Individual-oocyte transcriptomic analysis shows that genotoxic chemotherapy depletes human primordial follicle reserve in vivo by triggering proapoptotic pathways without growth activation.

Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle 'burn-out' as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.

Stage-III and -IV endometrial cancer: A single oncology centre review of 104 cases.

The objective of this study was to evaluate the clinicopathological characteristics, treatment and prognosis of advanced endometrial cancer (EC). Patients who underwent surgery for advanced EC between January 1995 and December 2012 were retrospectively reviewed. Patients with missing data, concurrent cancers or uterine sarcomas and those who did not undergo surgery were excluded. The effects of clinicopathological factors on progression-free survival (PFS) and overall survival (OS) were analyzed. A total of 104 patients were included. Most presented with endometrioid histology (74%) and stage-III disease (87.5%), and 76.9% underwent optimal cytoreduction. A multivariate analysis confirmed that lymphovascular space invasion (LVSI) is an independent poor prognostic factor for PFS [odds ratio (OR): 21.37, p = 0.005] and OS [OR: 8.09, p = 0.044]. Suboptimal cytoreduction is another independent poor prognostic factor for PFS [OR: 5.68, p < 0.001]. Our study demonstrated that LVSI and optimal cytoreduction are the most significant factors affecting the survival of advanced EC patients.

The Effect of Long Term Pre/postnatal Low/high Dose Nicotine Exposure on Tissue Oxidant/antioxidant Status and DNA Damage in Rats.

BACKGROUND: Most women do not stop smoking either during pregnancy or in the lactation period. This study was carried out to investigate the effect of long term per oral pre/postnatal low/high dose nicotine exposure on fetal plasma/tissue oxidant-antioxidant status in rats. METHODS: The study groups were composed of pups whose parents used or did not use nicotine in pregnancy and lactation period. The pups were divided into 3 groups, each consisting of 10 rats; the control group (normal drinking water), low and high dose nicotine groups according to the dose of nicotine (0.4 mg/kg and 6.0 mg/kg BW/day, respectively) given per oral in drinking water. At the end of the 12(th) month, tissue/hemolysate/plasma oxidant-antioxidant status parameters and 8-hydroxy-2-deoxy-guanosine levels were measured. RESULTS: Plasma cotinine levels were higher in nicotine groups compared to controls (p<0.01). A significant increase in liver malonyldialdehyde levels (p<0.05) and a significant decrease in kidney superoxide dismutase activities (p<0.05) were determined in both nicotine groups compared to controls while no statistically significant difference was found in the other parameters. CONCLUSION: This investigation showed that long term nicotine exposure during-after pregnancy may have an adverse effect on vital organs of the offspring via impairing tissue oxidant/antioxidant balance. Liver and kidney seem to be the mostly affected organs possibly due to their major roles in nicotine metabolism.

Fertility preservation options in women with endometriosis.

Endometriosis is a common, chronic condition in reproductive age women. Although some women may be asymptomatic, most women present with dysmenorrhea, dyspareunia, pelvic pain and/or infertility. Despite the fact that a causal relationship between endometriosis and infertility has not been clearly established, the fecundity rate of untreated women with endometriosis is lower than normal couples. However, suppressive medical therapy for endometriosis has not been shown to improve fecundity rates and may only result in a delay in the use of more effective treatments to achieve pregnancy. In the other hand, surgery for severe endometriosis can be useful to treat infertile women, but several studies reported a lower ovarian reserve after excision of ovarian endometriomas, due to incidental excision of normal ovarian tissue together with the endometrioma wall. Therefore, fertility preservation procedures should be considered to reproductive-age women at risk of impaired fertility related to endometriosis progression or endometriosis surgical treatment. The purpose of this document was to review the current literature regarding fertility preservation techniques for patients diagnosed with endometriosis.

Ectopic pregnancy; risk factors and comparison of intervention success rates in tubal ectopic pregnancy.

OBJECTIVE: The assessment of ectopic pregnancy, its risk factors and comparison of the treatment modalities. MATERIAL AND DESIGN: Between January 2002 and July 2009, 254 ectopic pregnancies were reviewed retrospectively at the Department of Obstetrics and Gynecology, Ege University. Complaints of patients, localizations of ectopic pregnancy and comparison of patients, whether they had medical therapy or surgery, were evaluated. Metotrexate (50 mg/m2 IM) was used in hemodinamically stable patients (hCG concentrations of patients varied between 450 IU/1 and 3660 IU/1). Patients with fetal cardiac activity and serum hCG concentrations higher than 5000 UI1, were treated surgically. Serum hCG concentrations were measured until the hormone was undetectable (< 1 IU/1). RESULTS: Tubal ectopic pregnancy consisted of 95% of ectopic pregnancies in this trial. The most frequently seen symptom was abdominopelvic pain (77%). Ectopic pregnancy occurred in patients including those with a history of pelvic surgery (12%), previous ectopic pregnancy (6%), usage of intrauterine devices (6%), history of infertility (5.5%) and history of pelvic inflamatory disease (4%). While hemodynamically stable, 83 patients were given single dose methotrexate (50 mg/m2), and 165 patients were treated surgically. Totally 93 salpingectomies and 54 salpingostomies were performed. Of 83 patients administered single dose methotrexate, 69 were successfully treated with one course, six patients needed a second course and surgical intervention was performed in eight patients. On the other hand, of patients that underwent surgery, seven of the salpingostomy group needed methotrexate for persistent trophoblasts and three of this group were reoperated. The tube was preserved in 49 patients in the salpingostomy group (90.7%) versus 75 (92.8%) in the methotexate group (p: 0.916). When undetectable hCG levels following initial therapy were considered, no significant difference was found between the two treatment groups (p: 0.804). DISCUSSION: In selected patients with low serum hCG concentrations systemic methotrexate is a good alternative. Early diagnosis of ectopic pregnancy improves medical therapy. Although salpingectomy solves the problem definitely, comprehensive studies are required concerning future fertility of salpingectomy patients compared with salpingostomy patients.

