FSH = 6
Started on medications and menstrual cycle shortened to every 40 days after 4 months. Started on clomiphene citrate 50 mg days 3 to 7 and conceived after 2nd cycle of treatment
Initial transvaginal ultrasound picture showed antral follicle count of 23. Ovulated with two maturing follicles.
Conceived with singleton pregnancy – ongoing at 20 weeks gestation.
c) Questions asked and Observations:
During the study we addressed the following questions and made some important observations:
i) Are hypothyroid female patients associated with infertility? Are their thyroid status managed prior to fertility treatment (e.g. IVF and ICSI)?
Observations: Hypothyroidism is exactly associated with infertility and decreases the success rate of ART. TSH, free T3 and free T4 are routinely checked prior to initiating therapy. If hypothyroidism is suspected from the above results, antithyroid antibodies are measured additionally. The levels of thyroid hormones are normalized prior to ART to get more efficiency in ART.
ii) Patients with hypothyroidism respond poorly to gonadotropic stimulation. Does maintenance of euthyroid state confer normal follicular response to gonadotropin?
Observations: Keeping appropriate levels of thyroid hormone is important not only for normal follicular response to gonadotoropin but also prime oocyte quality. The response to gonadotropin in women suffering from thyroid disorders is improved with the restoration of euthyroid state.
iii) Is the ovarian response to gonadotropic stimulation in thyroid hormone- treated hypothyroid patient follicular-stage specific?
Observations: The ovaries of hypothyroid women do not response well to the gonadotropic stimulation compared to those in women with normal thyroid function. However, good ovarian response is evident and initial follicular development is more rapid if thyroid hormone levels are maintained within normal range.
iv) What level of thyroid hormones is optimal?
Observations: This is a controversial topic. We know that at the extremes of hypo- and hyperthyroidism, fertility is severely affected, but an optimal value has yet to be agreed to. It might be possible that oocyte quality is not always improved enough to get a pregnancy or keep a good pregnant status even if thyroid status backs to normal range after the thyroid replacement therapy.
d) Conclusions:
Adequate thyroid hormone and FSH levels are needed to maintain the ovarian follicular growth. Basic research provides important information on the mechanism of hormonal interaction in the treatment strategy and offer important insight for the development of new treatment strategies for infertile women.
VI. Unanswered questions for future investigations
1. How does T3 act on oocyte and upregulate GDF-9 if it has a direct effect on oocyte in rat preantral follicle stage? Is it via thyroid hormone receptor in oocyte by the direct stimulation of thyroid hormone response element?
2. Does the interaction of FSH and T3 increase not only the FSHR mRNA level but also the FSHR protein level in preantral follicles?
3. Is the interation of FSH and T3 increase the FSHR expression level owing to the increase of cAMP in the FSHR function or the increase of the number of FSHR ?
4. Is there any difference in the follicular growth, the number of follicle, FSHR expression level in granulosa cell and GDF-9 expression level in oocyte respond to the gonadotropin between the infertile women without thyroid hormone deficiency and those with hypothyroidism under ART?
5. Could the FSHR mRNA expression in granulosa cells and GDF-9 level in follicular fluid by the follicle aspiration during ART be good biomarkers of human oocyte quality and response to hormone?
6. Could the infertile women taken gonadotropin treatment and hypothyroid women already treated with the thyroid hormone replacement therapy plus gonadotropin treatment have the similar good oocyte quality to get a success in ART?
7. How long after thyroid function is stabilized is Oocyte development after gonadotropin optimized?
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This was pretty informative.
This was pretty informative. I was unaware of T3's affect on folliculogenesis. Is there an effect of hyperthyroidism which is similar to the effect of hypothyroidism? or is it the opposite effect?
Depending on the degree of
Depending on the degree of hyperthyroidism, in mild the woman can respond normally to gonadotropins but egg quality (as determined by pregnancy rates) is reduced. In more classical forms of hyperthyroidism, women are refractory to gonadotropins and become oligomenorrheic or amenorrheic.
