Effect of controlled ovarian hyperstimulation on puberty and estrus in mice offspring.

Controlled ovarian hyperstimulation (COH) is widely used for the treatment of infertility, while the long-term effects of COH on the reproductive function in female offspring are currently unknown. Based on the fact that COH could cause high E2 levels in women throughout pregnancy and excess estrogenic exposure during fetal development is harmful to subsequent adult ovarian function, we assumed the hypothesis that COH disrupts reproductive function in female offspring. To test this hypothesis, COH was induced in mice to obtain female offspring by pregnant mare serum gonadotropin (PMSG) and HCG, and then we evaluated pubertal transition, serum levels of E2, anti-Mullerian hormone (AMH), FSH and LH, mRNA expressions of Esr1, Amhr2, Fshr and Lhcgr in ovaries, number of follicles and ovarian histology. We also investigated the apoptosis of follicles by TUNEL; the mRNA expressions of Fas, FasL, Bax, Bcl2, and caspase 3, 8 and 9 by quantitative real-time PCR; and the protein expressions of cleaved-caspase (CASP) 3, 8 and 9 by Western blot. Moreover, we further observed estrous cyclicity in young adult offspring, performed follicle counting and measured the level of AMH in both serum and ovary. COH could induce detrimental pregnancy outcomes, as well as delayed pubertal transition and irregular estrous cycle due to the aberrant growth and maturation of follicles in female offspring. Our novel findings add new evidence to better understand the potential risks of COH on the reproductive function in female offspring, raising the awareness that COH could exert adverse effects on female offspring, rather than just obtain more oocytes for fertilization.

'The child that tiire doesn't give you, God won't give you either.' The role of Rotheca myricoides in Somali fertility practices.

The paper introduces the Baanashada Dumarka, a Somali fertility therapy carried out by a spirit medium, known locally as 'Alaqad. Baanashada is aimed at women whose fertility issues are believed to be caused by spirits. The study also explores a component of the Baanashada, namely, the use of tiire (Rotheca myricoides), or the butterfly bush. Although Rotheca myricoides is known to possess a number of medicinal components as confirmed by studies of modern science, so far, there exist no studies on its potential (or lack of) fertility effects. Hence, the alleged fertility benefits of the butterfly bush need examining. In 2008 a British Somali woman died of herbs placed in her cervix by a traditional healer in Somaliland. This piece of information indicated not only the role of herbal medicine in fertility practices, but also the popularity of traditional reproductive medicine beyond border, class or educational background. Yet, current research into Somali women's health focuses mainly on Female Genital Mutilation (FGM), examined often without the context of wider cultural practices. This paper, however, suggests that rituals, beliefs and material culture play a paramount role in women's practices. For example, as explored elsewhere, the wagar, a wooden and sacred object made of the African olive, is critical for fertility practices. The current paper illuminates further the significance of reproduction practices in Somali society and the potential continuity of traditions associated with the perpetuation of kinship. It concludes that fertility rituals are part of a wider context of interaction with sacred landscapes, objects and archaeological sites, often associated with past legends in the Horn of Africa.

Impaired mitochondrial function in murine oocytes is associated with controlled ovarian hyperstimulation and in vitro maturation.

The present study was designed to determine whether controlled ovarian hyperstimulation (COH) and in vitro maturation (IVM), two common clinical procedures in human IVF treatment, have an impact on mitochondrial DNA (mtDNA) copy number and mitochondrial function in oocytes. Matured mouse oocytes recovered following COH, IVM and natural cycles (NC), which simulated those treatments in human clinic IVF treatment. The copies of mtDNA, the activity of mitochondria as determined by inner mitochondrial membrane potential and oocyte adenosine trisphosphate (ATP) content, pattern of mitochondrial distribution, reactive oxygen species (ROS) levels and the integrity of the cytoskeleton were evaluated in oocytes. Significant differences were detected between COH and NC groups in all measures, except the pattern of mitochondrial distribution and ROS levels. There were also significant differences detected between IVM and NC treatment groups in the copies of mitochondrial DNA, the level of ROS and the integrity of the cytoskeleton in oocytes. In conclusion, the results of this investigation indicate that non-physiological COH and IVM treatments inhibit mtDNA replication, alter mitochondrial function and increase the percentage of abnormal cytoskeleton and ROS production. Damage related to the mitochondria may partly explain the low efficiency of IVF and high rate of embryonic loss associated with these clinical procedures.

Cytotoxic effects of nasal buserelin on nasal mucosal tissue in rabbits.

