Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

BACKGROUND: Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. OBJECTIVE AND DESIGN: In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. RESULTS: The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

Human oocyte morphometry before and after cryopreservation: A prospective cohort study.

The cryopreservation of human oocytes is an important strategy to spare fertility in women submitted to gonadotoxic therapy, ovarian surgery, or even to allow gestation by assisted reproduction technology after natural ovarian senescence. Methods to predict oocyte resistance to cryopreservation are still based on qualitative morphological assessment. In this study we evaluated whether morphometric characteristics of mature oocytes before vitrification and after warming are related to successful fertilization by intracytoplasmic sperm injection (ICSI). This was a prospective cohort study including 28 infertile women and 71 oocytes. Morphometric assessments included oocyte diameter, perivitelline space (PS), zona pellucida (ZP) and first polar body (PB). Out of 49 warmed oocytes, 27 (55%) survived cryopreservation and their pre-vitrification measures were similar to those of the 22 oocytes that perished. However, the oocytes that eventually failed to be fertilized had undergone more enlargement of the total diameter (p = 0.029) and shrinking of the PS (p = 0.033) after cryopreservation, compared to oocytes that were successfully fertilized. These findings suggest that the morphometric characteristics of fresh oocytes do not predict their survival to vitrification, while fertilization failure is associated with oocyte enlargement and PS shrinking after cryopreservation.

BMP-4 increases activin A gene expression during osteogenic differentiation of mouse embryonic stem cells.

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with ß-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p < 0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.

Rapid thawing human sperm does not affect basic parameters in normozoospermic men: a double-blind prospective study.

PURPOSE: To compare sperm recovery from slow versus rapid thawing technique using thirty-eight normozoospermic human sperm samples, as follows. Twentyone samples from men taking part in routine infertility screening exams (infertile group) and seventeen from proven fertile volunteer men with at least one child (fertile group). MATERIALS AND METHODS: After analysis of motility, concentration, strict morphology and functional integrity of membranes, sperm was divided into two aliquots of 0.5 mL each and frozen in TyB-G medium. Samples were thawed at room temperature (25 ± 2° C) for 25 minutes (slow thaw) or in a water bath at 75° C for 20 seconds followed by water bath at 37° C for 3 minutes (rapid thaw). After thawing, motility, strict morphology and functional integrity of membranes were evaluated by a blinded investigator. The results were expressed as mean ± standard deviation for parametric variables and analyzed using Student's t-test. Data with unpaired non-parametric variables were expressed as median (interquartile range) and analyzed by the Mann-Whitney test. Wilcoxon test was used to analyze non-parametric paired variables. RESULTS: There was no significant difference between techniques for total and progressive motility, percentage of normal morphological forms, hypoosmotic swelling test. CONCLUSIONS: Although the rapid thawing protocol was completed in a shorter time (three minutes and 20 seconds versus 25 minutes, respectively), it wasn't harmful since both techniques showed comparable spermatozoa recovery. Additional research is needed to confirm its safety in clinical research before introducing this methodology in routine assisted reproduction.

Assessment of fertility protection and ovarian reserve with GnRH antagonist in rats undergoing chemotherapy with cyclophosphamide.

BACKGROUND: Reproductive function following chemotherapy is of increasing importance given that survival rates are improving. We assessed whether a gonadotropin-releasing hormone antagonist (GnRHant; cetrorelix) could promote ovarian protection against damage due to chemotherapy. METHODS: Forty-two female Wistar rats were used in this study. Animals were divided into four groups: group I (n=9) received placebo twice; group II (n=12) received placebo+cyclophosphamide (CPA); group III (n=12) received GnRHant+CPA; and group IV (n=9) received GnRHant+placebo. After medication, the estrous cycle was studied through vaginal smears. Rats were mated, pregnancy was documented and the number of live pups evaluated. Afterwards, rat ovaries were removed and prepared for histological studies. The ovarian cross-sectional area was measured and follicles were counted. RESULTS: Cyclic changes in vaginal smears were observed in all but one animal after treatment, but group II had a significantly lower rate of animals with proestrus or estrus (p<0.01). The offspring was markedly reduced by CPA treatment (group II, 3.00+/-1.33 pups vs. group I, 11.44+/-0.78 pups, p<0.01) and this effect was partly reversed by pre-treatment with GnRHant (group III, 7.00+/-1.31 pups). The ovarian cross-sectional area was not significantly different between groups, neither was the number of individual follicle types. However, rats in Group IV had a higher total number of ovarian follicles than those in the control group (17.1+/-1.22 vs. 10.9+/-0.70, p<0.05). CONCLUSION: The use of a GnRHant before CPA chemotherapy provided protection of fertility.

Hemoglobin levels predict quality of life in women with heavy menstrual bleeding.