Jo Watson RN, MScN,
Jo Watson RN, MScN, ACNP
Director, Obstetrics and Gynaecology
Sunnybrook Health Sciences Centre
Thanks for your presentation. I appreciate how you continued your lab learning in a clinical setting. What will be your next step in you learning process?
Thyroid hormone has been
Thyroid hormone has been recognized that it might have a direct role in ovarian physiology via receptors in granulosa cells. However, the mechanism of action of thyroid hormone on follicle development is not fully understood.
To make a further investigation in the mechanism, my next step is to examine whether thyroid hormone has a direct action on GDF-9 expression in oocyte via thyroid hormone receptors or acts via some granulosa cells factor(s) which regulates the expression of GDF-9 in the oocyte.
In addition, I am planning to evaluate whether the increased granulosa cell FSH receptor mRNA abundance in response to T3 is of physiological significance. Specifically, I will examine the changes in cAMP response of follicles previously cultured with T3 and FSH and subsequently challenged with FSH. If the increase in FSH receptor is functional, one would expect that a further challenge with FSH would result in greater increase in cAMP production by the follicles.
Hi Noriko, It was great to
Hi Noriko,
It was great to see that you had some good results in the lab and I was particularly interested in the finding that it is oocyte-derived GDF-9 that is important rather than injection from an external source. What do you make of that finding? Since I am not familiar with this area, I am wondering if it is to be expected or whether it was somewhat surprising.
Being from a psychology background, I am curious about the type of statistics that you used. I see that many aspects of your project were significant at p<.001, etc. but I wondering how you went about analyzing the data.
I also found myself thinking about ways to expand the clinical investigations aspect of your research and was considering something similar to #4 and #6 in your unanswered questions. I was thinking that there must be some way to compare groups of women, in addition to individual case studies, to answer these questions. I was wondering whether something like a retrospective review of medical charts could answer these questions initially, and whether this would provide some additional insights.
Thanks for sharing your research with us,
Alex.
My results showed that
My results showed that depletion of oocyte GDF-9 (knockdown by intra-oocyte injection of GDF-9 MO) suppressed preantral follicular growth. Although another oocyte-derived factor such as BMP-15 may also play a role in the regulation of preantral growth, this does not explain the current findings. If preantral follicular growth was not increased by the addition of GDF-9 and no significance difference could be seen between follicular growth of GDF-9 MO group and that with additional GDF-9 group, meaning that it might be possible that GDF-9 MO knockdowned the other factors in oocyte.
Concerning the statistic analysis in the project, data from at least three independent experiments were subjected to one- or three-way (repeated-measure) ANOVA Differences between experimental groups were determined by the Tukey or Bonferroni post-test.
Clinical studies are theoretically possible and very interesting. The problem is that there is a medical duty to care so that when the condition is discovered, patients are treated and then referred for fertility assessment, thereby introducing a bias in the true prevalence of hypothyroidism in infertility disorders. The better way is an animal model in a subhuman primate. Retrospective studies have been reported but are compromised by the fact that different clinicians treat the same problem in different ways.
Hello Noriko, thank you for
Hello Noriko,
thank you for sharing your research and the promising results with us. Congratulations!
What do you think about a possible teleological relevance of thyroid control over folliculogenesis?
All the best,
Martin
The infertility women have a
The infertility women have a wide range of the causes. Although thyroid hormone dysfunction is one of key causes for infertile women, generally the proper treatments would be selected according to the causes, and ART is the ultimate tool.
Keeping thyroid hormone at a normal level is needed for the normal reproductive function and has two important meanings for infertile women. One is that thyroid hormone acts on ovaries directly to regulate follicular development. The other is treating thyroid dysfunction could reverse menstrual abnormalities and thus improve fertility.
Major promise to proceed to the process of ART is to get prime eggs with good quality by controlling thyroid hormone level. It would be helpful to improve the success rate of ART for infertile women.
Thank you for your answer
Thank you for your answer Noriko.