To investigate the cytotoxic effects of nasal buserelin on rabbit nasal mucosal tissue, twenty-four female rabbits were studied prospectively. The rabbits were divided into 4 groups including 6 rabbits. The rabbits' left noses were included in the all study groups: 150 µg/puff/day of buserelin acetate was administered topically twice daily during 21, 42 and 63 days. Saline was administered topically twice daily to the left nasal cavity in the control group. The nasal septal mucosal stripe tissue was carefully removed from underlaying cartilage after sedation. HE staining, Masson's trichrome, toluidine blue and TUNEL staining were used to evaluate mucosal changes. Each preparation was investigated via apoptotic cells, and they were accounted. Kruskal-Wallis test was used to evaluate nonparametric comparison of apoptotic cells. Mononuclear cells have been raised in the sub-epithelial connective tissue, nucleuses of epithelial cells in the apical region were pyknotic, and apoptotic cells were determined on 21-day group. In the 42-day group, nasal epithelial tissue was similar to 21-day group and epithelial cells including pyknotic nucleus were present in this group, too. In the 63-day group, epithelial cells were light colored. Venous sinuses in the sub-epithelial connective tissue were wide but not congested and not raised collagen filaments. In the intra-epithelial tissue, some of cells were TUNEL (+). Apoptotic cells were fewer in the control group according to 21-day group. In 42- and 63-day groups, these cells were fewer than in 21-day group. Numerical difference was present between the groups, but statistical significance was not found between the groups. We concluded that nasal buserelin cytotoxicity was not potent in the nasal cavity in rabbits. We use nasal buserelin in all indications with confidence.

Effects of tocotrienol from Bixa orellana (annatto) on bone histomorphometry in a male osteoporosis model induced by buserelin.

INTRODUCTION: Osteoporosis is a debilitating skeletal side effect of androgen deprivation therapy based on gonadotropin-releasing hormone (GnRH) agonist in men. Tocotrienol from Bixa orellana (annatto) has been demonstrated to offer protection against osteoporosis by exerting anabolic effects on bone. Thus, it may prevent osteoporosis among GnRH agonist users. OBJECTIVE: This study aimed to determine the effectiveness of annatto-tocotrienol on the bone turnover markers and bone histomorphometry in a model of male osteoporosis induced by buserelin (a GnRH agonist). METHODS: Forty-six three-months-old male Sprague-Dawley rats (three months old; 300-350 g) were randomly divided into six groups. The baseline control group (n = 6) was sacrificed at the onset of the study. The normal control group (n = 8) received corn oil (the vehicle of tocotrienol) orally daily and normal saline (the vehicle of buserelin) subcutaneously daily. The buserelin control (n = 8) received corn oil orally daily and subcutaneous buserelin injection 75 µg/kg/day daily. The calcium control (n = 8) received 1% calcium in drinking water and subcutaneous buserelin injection 75 µg/kg/day. The remaining rats were treated with two different treatments, i.e., (1) oral annatto tocotrienol at 60 mg/kg/day plus subcutaneous buserelin injection 75 µg/kg/day (n = 8); (2) oral annatto tocotrienol at 100 mg/kg/day plus subcutaneous buserelin injection 75 µg/kg/day (n = 8). The rats were injected with calcein twice before being sacrificed to label the bones. The rats were euthanized, and their blood and right femur were harvested at the end of the treatment for bone turnover markers and bone histomorphometry examination. RESULTS: Both serum osteocalcin and C-telopeptide of type 1 collagen were not significantly different between treated groups and buserelin control (P > 0.05). The buserelin control group had a significantly lower bone volume and higher eroded surface compared with the normal control group (P < 0.05). Both groups treated with annatto tocotrienol (60 mg/kg/day and 100 mg/kg/day) had significantly higher bone volume, trabecular thickness and osteoblast number, as well as a significantly lower single-labelled surface compared with the buserelin control (P < 0.05). Only rats treated with annatto tocotrienol 60 mg/kg/day had a significantly higher double-labelled surface compared with buserelin control (P < 0.05). CONCLUSION: Annatto tocotrienol can prevent trabecular bone loss by increasing the mineralising surface and osteoblasts number. Thus, it has a potential role in preventing bone loss in men using GnRH agonist.

Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte.

