Letrozole increases preantral follicle growth and decreases estradiol production without impairing follicle survival.

BACKGROUND: Letrozole has been reported to be effective in treating anovulation, preventing ovarian hyperstimulation syndrome (OHSS), and retrieving oocytes in breast cancer patients. However, the role and mechanism of letrozole in follicular development remain unclear. RESULTS: We treated mouse preantral follicles with various treatments; we found no significant difference in follicle survival rates in the letrozole (LET) group compared with the control group, but the average diameter of follicles in the LET group tended to be larger (CTRL vs. LET 30, p = 0.064; CTRL vs. LET 100, p = 0.025). The estradiol concentrations in culture media of the LET group were significantly lower than those observed in the control group (CTRL vs. LET 30, p = 0.038; CTRL vs. LET 100, p = 0.025). We further found a marked increase in follicle-stimulating hormone receptor (FSHR) gene expression in response to letrozole treatment (CTRL vs. LET 30, p = 0.075; CTRL vs. LET 100, p = 0.034). This result suggested that increased FSHR expression promotes follicle development. Letrozole inhibited aromatase activity, but the effect was limited. Letrozole did not significantly reduce vascular endothelial growth factor (VEGF) gene expression. CONCLUSIONS: Letrozole may promote follicle development by increasing the expression of FSHR. Letrozole may be useful for fertility preservation of patients with estrogen-dependent cancers such as breast cancer and various other cancers. Whether letrozole has a direct effect in reducing OHSS requires further investigation.

Pioglitazone suppresses excessive follicular development in murine preantral follicles.

Polycystic ovary syndrome (PCOS) is an endocrine disease that is common in women in their reproductive period. Patients with this disease suffer from anovulation and hyperandrogenism. Ovulation induction with exogenous gonadotropin often causes ovarian hyperstimulation syndrome because many small antral follicles pause in their growth. Treatment with insulin sensitizers is reportedly effective for both anovulation associated with PCOS, and suppression of excessive follicular growth; however, the underlying mechanism of action remains unknown. Although pioglitazone is known as an insulin sensitizer, it also has a potent modulator of cell growth and apoptosis irrespective of insulin resistance. To clarify the effect of pioglitazone on follicular growth, we performed in vitro culture of murine preantral follicles. Secondary follicles (100-160 µm in diameter) isolated from 6-week-old ICR mice were individually cultured for 13 days. Culture conditions were as follows: 1) follicle-stimulating hormone (FSH; 33 mIU/mL; control), 2) FSH plus dihydrotestosterone (DHT; 500 ng/mL), 3) FSH plus pioglitazone (5 ng/mL), and 4) FSH plus DHT/pioglitazone. Survival rate and follicle diameter were evaluated, and concentrations of estradiol (E2) and vascular endothelial growth factor (VEGF) in culture media were measured. mRNA expression of various growth-promoting factors and Vegf within follicles were also assessed. Although no significant differences were observed with regard to survival rate, follicle diameters on day 13 were significantly different.Compared with the control group, the DHT group showed enhanced growth, while groups administered pioglitazone showed stagnation of the accelerated growth induced by DHT. Although DHT treatment enhanced the expression of bone morphogenetic protein 2 (Bmp2) mRNA, pioglitazone exposure suppressed induction of Bmp2 mRNA by DHT. Vegf mRNA and protein expression were also significantly reduced when pioglitazone was added to culture media containing DHT.Administration of pioglitazone negatively affected follicular growth and VEGF levels, which may suppress excessive follicular growth and prevent ovarian hyperstimulation syndrome.

Pioglitazone is effective for multiple phenotyepes of the Zucker fa/fa rat with polycystc ovary morphology and insulin resistance.