Vulvar endometrioma: a case report.

Endometriosis is a benign and common disorder that is characterized by ectopic endometrium outside the uterus. Extrapelvic endometriosis, like of the vulva, is rarely seen. We report a case of a 47-year-old woman referred to our clinic due to complaints of a vulvar mass and periodic swelling of the mass at the time of menstruation. During surgery, the cyst ruptured and a chocolate-colored liquid escaped onto the surgical field. The cyst was extirpated totally. Hipstopathological examination showed findings compatible with endometriosis. She was asked to follow-up after three weeks. The patient had no complaints and the incision field was clear at the follow-up.

The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone.

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

PPARalpha-dependent induction of liver microsomal esterification of estradiol and testosterone by a prototypical peroxisome proliferator.

Fatty acyl-coenzyme A:estradiol acyltransferase in liver microsomes catalyzes the formation of estradiol fatty acid esters. These estrogen esters are extremely lipophilic and have prolonged hormonal activity because they are slowly metabolized and slowly release estradiol. Our previous studies showed that treatment of female rats with clofibrate or gemfibrozil (peroxisome proliferators commonly used as hypolipidemic drugs) markedly stimulated the liver microsomal esterification of estradiol. Although clofibrate administration is a potent inducer of liver microsomal fatty acyl-coenzyme A:estradiol acyltransferase in rats, it is a poor inducer in mice. In contrast to these observations, Wy-14,643 (an exceptionally potent prototypical peroxisome proliferator) is a strong inducer of fatty acyl-coenzyme A:estradiol acyltransferase in mice. To explore the role of PPARalpha in the induction of fatty acyl-coenzyme A:estradiol acyltransferase and fatty acyl-coenzyme A:testosterone acyltransferase activities by peroxisome proliferators, we fed 0.1% Wy-14,643 to female wild-type and PPARalpha null mice for 11 d. The liver microsomal acyl-coenzyme A:estradiol acyltransferase and acyl-coenzyme A:testosterone acyltransferase activities were increased 4- to 5-fold in wild-type mice fed Wy-14,643, but no increase was observed in null mice. These results demonstrate that induction of acyl-coenzyme A:estradiol acyltransferase and acyl-coenzyme A:testosterone acyltransferase activities by a prototypical peroxisome proliferator is dependent on PPARalpha.

Clotrimazole is a selective and potent inhibitor of rat cytochrome P450 3A subfamily-related testosterone metabolism.

In this study, clotrimazole (CTZ) and ketoconazole (KTZ) were evaluated for their inhibition of testosterone metabolism catalyzed by rat hepatic microsomes differentially expressing certain cytochrome P450 enzymes. The objective was to compare the inhibitory potencies using hepatic microsomes from adult female rats treated with dexamethasone (F-DEX) and hepatic microsomes from vehicle-treated adult male rats (M-VEH), which are known to contain high levels of isozymes CYP3A1 (3A23) and 3A2, respectively. The results demonstrate that CTZ is a very potent and selective inhibitor of the 6beta-hydroxylation of testosterone, a CYP3A-mediated reaction, in all rat metabolic systems tested. The IC(50) value was 9.7 nM in F-DEX, and 6.7 nM in M-VEH for CTZ. The in vitro inhibitory potency for CTZ significantly exceeds the same parameters for KTZ, a well established specific inhibitor of human CYP3A-mediated reactions. It was found that the IC(50) values of KTZ in F-DEX and M-VEH were 69 and 780 nM, respectively. These values for KTZ are 10-fold and 100-fold higher, respectively, than for CTZ. CTZ, at the concentration that inhibits 90% and more of CYP3A-mediated reactions (40 nM), has less than a 10% inhibitory effect on the activities of other rat liver enzymes, such as CYP1A1, -1A2, -2A1, -2B1, -2B2, -2C11, and -2E1. In summary, CTZ is a more potent and selective inhibitor of all CYP3A-mediated reactions than KTZ in rat hepatic microsomes.