My question was rather about the teleological than physiological meaning of thyroid control over folliculogenesis. I.e., why does woman's body 'need' such a control system? Is there a way we can find a bigger picture/physiological model where these findings might fit into?
As an example I think of the recent developments in neuroimmunology that have taught us how such systems oriented thinking can produce amazing new insights into joint function and connectivity of immune and nervous systems that turned out highly relevant for medicine.
Another question I wondered about is whether did you or do you intend to consider accounting for autoimmune thyroid disorders? I.e., how antibodies produced in such disorders may or may not interact/deteriorate the folliculogenesis on top of the main T3 deficiency mediated impairment.
Thank you again,
Martin
Teleology speaks to the
Teleology speaks to the intension of Graves’ disease and the purpose of a finding, but it is always speculation. It is known that hypothyroid women that are untreated are more likely to give birth to cretin children or children with thyroid gland hyperplasia (goitre). These children could be mentally challenged (retarded) if not treated with thyroid hormones. Although hypothyroidism does not prevent pregnancy, it does reduce the chances - hence a teleological reason why altered thyroid function can affect folliculogenesis.
With regard to autoimmune thyroid disorders, the clinical relevance of thyroid autoimmunity (TAI) in infertility remains somewhat controversial. It might be because most studies were retrospective, perhaps introducing biases in the observed results. There is a significant association between the presence of the TAI and an increased risk of subsequent miscarriage in the first trimester of pregnancy rather than infertility. Actually TAI does not seem to interfere with pregnancy rate after ART. It was reported that the presence of these auto-antibodies did not reduce the chance of pregnancy after ovulation induction.
So far, I have not intended to investigate the effect of TAI on folliculogenesis. As you raised a question, it is an interesting point to know whether infertility in the women with TAI is directly related to the presence of circulating thyroid antibodies or secondary hypothyroidism caused by TAI in terms of folliculogenesis. In women of fertile age, TAI is the most common cause of hypothyroidism, while TAI could be present without thyroid dysfunction and is thus undiagnosed. Through measuring thyroperoxidase antibodies (TPO-Ab) and thyrogloblin antibodies (TG-Ab), several investigations have shown an association between TAI and infertility. In addition, the association was observed between TAI and endometriosis, a major cause of infertility. Women with thyroid antibodies are deficient in cellular immunity.
Excellent work, Noriko.
Excellent work, Noriko. There are some very good questions submitted and you have answered them well.
If I may throw my own thought into the mix: It is fairly well established that the concentration/dose of FSH to which follicles are exposed can have quite dramatic effects on the developmental competence of the enclosed oocyte. Do ART programs (and researchers) need to be more careful with the FSH dose used in women with thyroid hormone dysfunction? This follows on your point 6 under "Unanswered questions for future investigations" regarding oocyte quality and I think is an important aspect that must always be kept in mind as the ultimate goal of any manipulations applied to promote follicle growth.
Barbara Vanderhyden
Both FSH and thyroid hormone
Both FSH and thyroid hormone appear to be necessary to achieve maximum fertilization rates. It has been known that thyroid hormone has an important role in oocyte physiology (Cramer et al. J Assist Reprod Genet. 20: 210-215, 2003).
I agree with you that we should be more careful of FSH dose because there is a known effect. It would be desirable that the maximum effect for follicular growth is induced by the minimum requirements of concentration/dose of FSH and thyroid hormone. Over-stimulation on ovaries or meaningless manipulation for follicular growth should be avoided. Indeed, rodent studies carried out in Vanderhyden’s laboratory (Thomas et al. Endocrinology 146:941-949, 2005) and clinical investigation (human) by Leader and his colleagues (Out et al. Hum Reprod. 14: 622-627, 1999) indicate that the growth of the oocytes and follicular growth is significantly more effective and efficient following stimulation with low than high dose of gonadotropin (i.e. more is not always better). That is why thyroid function in all infertile women is carefully screened and is treated to normalize their thyroid dysfunction (i.e.hypo or hyperthyroid) before commencing with ART therapy with FSH stimulation.