Paclitaxel is evidence to reduce growing ovarian follicle growth in mice model study.

We aimed to investigate the effects of paclitaxel on ovarian follicle development and oocyte meiotic competence. Early secondary follicles were cultured individually with or without paclitaxel 2.5 × 10-10, 2.5 × 10-9, and 2.5 × 10-8 M for 12 days. Thereafter, the follicles were treated with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). Follicle morphology, oocytes meiotic maturation, immunofluorescence for α-tubulin of the oocytes, mRNA expression of growth differentiation factor 9 (GDF9), B-cell lymphoma 2 (Bcl2), and Bcl2-associated X (Bax) were examined every 4, 8, and 12 days of the culture period. We demonstrated that high dose paclitaxel treatment decreased follicle survival (p ≤ 0.05), while lower dose (2.5 × 10-9 M) reduced the survival compared to control after 12 days of culture. The number of oocytes at MII stage was not significantly different between control and paclitaxel groups (p > 0.05). Paclitaxel increased GDF9 expression in oocytes after 4 days of the culture (p ≤ 0.05). Bcl2 declined significantly compared to control after 8 days (p ≤ 0.05 for all groups), while Bax expression tended to be consistent (p ≥ 0.05 for all groups). To conclude, high concentration paclitaxel reduces follicle preantral follicle growth, while in lower concentration it decreases more growing follicle growth and survival.

Changes in the cortisol and oxytocin levels of first-time pregnant women during interaction with an infant: a randomized controlled trial.

BACKGROUND: During pregnancy, physiological, psychological, and social changes affect pregnant women's childcare anxiety and childrearing behavior. However, there are scarce reports on hormonal evaluation related to such anxiety and behavior. Herein, we evaluated changes in salivary cortisol (primary outcome) and oxytocin (secondary outcome) levels of first-time pregnant women when interacting with an infant and discussed the relation of these changes to the women's stress level. METHODS: This was a two-arm randomized controlled trial. Participants were randomly assigned using a web-based randomization system. The experimental group involved interaction with an infant for 30 min. The control group involved watching a DVD movie of an infant for 30 min. Saliva samples were collected at preintervention and postintervention. Saliva samples were assayed, and all data were compared between and within the groups using independent t-test and paired t-test with a two-sided 5% significance level. This study was approved by the Research Ethics Committee of St. Luke's International University. RESULTS: A total of 102 women were randomly assigned to the experimental (n = 51) and control (n = 51) groups. Finally, 38 women in the experimental group and 42 women in the control group were analyzed. The salivary cortisol level significantly decreased after the interventions in both groups (t = 4.57, p = 0.00; t = 5.01, p = 0.00). However, there were no significant differences in the salivary cortisol (t = 0.349, p = 0.73) and oxytocin (t = - 1.945, p = 0.58) levels between the two groups. CONCLUSIONS: The salivary cortisol level of first-time pregnant women significantly decreased in the experimental and control groups postintervention, although no significant difference was found between the two groups. Such decrease indicates stress reduction and release among these women. The absence of a significant increase in salivary oxytocin level in both groups may be related to the limitations of an insufficient number of samples that could be analyzed owing to the small saliva volume in some samples and the lack of adequate tactile stimulation of the intervention protocol. These results and procedural limitations provide useful insights into approaching subsequent studies aiming at continuously optimizing detection procedures. TRIAL REGISTRATION: UMIN000028471 (Clinical Trials Registry of University Hospital Information Network. July 31, 2017- Retrospectively registered.

Olfactory Exposure to ß-Caryophyllene Increases Testosterone Levels in Women's Saliva.