BACKGROUND: Hyperandrogenism and insulin resistance may be related to the etiology of PCOS. Zucker fa/fa rats with polycystic ovary are obese, have insulin resistance without diabetes mellitus or hyperandrogenism and can be utilized as PCOS model rats without effects of hyperandrogenemia. PCOS patients are reported to have elevated levels of serum anti-Mullerian hormone (AMH), which has an inhibitory action on folliculogenesis, and low levels of serum adiponectin, which blocks apoptosis and induces biological effects in some tissues. Pioglitazone, an insulin sensitizer, is administered to PCOS patients with insulin resistance to induce ovulation but the mechanisms by which this occurs have not been elucidated. METHODS: We purchased 4-week-old female fatty Zucker fa/fa rats as well as lean Zucker +/+ rats for use as control rats with normal insulin sensitivity. The Zucker fa/fa rats were administered pioglitazone (2.5 mg/kg body weight/day) or a vehicle every day for 14 days in separate groups. The Zucker +/+ rats were also administered the vehicle. After 2 weeks of treatment, they were euthanized and we obtained serum samples and both ovaries and determined the body weight, ovarian weight, and serum AMH, adiponectin, testosterone, and androstenedione levels. We also examined ovarian histology to check follicle numbers by using hematoxylin-eosin staining, and the number of atretic follicles using Tdt-mediated dUTP nick end labeling (TUNEL) methods. RESULTS: The Zucker fa/fa rats used as PCO model rats and Pioglitazone treated PCO model rats were significantly heavier than the Zucker +/+ control rats (p < 0.05) at 15 day old. Pioglitazone treatment did not influence body weight or ovarian weight in either group. However, the total number of follicles was significantly larger in the PCO model rats than in the control rats (P < 0.05). Although pioglitazone treatment appeared to decrease the total number of follicles in the PCO model rats, the decrease was not statistically significant. However, pioglitazone treatment significantly decreased the total number of atretic follicles and the rate of atreteic follicles in the PCO model rats (P < 0.05). The serum AMH level was significantly higher in the PCO model rats than in the control rats. Pioglitazone treatment significantly decreased the serum AMH level and significantly increased the serum adiponectin level in the PCO model rats (P < 0.05). Serum testosterone and androstenedione levels were quite low or undetectable in the 3 groups of rats, and were not influenced by pioglitazone treatment. CONCLUSION: In this study, pioglitazone treatment reduced the serum AMH level and increased the serum adiponectin level in PCO model rats. These effects are related to reduction of the total number of atretic follicles and rate of atretic follicles. This proves that pioglitazone treatment improves healthy follicle growth in these PCO model rats with insulin resistance.

Long-term treatment with dehydroepiandrosterone may lead to follicular atresia through interaction with anti-Mullerian hormone.

BACKGROUND: Hyperandrogenism is the primary manifestation of polycystic ovary syndrome (PCOS), which appears to be caused by excess exposure to androgen. As such, androgenized animal models have been developed and investigated to study the etiology of PCOS. Anti-Mullerian hormone (AMH) is known to be associated with follicle growth, and its levels are two to three times higher in women with PCOS than in those with normal ovaries. We studied how duration of androgen administration affects folliculogenesis and AMH expression. METHODS: We divided 30 immature (3-week-old) Sprague Dawley rats into six groups. Three groups were injected each evening with dehydroepiandrosterone (DHEA) (6 mg/100 g body weight/0.2 ml sesame oil) for 7, 15 or 30 days, respectively. The three control groups were injected with 0.2 ml of sesame oil for the corresponding lengths of time. Resected ovaries were sectioned and examined to determine follicle numbers at each developmental stage, and immunostained to assess AMH expression. RESULTS: On day 7, follicle numbers and AMH expression levels at each developmental stage of follicle growth were similar in the respective control and DHEA groups. On day 15, the total follicle number (P = 0.041), the percentage of primordial follicles (P = 0.039) and AMH expression were significantly greater in the DHEA than the control group. On day 30, the percentages of primordial (P = 0.005), primary (P = 0.0002) and atretic (P = 0.03) follicles were significantly greater in the DHEA group, whereas the percentage of intermediary follicles (early pre-antral, late preantral, and early antral follicles) was significantly lower in the DHEA group (P = <0.0001). AMH expression in DHEA-treated rats on day 30 was seen exclusively in the primordial (P = 0.0413) and late antral follicles (p = 0.028). CONCLUSIONS: Androgen administration increases AMH production in a process that regulates the growth of primordial follicles. That is, androgen-induced AMH expression provides local negative feedback to folliculogenesis augmented by androgen.

Assisted reproductive technique increases the risk of placental polyp.

The purpose of this study was to clarify the risk factors and outcomes of placental polyp. This retrospective study was conducted on 1645 patients delivered or aborted in Sapporo Medical University from 2007 through 2011. Transvaginal color Doppler ultrasonography, hysteroscopy, contrast-enhanced MRI or 3D-CT angiography were performed. There were 1532 deliveries and 113 abortions. Seventy-one (4.3%) were ART-conceived and the remaining 1574 (95.7%) were non-ART pregnancies. Fifteen (0.91%) cases were confirmed as having placental polyp. Nine cases of placental polyp were identified among the 1574 (0.57%) as non-ART-related pregnancies, and 6 were identified among the 71 (8.5%) as ART-related pregnancies. Thus, pregnancies achieved through ART showed 20x greater incidence of complicating placental polyp than pregnancies achieved through without ART (p = 9.02 × 10(-6); odds ratio, 19.59; 95% confidence interval, 5.27-72.84, logistic regression analysis). Evaluation of blood flow within the polyp showed that in five of seven patients with low blood flow, the polyps spontaneously dropped off 79-115 days postpartum. Thus, ART-related pregnancies may be a risk factor of placental polyp, and spontaneous drop-off of the polyp is often observed in cases with low blood flow within the mass.