OBJECTIVE: To study the effect of artificial oocyte activation (AOA) on chromosome segregation errors in the meiotic divisions. DESIGN: Prospective cohort study with historical control. SETTING: Private/academic IVF centers. PATIENT(S): Fifty-six metaphase II oocytes were donated from 12 patients who had undergone IVF between June 2008 and May 2009. INTERVENTION(S): Oocytes were activated by 40 minutes' exposure to 100 µM calcium-ionophore. The activated oocyte was tubed and analyzed by array comparative genomic hybridization and/or single-nucleotide polymorphism genotyping and maternal haplotyping (meiomapping). A control sample of embryos derived from normally fertilized oocytes was included for comparison. MAIN OUTCOME MEASURE(S): Incidence of chromosome segregation errors in artificially activated and normally fertilized oocytes in relation to pronuclear evaluation. RESULT(S): Of 49 oocytes that survived the warming procedure, thirty-nine (79.6%) activated. Most activated normally, resulting in extrusion of the second polar body and formation of a single or no pronucleus (2PB1PN: 30 of 39, 76.9%; or 2PB0PN: 5 of 39, 12.8%). Twenty-seven of these were analyzed, and 16 (59.3%) were euploid, showing no effect of AOA on meiotic segregation. Single-nucleotide polymorphism analysis of normally activated oocytes confirmed normal segregation of maternal chromosomes. No difference in the proportion of meiosis II type errors was observed between artificially activated oocytes (28.6%; 95% confidence interval 3.7%-71.0%) compared with embryos obtained from normally fertilized oocytes (44.4%; 95% confidence interval 13.7%-78.8%). The abnormally activated oocytes, with ≥2PN (4 of 39, 10.3%) were diploid, indicating a failure to coordinate telophase of meiosis II with polar body extrusion. CONCLUSION(S): From this preliminary dataset, there is no evidence that AOA causes a widespread increase in chromosome segregation errors in meiosis II. However, we recommend that it be applied selectively to patients with specific indications.

Does prior infertility increase the risk of tubal pregnancy?

One hundred forty-nine patients who underwent surgery for tubal pregnancy at five hospitals in Seattle (WA) between 1975 and 1979 were interviewed to determine the risk factors for this disorder. Their responses were compared with those of 706 control women who had conceived an intrauterine pregnancy during the same time period during which the tubal pregnancies occurred. A higher proportion of cases reported a history of infertility (attempt to conceive without success for a period of at least 1 year) than controls (relative risk [RR] = 2.5; 95% confidence interval [CI] = 1.7-3.7). Women who were diagnosed in the investigation of their infertility as having tubal or ovulatory dysfunction had relative risks of tubal pregnancy of 5.8 (95% CI = 2.1-16.4) and 3.4 (95% CI = 1.3-8.5), respectively. The average time over which subjects had attempted to conceive before index pregnancies that were planned was longer among cases (15.4 months) than among controls (6.9 months). These results support the hypothesis that a history of infertility predisposes women to an increased risk of tubal pregnancy. The authors also found that, among infertile women, about twice as many cases (14.3%) as controls (6.8%) were current fertility drug users (RR = 3.1; 95% CI = 1.1-9.1), which suggests that the use of fertility drug(s) may further increase the risk of tubal pregnancy.

[Assessment of genetic toxicity of the exposure to clomiphene citrate, with various bacterial test systems]. / Valoración de riesgo gentóxico de exposición a citrato de clomifeno, con diferentes sistemas de prueba bacterianos.

Clomiphene citrate (CC) induces framshift mutations on the Salmonella typhimurium Ames strains TA1538, TA97 and TA100 employing in vitro metabolic activation with S9 aerochlor 1254 induced rat livers, but not base pair substitution mutations with neither the standard or the preincubation method. CC induced genolethal DNA damages on the Escherichia coli PolA-/PolA+ with S9 on the preincubation method or without S9 on the disk diffusion one. The severe primary DNA damages produced b CC was verified by the SOS induction on the lysogenic lambda phage induction with De Marini (1988) method and the induction of colicin E1 plasmid on E. coli. These results are suggestive that CC may be an adduct forming compound which is able to inhibit replication if the cell lacks DNA polymerase, or it may produce framshift mutations after replications. CC induced damages could be large lesions conducing to unicatenary DNA strains, that are able to induce the lexA regulated genes. So, the use of this ovulation inductor is a risk of genotoxic damage and it is advisable to do a risk-benefit evaluation in any particular case before its prescription.

Tubo-ovarian dysplasia in relationship with ovulation induction in rats.