INTRODUCTION: From previous studies, we hypothesized that olfactory exposure to ß-caryophyllene stimulates women's libido. However, Japan's sex culture is so closed that it is difficult to test this possibility without accumulating scientific evidence. Therefore, it is necessary to measure the concentration of sex-related hormones in saliva, an experimental technique that is relatively easy to obtain research permission, and to obtain a scientific basis to convince ethics committee reviewers. AIM: The aim of this study is to investigate whether ß-caryophyllene increases salivary testosterone concentrations associated with libido and vaginal sensation during intercourse in women. METHODS: 19 women in the follicular phase of the menstrual cycle participated in the study. The subjects then sat in front of the odor exposure device we had created. Each subject was exposed to dipropylene glycol for 20 minutes, followed by 3% ß-caryophyllene for 20 minutes. Saliva was collected 4 times: before and after control exposure, and before and after ß-caryophyllene exposure. MAIN OUTCOME MEASURE: Salivary testosterone and estrogen concentrations were measured with a competition ELISA. RESULTS: ß-caryophyllene significantly increased the salivary concentration of testosterone (control vs ß-caryophyllene; 0.97 ± 0.05 vs 1.13 ± 0.03, P = .00, 95% confidence interval of control: 0.84-1.09, 95% confidence interval of ß-caryophyllene: 1.04-1.20) but not estrogen (control vs ß-caryophyllene; 1.05 ± 0.03 vs 1.07 ± 0.04, P = .69, 95% confidence interval of control: 0.96-1.12, 95% confidence interval of ß-caryophyllene: 0.98-1.15). STRENGTHS & LIMITATIONS: The personal preferences of the subjects and the order of exposure may have affected the results. CONCLUSION: ß-caryophyllene may be a remedy with fewer side effects for women with decreased libido. We believe that ß-caryophyllene may be a remedy for women with decreased libido. However, this hypothesis must be tested by further clinical studies. Wataru Tarumi, Kazuyuki Shinohara. Olfactory Exposure to ß-Caryophyllene Increases Testosterone Levels in Women's Saliva. J Sex Med 2020;8:525-531.

Women's body odour during the ovulatory phase modulates testosterone and cortisol levels in men.

A growing body of evidence suggests that men may perceive women's bodily odour to be more attractive during the high-fertility ovulatory phase than during other phases in the menstrual cycle. In particular, women's bodily odour may influence important aspects of male mating behaviour, but the precise nature of this phenomena remains to be elucidated. Twenty-six men and five women participated in the study. Each woman wore a cotton T-shirt during the night for 3 days during the ovulatory phase, after which the regions of the T-shirt that had been in contact with the woman's chest, armpits, and back, were cut out of the garment. We evaluated the changes in testosterone and cortisol levels in the saliva of men who smelled these cloth pieces. The odour emitted from the backs of women in the ovulatory phase was found to increase testosterone secretion in men, whereas the odour emitted from the chests of women in the ovulatory phase reduced cortisol secretion in men. These results suggest that the odour of specific body parts of women modulate unconscious physiological reactions in men.

The Effects of Essential Oil on Salivary Oxytocin Concentration in Postmenopausal Women.

Objectives: The aim of this study was to find essential oils that have increased the oxytocin concentration in postmenopausal women. Methods: Fifteen postmenopausal women participated in this study and the effects of 10 different essential oils were investigated. The essential oils included rose otto, sweet orange, lavender, neroli, frankincense, jasmine absolute, ylang ylang, roman chamomile, clary sage, and Indian sandalwood. The subjects were exposed to the control first for 20 min, followed by exposure to an essential oil for 20 min. Each subject received exposure to only a single kind of essential oil per day. Saliva was collected four times for each patient: immediately before and immediately after control exposure, and immediately before and immediately after essential oil exposure. The oxytocin concentration in the saliva was measured using a competitive ELISA kit. Results: The results showed that salivary oxytocin concentrations increased significantly more after exposure to lavender, neroli, jasmine absolute, roman chamomile, clary sage, and Indian sandalwood than after exposure to the control odor. Conclusions: The aroma of certain essential oils may elicit increased secretion of oxytocin in postmenopausal women. This study suggests that olfactory stimulation with any of a number of essential oils increases salivary oxytocin concentrations, which may inhibit aging-induced reduction in muscle mass and function in women.

Determination of Follicular Localization in Human Ovarian Cortex for Vitrification.

PURPOSE: To determine the optimal follicle localization for ovarian vitrification in adolescent and young adult (AYA)-aged (between 15 and 39 years of age) patients with cancer or primary ovarian insufficiency (POI). METHODS: In total, ovaries from 24 women were included in our study. These include women who received ovariectomy for fertility preservation before gonadotoxic treatments for cancer (n = 4), or for the treatment of POI by the in vitro activation method (n = 8), and other women and infants (0-3 years of age) whose ovaries were autopsied (n = 12). Before cryopreservation, a portion of the ovary sampled from cancer and POI patients was used for histological analysis. Depths of follicles from the surface of ovarian cortices were then measured by using digital imaging software. The locations of the follicles at different developmental stages in the ovarian cortex were noted. RESULTS: The mean depth at which the primordial and primary follicles were located was 271 µm in infants. This was deeper in women in their twenties, thirties, and forties (501, 462, and 493 µm, respectively). The majority of secondary follicles were located <1000 µm from the ovarian surface (mean depth, 639 µm). In regard to patients with POI, the mean depth of primordial and primary follicles was 566 µm, whereas 70% of secondary follicles were located >1000 µm deep. CONCLUSION(S): These findings suggest that <1 mm is a potential optimum thickness of normal ovarian tissue for vitrification and a requirement that thicker ovarian cortices include secondary follicles in POI patients.

