M.A. Fierro

In vitro fertilization pregnancy rates in levothyroxine-treated women with hypothyroidism compared to women without thyroid dysfunction disorders.

BACKGROUND Untreated hypothyroidism can lead to ovulatory dysfunction resulting in oligo-amenorrhea. Treatment with levothyroxine can reverse such dysfunction and thus should improve fertility. The purpose of this retrospective study was to assess whether in vitro fertilization (IVF) pregnancy rates differ in levothyroxine-treated women with hypothyroidism compared to women without thyroid dysfunction/disorders. METHODS Treated hypothyroid and euthyroid women undergoing IVF at an academic IVF center were studied after Institutional Review Board approval. Women with hypothyroidism were treated with levothyroxine 0.025-0.15 mg/day for at least 3 months to maintain baseline thyrotropin (TSH) levels of 0.35-4.0 μU/mL prior to commencing IVF treatment (HYPO-Rx group). Causes of infertility were similar in both groups with the exception of male factor, which was more common in the HYPO-Rx group. The main outcomes studied were implantation rate, clinical pregnancy rate, clinical miscarriage rate, and live birth rate. RESULTS We reviewed the first IVF retrieval cycle performed on 240 women aged 37 years or less during the period January 2003 to December 2007. Women with treated hypothyroidism (n=21) had significantly less implantation, clinical pregnancy, and live birth rates than euthyroid women (n=219). CONCLUSIONS We conclude that, despite levothyroxine treatment, women with hypothyroidism have a significantly decreased chance of achieving a pregnancy following IVF compared to euthyroid patients. A larger prospective study is necessary to assess confounding variables, confirm these findings, and determine the optimal level of TSH prior to and during controlled ovarian hyperstimulation for IVF.

IGF1R Signaling Is Necessary for FSH-Induced Activation of AKT and Differentiation of Human Cumulus Granulosa Cells

CONTEXT FSH is routinely administered to in vitro fertilization patients to induce follicle maturation. During this process, granulosa cells differentiate and acquire specific functional characteristics that are required to coordinate ovulation and oocyte maturation. OBJECTIVE The objective of the study was to gain insight into the molecular mechanisms regulating human granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus and mural granulosa cells were isolated from the follicular aspirates of in vitro fertilization patients and analyzed immediately or cultured in serum-free media in the presence of FSH, IGFs, or an inhibitor of type I IGF receptor (IGF1R) activity. MAIN OUTCOME We quantified the mRNA and protein levels of steroidogenic enzymes, components of the IGF system, and gonadotropin receptors; measured 17β-estradiol levels; and examined the activation of intracellular signaling pathways to assess the granulosa cell differentiation as well as the FSH and IGF actions in both cumulus and mural cells. RESULTS In freshly isolated cells, LH receptor (Lhr) and steroidogenic acute regulator (Star) were expressed at lower levels in cumulus than mural cells, whereas FSH receptor (Fshr) and anti-Müllerian hormone (Amh) were expressed at higher levels in cumulus than mural cells. In vitro, the expression of Igf2, the differentiation markers Lhr, Star, and Cyp19a1 (aromatase) as well as 17β-estradiol production remained low in untreated cumulus cells but increased significantly after FSH treatment. Strikingly, this stimulatory effect of FSH was abolished by the inhibition of IGF1R activity. FSH-induced activation of v-akt murine thymoma viral oncogene homolog 3 (AKT) required IGF1R activity, and overexpression of constitutively active AKT rescued the induction of differentiation markers and 17β-estradiol production by FSH in the presence of the IGF1R inhibitor. CONCLUSIONS The cumulus cell response to FSH resembles the differentiation of preantral to preovulatory granulosa cells. This differentiation program requires IGF1R activity and subsequent AKT activation.

FSH Regulates IGF-2 Expression in Human Granulosa Cells in an AKT-Dependent Manner.

CONTEXT IGF-2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF-2 gene or the interaction of IGF-2 and FSH during follicle development. OBJECTIVE To examine the mechanisms involved in the regulation of the IGF-2 gene by FSH and the interplay between FSH and IGF-2 during granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus granulosa cells were separated from cumulus-oocyte complexes isolated from the follicular aspirates of in vitro fertilization patients and cultured for in vitro studies. MAIN OUTCOME Protein and mRNA levels of IGF-2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF-2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF-2. RESULTS FSH significantly enhanced IGF-2 expression after 8 hours of treatment and at low doses (1 ng/mL). Reciprocally, IGF-2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF-2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF-2 promoter usage in human cumulus cells showed that the IGF-2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter-derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF-2 transcripts was blocked by cotreatment with inhibitors of AKT or IGF-1 receptor (IGF-1R). The inhibitory effect of the IGF-1R inhibitor on FSH-induced IGF-2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct. CONCLUSIONS FSH is a potent enhancer of IGF-2 expression in human granulosa cells. In return, IGF-2 activation of the IGF-1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF-2 that is critical for human granulosa cell proliferation and differentiation.

Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect on AMH mRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase in AMH mRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.