Compatible cut-off values for luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio in diagnostic criteria of the Japan Society of Obstetrics and Gynecology for polycystic ovary syndrome.

AIM: The abnormal secretion of luteinizing hormone (LH) is one of the typical features of polycystic ovary syndrome (PCOS) and adopted in the diagnostic criteria of the Japan Society of Obstetrics and Gynecology (JSOG). We investigated cut-off values for LH and the LH/follicle-stimulating hormone (FSH) ratio in resent two measurement systems for the diagnosis of PCOS. METHODS: Ninety-nine controls and 106 patients with PCOS were enrolled. Serum LH and FSH levels were measured using an electrochemiluminescence immunoassay (ARCHITECT) and chemiluminescence immunoassay (ECLusys). We examined the distribution of the measured levels, selected the conversion closest to the standard normal distribution in the control group, and calculated mean + 1 SD values for LH and the LH/FSH ratio as candidates. Cut-off values coincided with the medians of the candidates in the two assay systems using a regression equation. We calculated the endocrinological abnormality rate in PCOS according to the JSOG criteria by abnormal LH secretion and elevated T. RESULTS: Cut-off values for LH (mIU/mL) and the LH/FSH ratio were 7.1 and 1.21, respectively, in ARCHITECT, and 9.9 and 1.51, respectively, in ECLusys. The detection rates of endocrinological abnormalities in PCOS were 72.2% and 70.6% in the nonoverweight/obese PCOS group and overweight/obese PCOS group, respectively, in ARCHITECT, and 69.4% and 73.5%, respectively, in ECLusys. CONCLUSION: We obtained cut-off values of LH and the LH/FSH ratio for diagnostic criteria of JSOG criteria for PCOS, that were highly compatible between two major assay systems. These cut-off values will contribute to the diagnosis of PCOS in Japan and presumably in women of Asian ethnicities.

A novel PCOS rat model and an evaluation of its reproductive, metabolic, and behavioral phenotypes.

BACKGROUND: Although animal models of PCOS have been used in many studies, none of them can reproduce both the reproductive and metabolic phenotypes of PCOS. In addition, behavioral parameters have not been evaluated in PCOS animal models. PURPOSE: We tried to produce an improved rat model of PCOS, and the reproductive, metabolic, and behavioral phenotypes of the model rats were evaluated. METHODS: Female rats were implanted with silicon tubes containing oil-dissolved dihydrotestosterone (Oil-DHT) as a new PCOS model. Their phenotypes were compared with those of conventional PCOS model rats (DHT), into which tubes containing crystalline DHT were implanted, and non-DHT-treated rats (control). RESULTS: Both the Oil-DHT and DHT rats showed greater body weight gain, food intake, and fat depot weight than the control rats. Furthermore, these groups showed fewer estrous stages and increased numbers of cystic follicles. The DHT rats exhibited lower ovarian and uterine weights than the control rats, whereas no such changes were observed in the Oil-DHT rats. The Oil-DHT and DHT rats showed less locomotor activity in the light phase than the control rats. CONCLUSIONS: Our proposed PCOS model reproduced both the reproductive and metabolic phenotypes of PCOS and may have potential for PCOS research.

The effects of androgens on metabolic functions in females.

The metabolic effects of androgens and their underlying mechanisms in females have been revealed by recent studies. An excess of androgens can have adverse effects on feeding behavior and metabolic functions and induce metabolic disorders / diseases, such as obesity, insulin resistance, and diabetes, in women and experimental animals of reproductive age. Interestingly, these effects of androgens are not observed in ovariectomized animals, indicating that their effects might be dependent on the estrogen milieu. Central and peripheral mechanisms, such as alterations in the activity of hypothalamic factors, reductions in energy expenditure, skeletal muscle insulin resistance, and ß-cell dysfunction, might be related to these androgens' effects. J. Med. Invest. 68 : 228-231, August, 2021.

Effects of gonadal status and the estrogen milieu on hypothalamic oxytocin gene expression and serum oxytocin levels in female rats.

Oxytocin (OT) and its receptor (OTR) play various roles in the central and peripheral regulation of appetite and body weight. Previously, we have shown that the administration of OT markedly decreased appetite and body weight gain in ovariectomized (OVX) obese rats. In addition, recent studies have shown that the endogenous OT system is also affected by endogenous or exogenous estrogen. In this study, we showed that ovariectomy decreased rats' hypothalamic OT/OTR mRNA and serum OT levels, but did not affect their visceral fat OTR mRNA levels. The chronic administration of estradiol (E2) abrogated these ovariectomy-induced changes; i.e., it increased the rats' hypothalamic OT/OTR mRNA and serum OT levels, and may be associated with reductions in food intake and body weight gain. In addition, acute E2 administration increased the rats' hypothalamic OTR mRNA and serum OT levels, but did not affect their hypothalamic OT mRNA levels. Taken together, these results suggest that endogenous OT and/or OTR expression might be positively regulated by E2 and that the suppressive effects of E2 on appetite and body weight gain might be mediated, at least in part, by the OT system. Thus, we consider that OT might be a target hormone to pursue subsequent interventions of menopause for menopause-induced metabolic disorders.