Cresyl Violet Stains Mast Cells Selectively: Its Application to Counterstaining in Immunohistochemistry.

The thiazine dye toluidine blue (TB) is well known to stain mast cells and hyaline cartilage metachromatically, and thus is mostly often used for their identification. However, TB is not suitable for counterstaining in immunohistochemistry, because of its high-background staining in the cytoplasm of other cell species and in extracellular structures. To expand the knowledge about dyestuffs staining mast cells in consideration with their usage in immunohistochemistry, we determined the stainability of several thiazines and oxazines, which are structurally related compounds to TB, using sections of mast cell-containing tissues. We found that all azine dyes used metachromatically stained mast cells and cartilage. Among these dyes, an oxazines cresyl violet (CV) stained mast cells with lower background, suggesting that those are useful for detecting mast cells and for counterstaining in immunohistochemistry. To ascertain its utility, CV was used in immunostaining of bHSDs in sections from adult rat ovary. Immunopositive signals reflected by DAB development in brown were clearly detected even after CV staining. We conclude that, similar to thiazines, oxazines stain mast cells metachromatically, and that of these, CV is more useful as a counterstain in immunohistochemistry than TB.

Effects of essential oil exposure on salivary estrogen concentration in perimenopausal women.

OBJECTIVES: The menopausal transition is the time from the onset of menstrual changes until one year after the final menstrual period. During this phase, perimenopausal women experience a variety of health-related symptoms, which seemingly derive from declining level of estrogen secretion. It has long been recognized that some essential oils have the efficacy of alleviating menopausal symptoms. On the basis of this, it is possible that these essential oils have the potency to facilitate estrogen secretion in women. The present study investigated this possibility by examining if the olfactory exposure to the essential oil increase salivary estrogen concentration. METHODS: We tested the effect of ten essential oils; clary sage, frankincense, geranium, lavender, jasmine absolute, neroli, rose otto, ylang ylang, orange and roman chamomile, which are thought to relieve perimenopasal symptoms. RESULTS: The results have shown increase of salivary estrogen concentration induced by exposure to geranium and rose otto compared to control odor. CONCLUSION: Together with the previous studies, the present study may give support to the notion that olfactory exposure to some essential oils can influence salivary concentration of estrogen.

The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

Effects of 5α-dihydrotestosterone and 17ß-estradiol on the mouse ovarian follicle development and oocyte maturation.

We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17ß-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10(-6) M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10(-6) M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation.

Involvement of hyaluronan synthesis in ovarian follicle growth in rats.

Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover, the origin of follicular HA is unknown. To clarify the localization of HA and its synthases in small growing follicles, involvement of HA in follicle growth, and gonadotropin regulation of HA synthase (Has) gene expression, in this study, perinatal, immature, and adult ovaries of Wistar-Imamichi rats were examined histologically and biochemically and by in vitro follicle culture. HA was detected in the extracellular matrix of granulosa and theca cell layers of primary follicles and more advanced follicles. Ovarian HA accumulation ontogenetically started in the sex cords of perinatal rats, and its primary site shifted to the intrafollicular region of primary follicles within 5 days of birth. The Has1-3 mRNAs were expressed in the ovaries of perinatal, prepubertal, and adult rats, and the expression levels of Has1 and Has2 genes were modulated during the estrous cycle in adult rats and following administration of exogenous gonadotropins in immature acyclic rats. The Has1 and Has2 mRNAs were predominantly localized in the theca and granulosa cell layers of growing follicles respectively. Treatments with chemicals known to reduce ovarian HA synthesis induced follicular atresia. More directly, the addition of Streptomyces hyaluronidase, which specifically degrades HA, induced the arrest of follicle growth in an in vitro culture system. These results indicate that gonadotropin-regulated HA synthesis is involved in normal follicle growth.

The rat oocyte synthesises melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesised enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole O-methyltransferase). Melatonin directly affects ovarian functions and previous studies have suggested that melatonin is synthesised in the ovary. In the present study, we examined whether AANAT and ASMT are expressed in the adult rat ovary. Reverse transcription-polymerase chain reaction analyses demonstrated that both AANAT and ASMT mRNAs are expressed in the ovary. Western blotting for AANAT protein showed that the ovary, like the pineal gland, contains this enzymatic protein with a molecular mass of 24kDa. Immunohistochemistry revealed that the AANAT protein is localised to the oocyte, corpus luteum and medulla, including mast cells. AANAT protein was found in oocytes at all stages of follicular development, and its levels in oocytes increased progressively throughout follicular development. Furthermore, isolated oocytes metabolised exogenous serotonin to melatonin. These findings demonstrate that melatonin is synthesised from serotonin in oocytes. Melatonin synthesised in the oocyte may be implicated in its own growth or maturation, for example, by acting as a calmodulin antagonist or an antioxidant.

Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence.

OBJECTIVE: To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. DESIGN: Experimental study. SETTING: St. Marianna University School of Medicine. ANIMAL(S): Prepubertal (14-day-old) BDF1 female mice. INTERVENTION(S): Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10(-11) to 10(-5) M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Diameters and morphology of follicles and oocytes; E(2) and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. RESULT(S): Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E(2) and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. CONCLUSION(S): These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

Acute reproductive toxicity of 3,3'-iminodipropionitrile in female rats.

A potent neurotoxin 3,3'-iminodipropionitrile (IDPN), which is an occupational exposure hazard in industry, induces persistent behavioral abnormalities in experimental animals; however, its reproductive toxicity has not been determined. Therefore, we assessed the toxicity of IDPN in the reproductive system of female rats. A single intraperitoneal injection of IDPN (1000 mg/kg body weight) into female Wistar-Imamichi rats caused acute estrous cycle arrest at diestrus for up to 15 days. The arrest was accompanied by follicular atresia, and following arrest, the estrous cycle and ovarian morphology recovered. Ovarian mRNA levels of growth differentiation factor 9 and Fas ligand, a cell death marker, transiently increased following IDPN injection, but eventually they returned to basal levels. IDPN added to in vitro cultures of ovarian follicles also induced the expression of these genes, indicating that IDPN directly promoted ovarian cell death.

Gonadotropins up-regulate the expression of enolase 2, but not enolase 1, in the rat ovary.

It has been demonstrated that the glycolytic enzymes, enolase 1 (ENO1) and enolase 2 (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.

Cryopreservation of ovarian tissue after pretreatment with a gonadotropin-releasing hormone agonist.

PURPOSE: The purpose of this study was to investigate whether slow-rate freezing or vitrification is better for cryopreservation of ovary tissues pretreated with gonadotropin-releasing hormone agonist. METHODS: In this nonclinical study performed in rats, leuprorelin acetate was administered to female Wistar rats, aged 6-8 weeks. After confirming arrest of the estrous cycle by examination of vaginal smears, ovarian tissue was cryopreserved by vitrification and slow-rate freezing prior to thawing and autotransplantation. The time required for estrous cycle recovery was assessed from vaginal smears in each group starting from day 1 of transplantation. Estradiol levels were also monitored after transplantation. RESULTS: The estrous cycle recovered after transplantation of ovarian tissue frozen by either method, but recovery was significantly faster after transplantation of vitrified tissue. The estradiol level also recovered by 10 days after transplantation. CONCLUSIONS: Ovarian function was restored after transplantation of tissue preserved by either vitrification or slow-rate freezing after pretreatment with leuprorelin acetate. This method may be applicable for patients scheduled to undergo cryopreservation of ovarian tissue before chemotherapy.

Ovarian toxicity of paclitaxel and effect on fertility in the rat.

AIM: Taxanes are regarded as key chemotherapy agents for breast cancer and gynecologic malignancies; therefore the ovarian toxicity of paclitaxel (PTX) is a matter of importance to younger women with malignancies. We examined the ovarian toxicity of PTX and its influence on fertility in rats. METHODS: Female Wistar rats aged 6-8 weeks received five doses of PTX at 3-day intervals. Ovarian toxicity was assessed by counting follicles, corpora lutea and atretic follicles, as well as by detecting apoptosis and measuring serum levels of estradiol (E2) and progesterone (P4). Fertility was assessed by mating females immediately or 24 days after PTX treatment. The number of fetuses, implantations and resorption sites was counted in each group. RESULTS: Exposure to PTX caused a decrease of antral follicles, but not primordial or pre-antral follicles. The number of corpora lutea showed a significant decrease, while follicular atresia was increased significantly. Apoptosis was only detected in antral follicles. Serum E2 levels were lower than in control rats, but not significantly, while P4 levels did not differ from those in control rats. Rats mated immediately after PTX treatment showed a significant decrease of fetuses and implantations, but these effects were not detected in rats mated at 24 days after treatment. CONCLUSION: Our findings suggest that the ovarian toxicity of PTX is mild and transient. Use of PTX may help to maintain the fertility of younger women because the fertility of rats was not influenced at 24 days after exposure to this drug.