Excessive androgen exposure in female-to-male transsexual persons of reproductive age induces hyperplasia of the ovarian cortex and stroma but not polycystic ovary morphology.

STUDY QUESTION: Does administration of androgen to female-to-male transsexual persons (FTMs) of reproductive age induce polycystic ovary (PCO) morphology? SUMMARY ANSWER: Administration of high-dose androgen to women causes pathognomonic changes to the ovarian cortex and stroma that resemble Stein-Leventhal syndrome but it does not induce PCO morphology. WHAT IS KNOWN ALREADY: Androgen is thought to play a key role in follicular development and to be involved in the pathophysiology of polycystic ovary syndrome (PCOS). In several experimental models, animals given high-dose androgen show ovarian changes similar to those in women with PCOS. In previous human studies, the ovaries of FTMs who received androgen for long periods also exhibited PCO morphology. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective case-control study of women, all without PCOS, undergoing salpingo-oophorectomy. The case group consisted of 11 FTMs taking testosterone (duration range: 17 months to 14 years), while the control group consisted of 10 patients with gynaecologic malignancies who did not receive testosterone. PARTICIPANTS/MATERIALS, SETTING, METHODS: Resected ovaries from both groups were compared histologically with respect to changes in the cortex and stroma, and the number of follicles at each maturation stage. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, the FTM group had a thicker ovarian cortex (P = 0.0001), and more hyperplastic collagen (P = 0.001), ovarian stromal hyperplasia (P = 0.003) and stromal luteinization, i.e. luteinized stromal cells with a small dark central nucleus surrounded by clear cytoplasm (P = 0.004). Isolated clusters of such stromal luteinized cells were found only in the testosterone-treated ovaries of FTMs. The number of primordial follicles was similar in the two groups (P = 0.22). The numbers of early stage (primary, pre-antral and early antral) follicles, which are dependent on androgen, were also similar (P = 0.81), as were the numbers of antral follicles (P = 0.97). In contrast, significantly greater numbers of atretic follicles were seen in the FTM than that in the control group (P = 0.01). LIMITATIONS, REASON FOR CAUTION: In addition to the low numbers in the study groups, the histological changes in FTMs were investigated after testosterone administration, therefore it is possible that some of the observed changes were already present, before androgen administration. WIDER IMPLICATION OF THE FINDINGS: Our results are at variance with those of earlier studies and suggest that excessive androgen exposure in women of reproductive age may not be a factor in the pathogenesis of PCOS.

Weight reduction and pioglitazone ameliorate polycystic ovary syndrome after removal of a Sertoli-stromal cell tumor.

This report presents an unusual case of Sertoli-stromal cell tumor and polycystic ovary syndrome successfully treated with weight reduction and an insulin-sensitizing agent. A 22-year-old woman, gravida 0, para 0, visited our hospital for the first time with a 12-year history of secondary amenorrhea and hypertrichosis. Transvaginal ultrasonography revealed a solid tumor in the right ovary. Right salpingo-oophorectomy was performed and pathological examination confirmed a Sertoli-stromal cell tumor. The patient's serum androgen levels declined postoperatively, but remained above normal. Pioglitazone treatment for 6 months also significantly reduced serum androgen levels, but they still remained above normal. However, after losing 12 kg of body weight, the patient's serum androgen levels declined to normal, and spontaneous menstruation became regular. Weight reduction with pioglitazone is an effective means of treating hyperandrogenism.

Distinctive features of female-to-male transsexualism and prevalence of gender identity disorder in Japan.

INTRODUCTION: The prevalence of transsexualism is thought to differ among socio-geographic backgrounds, and little is known about its prevalence in Japan. Polycystic ovary syndrome (PCOS), which is known to be associated with insulin resistance and metabolic syndrome, is often seen in female-to-male (FTM) transsexual patients. Consequently, detection of PCOS is an important part of health care for these individuals. AIM: The purpose of this study was to assess the prevalence of transsexuality in Japan, as well as the incidences of PCOS and insulin resistance among Japanese FTM transsexual patients. METHODS: One hundred four male-to-female (MTF) and 238 FTM Japanese transsexual patients were studied. Medical histories, including histories of menstrual cycling and hormone treatment, were taken. To exclude other diseases, such as congenital adrenal hyperplasia and hormone-secreting tumors, thorough medical assessments, including transvaginal or transrectal ultrasonography and measurement of serum hormone levels and insulin resistance indexes, were performed. MAIN OUTCOME MEASURES: The diagnosis of PCOS was based on the Rotterdam 2003 criteria. RESULTS: Based on demographic statistics, the prevalences of MTF and FTM transsexuality are about 3.97 and 8.20 per 100,000 people, respectively, making the MTF-to-FTM ratio about 1:2. Of the FTM transsexual patients studied, 128 had not taken hormones before their initial assessment (untreated group); the remaining 50 self-administered androgen. Among the untreated group, 32.0% were diagnosed with PCOS, 30.1% were insulin-resistant, and 31.1% showed hypoadiponectinemia. CONCLUSIONS: The sex ratio among Japanese transsexuals is different than among Caucasians. PCOS and insulin resistance are common findings in FTM transsexual patients at initial presentation.