OBJECTIVE: To assess tubo-ovarian dysplasia via morphologic and immunohistochemical study of rats exposed to ovulation stimulation protocols. DESIGN: Animal experimental study. SETTING: Academic research hospital. ANIMAL(S): 72 female Wistar rats divided into three groups. INTERVENTION(S): Stimulation protocols using follicle-stimulating hormone (FSH) or clomiphene citrate for 3, 6, or 12 cycles, after which the animals were killed. MAIN OUTCOME MEASURE(S): Ovarian and tubal dysplasia score and immunohistochemical assessment using p53 and Ki67. RESULT(S): The ovarian dysplasia score was statistically significantly higher after 12 stimulation cycles in the groups receiving FSH (group B) or clomiphene citrate (group C) compared with control (group A). The tubal dysplasia score was statistically significantly increased after only three stimulation cycles in groups B and C. The Ki67 proliferation marker was statistically significantly expressed in the ovaries from group C, and in the fallopian tubes from groups B and C. P53 was constantly low in all three groups. CONCLUSION(S): Ovulation stimulation may induce tubal and ovarian histopathologic and immunohistochemical abnormalities with a dose effect. The role of the fallopian tubes and their interaction with the ovaries require further study.

In vivo evaluation of the genotoxic effects of clomiphene citrate on rat reticulocytes: a micronucleus genotoxicity.

OBJECTIVE: To determine the genotoxic effects of clomiphene citrate (CC) on rat reticulocytesin vivo. METHODS: In this prospective, randomized, controlled study, rats were each assigned randomly to the CC 50, CC 100, CC 200, or control group and were given repeat doses of 0.16, 0.32 or 0.64 mg CC, or normal saline, respectively. Each study group received its CC dose in 2 ml of saline intraperitoneally for 5 days, while the control group received only 2 ml of saline. Each treatment cycle was repeated six times. Six months later, the rats were euthanized. Bone marrow tissues were removed, and pluripotent reticulocyte cells with micronuclei, nuclear buds, and binuclear abnormalities were analyzed using an in situmicronuclei assay under light microscopy. The proportion of micronucleated erythrocytes was measured. RESULTS: Fewer cells with nuclear buds and binuclear abnormalities were detected in the CC 50 group and controls. The CC 100 and 200 groups had significantly (p < 0.05) more nuclear buds and binuclear abnormalities compared with the CC 50 group and controls in the cytogenetic analysis of bone marrow stem cells. CONCLUSION: In rats, the micronucleus genotoxicity assay suggests a dose-dependent CC effect on genomic instability in bone marrow stem cells in vivo.

Oral administration of clomiphene to neonatal rats causes reproductive tract abnormalities.

Oral administration of clomiphene at 2, 4, or 8 mg/kg to 4-day-old rats caused multiple histopathological abnormalities of the reproductive tract in both male and female animals. No histopathological abnormalities were observed in 30-day-old male rats at any dose examined. In contrast, 30-day-old females showed hypertrophy of the myometrium at all doses examined, and hypertrophy of the luminal or glandular epithelium, and dilatation of the uterine lumen were observed in the highest dose group. In post-pubertal rats, histopathologically marked changes were observed in the testes and epididymides in males, and in the ovaries and uterus in females in the highest dose group. In addition, relative weight of male reproductive organs in the highest dose group was decreased as compared with that in the controls. These results suggested that early neonatal exposure to clomiphene induced marked reproductive tract abnormalities in males after puberty, as well as in females.

Prevention of osteopenia induced with a gonadotropin-releasing hormone agonist in rats.

We investigated the effects of conjugated estrogens as an add-back replacement drug, incadronate sodium as a bisphosphonate, and alfacalcidol as a vitamin D(3) analog on femoral bone mineral density (BMD) and bone mineral content (BMC) in female rats chronically treated with the gonadotropin-releasing hormone (GnRH) agonist leuprorelin acetate. The chemical castration of the rats by the administration of GnRH agonist for 16 weeks reduced the BMD values to 92.3%, 91.3%, and 93.3% of those of the normal control animals in the whole femur, metaphysis, and diaphysis of the femur, respectively. The BMC value was decreased to 91.0% of that of the normal control animals by the chronic GnRH agonist treatment. However, a simultaneous 8-week administration of conjugated estrogens, bisphosphonate, and vitamin D(3) analog markedly augmented the BMC values to 110.3%, 110.1%, and 114.4%, respectively, of those in the rats treated with the GnRH agonist alone. These findings indicate that antiosteoporotic agents could be useful for preventing induced osteopenia under the careful monitoring of biochemical markers of osteoblastic activity or bone resorption and BMD or BMC in patients undergoing GnRH treatment.