The effects of chronic oxytocin administration on body weight and food intake in DHT-induced PCOS model rats.

Polycystic ovary syndrome (PCOS) is commonly associated with metabolic disorders, which are exacerbated by obesity. Recent studies have revealed that oxytocin contributes to metabolic, appetite, and body weight regulation. In the present study, we evaluated the effects of chronic administration of oxytocin on body weight, food intake, and fat mass in a dihydrotestosterone-induced rat model of PCOS. Body weight, body weight change, and relative cumulative food intake were significantly lower in the oxytocin-treated PCOS rats than in the vehicle-treated control PCOS rats. Similarly, visceral adipocyte size was significantly smaller in the oxytocin-treated PCOS rats than in the vehicle-treated control PCOS rats. On the other hand, the numbers of cystic follicles in the ovary did not differ between the two groups. The chronic administration of oxytocin did not affect the rats' serum aspartate aminotransferase, alanine aminotransferase, or lactate dehydrogenase levels, indicating that it does not have adverse effects on hepatic function. These findings suggest that oxytocin could be a candidate drug for preventing the onset of obesity-related metabolic disorders in PCOS patients.

The reduction in sexual behavior induced by neonatal immune stress is not related to androgen levels in male rats.

PURPOSE: It is known that various types of stress in early life increase the incidence of diabetes, myocardial infarctions, and psychiatric disorders in adulthood. We examined the mechanism by which neonatal immune stress reduces sexual behavior in adult male rats. METHODS: Male rats were randomly divided into 3 groups: the control (n = 17), postnatal day 10 lipopolysaccharide (PND10LPS) (n = 31), and PND25LPS (n = 16) groups, which received intraperitoneal injections of LPS (100 µg/kg) or saline (injection volume: ≤0.1 ml/g) on postnatal days 10 and 25. In experiment 1, male rats (age: 11 to 12 weeks) were put together with female rats in a one-to-one setting for mating, and sexual behavior (mounting, intromission, and ejaculation) was monitored for 30 minutes. The serum levels of luteinizing hormone (LH) and testosterone (T) and the hypothalamic mRNA expression levels of factors related to sexual behavior were examined. After experiment 1 finished, the remaining 37 male rats were used for experiment 2: the control group (n = 8), PND10 LPS group (n = 21) and PND25LPS group (n = 8) these rats had been given an i.p. injection of the saline during the expriment1. All of the rats were orchidectomized at 14 weeks of age. After a 3-week recovery period, a silastic tube containing crystalline T was subcutaneously implanted into the back of each rat. The rats' sexual behavior, serum hormone concentrations, and hypothalamic mRNA expression levels were assessed. RESULTS: In experiment 1, preputial separation occurred significantly later in the PND10LPS group than in the control group. The frequency of sexual behavior was significantly lower in the PND10LPS group than in the control group. The serum T concentrations of the PND10LPS and PND25LPS groups were significantly lower than that of the control group, but the serum LH concentrations of the 3 groups did not differ significantly. The hypothalamic mRNA expression levels of progesterone receptor B (PRB) and gonadotropin-releasing hormone (GnRH) were significantly lower in the PND10LPS and PND25LPS groups than in the control group, whereas the hypothalamic PRA + B mRNA expression levels of the 3 groups did not differ significantly. In experiment 2, after T supplementation the frequency of sexual behavior was significantly lower in the PND10LPS and PND25LPS groups than in the control group, although there were no significant differences in the serum T or LH concentrations or the hypothalamic PRB, PRA + B, or GnRH mRNA expression levels of the 3 groups. CONCLUSION: In male rats, immune stress in the early neonatal period delayed sexual maturation, reduced sexual behavior, suppressed the serum T concentration, and downregulated the hypothalamic mRNA expression levels of GnRH and the PR in adulthood. The delayed sexual maturation was presumed to have been caused by the reduction in the serum T concentration. However, the rats that experienced neonatal stress exhibited reduced sexual behavior irrespective of their serum T concentrations.

The effects of chronic testosterone administration on hypothalamic gonadotropin-releasing hormone regulatory factors (Kiss1, NKB, pDyn and RFRP) and their receptors in female rats.

The effects of androgens on gonadotropin-releasing hormone (GnRH) secretion in females have not been fully established. To clarify the direct effects of androgens on hypothalamic reproductive factors, we evaluated the effects of chronic testosterone administration on hypothalamic GnRH regulatory factors in ovariectomized (OVX) female rats. Both testosterone and estradiol reduced the serum luteinizing hormone levels of OVX female rats, indicating that, as has been found for estrogen, testosterone suppresses GnRH secretion via negative feedback. Similarly, the administration of testosterone or estradiol suppressed the hypothalamic mRNA levels of kisspeptin and neurokinin B, both of which are positive regulators of GnRH, whereas it did not affect the hypothalamic mRNA levels of the kisspeptin receptor or neurokinin-3 receptor. On the contrary, the administration of testosterone, but not estradiol, suppressed the hypothalamic mRNA expression of prodynorphin, which is a negative regulator of GnRH. The administration of testosterone did not alter the rats' serum estradiol levels, indicating that testosterone's effects on hypothalamic factors might be induced by its androgenic activity. These findings suggest that as well as estrogen, androgens have negative feedback effects on GnRH in females and that the underlying mechanisms responsible for these effects are similar, but do not completely correspond, to the mechanisms underlying the effects of estrogen on GnRH.