Remarkable features of ovarian morphology and reproductive hormones in insulin-resistant Zucker fatty (fa/fa) rats.

BACKGROUND: Zucker fatty (fa/fa) rats are a well-understood model of obesity and hyperinsulinemia. It is now thought that obesity/hyperinsulinemia is an important cause of endocrinological abnormality, but to date there have been no reports on the changes in ovarian morphology or the ovarian androgen profile in rat models of obesity and insulin resistance. METHODS: In this study we investigated the effects of obesity and hyperinsulinemia on ovarian morphology and the hormone profile in insulin-resistant Zucker fatty rats (5, 8, 12 and 16 weeks of age, n = 6-7). RESULTS: Ovaries from 5-week-old fatty rats had significantly greater total and atretic follicle numbers, and higher atretic-to-total follicle ratios than those from lean rats. Ovaries from 12- and 16-week-old fatty rats showed interstitial cell hyperplasia and numerous cysts with features of advanced follicular atresia. In addition, serum testosterone and androstenedione levels significantly declined in fatty rats from age 8 to 16 weeks, so that fatty rats showed significantly lower levels of serum testosterone (12 and 16 weeks) and androstenedione (all weeks) than lean rats. This may reflect a reduction of androgen synthesis during follicular atresia. Serum adiponectin levels were high in immature fatty rats, and although the levels declined significantly as they matured, it remained significantly higher in fatty rats than in lean rats. On the other hand, levels of ovarian adiponectin and its receptors were significantly lower in mature fatty rats than in lean mature rats or immature fatty rats. CONCLUSIONS: Our findings indicate that ovarian morphology and hormone profiles are significantly altered by the continuous insulin resistance in Zucker fatty rats. Simultaneously, abrupt reductions in serum and ovarian adiponectin also likely contribute to the infertility seen in fatty rats.

Significance of matrix metalloproteinases in the pathophysiology of the ovary and uterus.

Matrix metalloproteinases (MMP) are capable of degrading a variety of extracellular matrix (ECM) proteins and are also involved in the processing of a number of bioactive molecules. Our findings indicate that the functions of MMP in the ovary and uterus are organ-specific and time-dependently vary during the reproductive cycle. Prolactin induces structural luteolysis indicated by loss of luteal weight, protein and DNA within 36 h after pretreatment with ergot alkaloid. MMP activation appears crucial for the selective depletion of protein during luteal involution, which entails loss of ECM accompanied by apoptosis. During GnRHagonist-induced luteolysis, this response was also associated with marked increases in MMP-2, which degraded collagen type IV, and MT1-MMP, which in addition to activating MMP-2 also degrades collagen type I, III and V. We also found that the level of MT1-MMP and MMP-2 expression in the human CL is greater during the late luteal phase than during either the early mid luteal phases or during gestation, respectively. That dehydroepiandrosterone (DHEA) treatment caused the formation of cysts from antral follicles in the ovaries of immature rats while depressing MMP-2 collagenolytic activity and enhancing lysyl oxidase expression highlights the importance of collagen degradation in the process of ovulation and suggests that changes in the activities of these enzymes play a key role in ovarian cystogenesis in polycystic ovary syndrome patients. Furthermore, immunohistochemical analyses showed that MT1-MMP and FasL co-localize with TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive apoptotic granulosa cells in rats treated with DHEA, that the Fas/FasL/Caspase-8 (death receptor-dependent) pathway is pivotal for follicular atresia and that increased levels of MT1-MMP likely play an important role in tissue remodeling during follicular atresia. After parturition, the uterus undergoes involution, a conspicuous feature characterized by a rapid reduction in the collagen content mediated by degradation of extracellular collagen bundles. Our findings strongly suggest that MT1-MMP, MMP-2 and MMP-9 are each time-dependently regulated and play important roles in tissue remodeling during postpartum uterine involution. (Reprod Med Biol 2006; 5: 235-243).

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats.

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis-related molecules (Fas, Fas ligand (FasL), Bcl-2, Bax). We gave 4-day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG-hCG-treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom+PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom+PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom+PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.