The effects of chronic testosterone administration on body weight, food intake, and adipose tissue are changed by estrogen treatment in female rats.

In females, estrogens play pivotal roles in preventing excess body weight (BW) gain. On the other hand, the roles of androgens in female BW, appetite, and energy metabolism have not been fully examined. We hypothesized that androgens' effects on food intake (FI) and BW regulation change according to the estrogens' levels. To evaluate this hypothesis, the effects of chronic testosterone administration in ovariectomized (OVX) female rats with or without estradiol supplementation were examined in this study. Chronic testosterone administration decreased BW, FI, white adipose tissue (WAT) weight, and adipocyte size in OVX rats, whereas it increased BW, WAT weight, and adipocyte size in OVX with estradiol-administered rats. In addition, chronic testosterone administration increased hypothalamic CYP19a1 mRNA levels in OVX rats, whereas it did not alter CYP19a1 mRNA levels in OVX with estradiol-administered rats, indicating that conversion of testosterone to estrogens in the hypothalamus may be activated in testosterone-administered OVX rats. Furthermore, chronic testosterone administration decreased hypothalamic TNF-α mRNA levels in OVX rats, whereas it increased hypothalamic IL-1ß mRNA levels in OVX with estradiol-administered rats. On the other hand, IL-1ß and TNF-α mRNA levels in visceral and subcutaneous WAT and liver were not changed by chronic testosterone administration in both groups. These data indicate that the effects of chronic testosterone administration on BW, FI, WAT weight, and adipocyte size were changed by estradiol treatment in female rats. Testosterone has facilitative effects on BW gain, FI, and adiposity under the estradiol-supplemented condition, whereas it has inhibitory effects in the non-supplemented condition. Differences in the responses of hypothalamic factors, such as aromatase and inflammatory cytokines, to testosterone might underlie these opposite effects.

Relationship between serum anti-Mullerian hormone and clinical parameters in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an ovulatory disorder that affects 6-10% of women of reproductive age. Serum AMH level may be an additional factor, or surrogate of PCOM, in the diagnostic criteria of PCOS. We evaluated the correlations between the serum AMH level and various endocrine and metabolic features in PCOS using the latest fully automated assay. Serum AMH level was compared between 114 PCOS patient (PCOS group) and 95 normal menstrual cycle women (Control group). Correlations between serum AMH level and various endocrine and metabolic factors were analysed in PCOS group. The serum AMH level was significantly higher in the PCOS group (8.35±8.19 ng/mL) than in the Control group (4.99±3.23 ng/mL). The serum AMH level was independently affected by age and the presence of PCOS on multiple regression analysis. Ovarian volume per ovary (OPVO) showed the strongest positive correlation (r=0.62) with the serum AMH level among related factors. On receiver operating characteristic (ROC) curve analysis, the cut-off value of AMH for the diagnosis of PCOS was 7.33 ng/mL, but this value did not have high efficacy (sensitivity 44.7%, specificity 76.8%). A cut-off value of 10 ng/mL had a high specificity of 92.6%, although the sensitivity was low (24.6%). The serum AMH level was elevated and reflected ovarian size in PCOS patients. The serum AMH level could be a surrogate for ultrasound findings of the ovaries in PCOS and might be useful for estimating ovarian findings without transvaginal ultrasound in the diagnosis of PCOS.

Kisspeptin mRNA expression is increased in the posterior hypothalamus in the rat model of polycystic ovary syndrome.

Hypersecretion of luteinizing hormone (LH) is a common endocrinological finding of polycystic ovary syndrome (PCOS). This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of gonadotropin-releasing hormone (GnRH). We evaluated the relationship between the hypothalamic-pituitary-gonadal axis (HPG axis) and kisspeptin using a rat model of PCOS induced by letrozole. Letrozole pellets (0.4 mg/day) and control pellets were placed subcutaneously onto the backs of 3-week-old female Wistar rats. Body weight, vaginal opening and vaginal smear were checked daily. Blood and tissues of ovary, uterus and brain were collected at 12-weeks of age. An hypothalamic block was cut into anterior and posterior blocks, which included the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), respectively, in order to estimate hypothalamic kisspeptin expression in each area. The letrozole group showed a similar phenotype to human PCOS such as heavier body weight, heavier ovary, persistent anovulatory state, multiple enlarged follicles with no corpus luteum and higher LH and testosterone (T) levels compared to the control group. Kisspeptin mRNA expression in the posterior hypothalamic block including ARC was higher in the letrozole group than in the control group although its expression in the anterior hypothalamic block was similar between groups. These results suggest that enhanced KNDy neuron activity in ARC contributes to hypersecretion of LH in PCOS and might be a therapeutic target to rescue ovulatory disorder of PCOS in the future.