OBJECTIVE: This study was designed to evaluate the effects of heavy menstrual bleeding (HMB) on the quality of life (QoL). METHODS: A prospective, observational study was conducted including 58 patients with HMB, aged 35 years or older, with a negative pregnancy test result, menstrual blood loss >80 ml, uterine volume up to 200 cc and negative endometrial biopsy. The QoL was evaluated by interview using the Short Form-36 (SF-36) questionnaire. Blood loss, measured by Pictorial Blood Loss Assessment Chart (PBAC), and hemoglobin levels were also assessed. Statistical analysis was performed using the Pearson coefficient correlation test. RESULTS: The age of the patients ranged from 35 to 52 years (42.8+/-0.2 years). Increase in monthly expenses, negative implications in conjugal life, work impairment and health-care utilization due to HMB were seen in 96.5, 94.7, 66.7 and 59.6% of the patients, respectively. Hemoglobin levels correlated to SF-36 physical and mental composites scores (p=0.020 and p=0.027, respectively). PBAC score was not correlated with the QoL (physical composite score: p=0.222 and mental composite score: p=0.642) or with hemoglobin levels (r=-0.065; p=0.278). Hemoglobin and QoL showed significant improvement after treatment (p<0.001). Hemoglobin level was the only independent predictor of the QoL measured by SF-36 physical (p=0.03) and mental (p=0.04) composites scores. CONCLUSIONS: HMB had significant repercussions in the social, medical and economic aspects of women. The impact on the QoL was associated with the hematimetric parameters.

Effect of a combination of ethinylestradiol 30 microg and drospirenone 3 mg on tolerance, cycle control, general well-being and fluid-related symptoms in women with premenstrual disorders requesting contraception.

PURPOSE: Positive effects on premenstrual symptoms have been observed with low-dose oral contraceptives. Drospirenone is a synthetic progestogen with antiandrogenic and antimineralocorticoid effects. This open-label, multicenter study evaluated the effects of a combination of ethinylestradiol 30 microg and drospirenone 3 mg on safety, cycle control, general well-being and fluid-related symptoms in women with premenstrual disorders requesting contraception. MATERIALS AND METHODS: A total of 241 healthy volunteers with symptoms of premenstrual disorder was enrolled in the study. Of the final sample, 203 completed the six-cycle treatment and were included in the efficacy analysis whereas 236 were included in the tolerability analysis. The subjects recruited to the study were required to fill up the Psychological General Well-Being Index (PGWBI). RESULTS: There was no significant change in body weight or blood pressure throughout the treatment. Adverse events reported by patients during treatment consisted of those already known to be associated with oral contraceptive use. PGWBI scores were significantly higher after six cycles of treatment compared with baseline values (p<.0001). A total of 198 (84.2%) subjects reported a great improvement in premenstrual symptoms. CONCLUSIONS: The results of this study confirm that oral use of a combination of ethinylestradiol 30 microg and drospirenone 3 mg provides good cycle control, is well tolerated and has a positive impact on symptoms of premenstrual disorder.

Prolactin inhibits oocyte release after gonadotropin stimulation in the rat: putative mechanism involving ovarian production of beta-endorphin and prostaglandin.

OBJECTIVE: To evaluate whether prolactin (PRL) is able to inhibit ovulation induced with exogenous gonadotropins in the rat and whether this effect could be mediated by the ovarian production of beta-endorphin, prostaglandin, and nitric oxide (NO). DESIGN: Controlled in vivo and in vitro experiments. SETTING: Academic research laboratories. ANIMAL(S): Immature female rats undergoing ovulation induction with equine gonadotropins and hCG. INTERVENTION(S): Prolactin (100 or 200 microg), PRL + the opioid antagonist naloxone (200 microg each), or placebo were injected SC 4 hours after hCG administration for ovulation induction. In the in vitro experiments, isolated preovulatory ovaries were incubated with or without PRL in a final concentration of 100 or 200 ng/mL. MAIN OUTCOME MEASURES(S): Number of oocytes ovulated in vivo, ovarian beta-endorphin, PGE(2) and NO(2)(-)/NO(3)(-) release, and NO synthase activity in vitro. RESULT(S): Prolactin reduced significantly the number of oocytes ovulated at the doses of 100 and 200 microg, and this effect was partially reversed by naloxone administration together with 200 mug PRL. PRL also induced a twofold increase in the ovarian release of beta-endorphin and a threefold decrease in the ovarian production of PGE(2). Ovarian NO synthase activity and the concentrations of NO(2)(-)/NO(3)(-) in the incubation medium were not modified by PRL. CONCLUSION(S): Prolactin is able to reduce the number of oocytes released and modulate ovarian beta-endorphin and PGE(2) release, which may account for its peripheral anovulatory effects. This local effect of PRL could interfere in the process of ovulation induction by exogenous gonadotropins.

The impact of a monophasic continuous estro-progestogenic treatment on Latin American menopausal women.

OBJECTIVE: The present study was undertaken to assess the impact, effectiveness and safety of a monophasic hormone replacement treatment (HRT) for continuous use with regards to the clinical effects, bleeding patterns and lipid profile of menopausal women in four Latin American countries. DESIGN: Three hundred and six postmenopausal women with natural menopause and uterus present were recruited. This was a multicentre prospective, clinical trial; the participating countries were Brazil (BR), Colombia (CO), Mexico (MX) and Argentina (AR). The study period was 12 months. The HRT regime was formulated in tablets containing 2 mg estradiol E2 and 1mg norethisterone acetate (NETA); one visit every 3 months was solicited. METHODS: HRT was given as one tablet every day without interruption for 1 year. Climacteric complaints, side-effects, reason for discontinuation, bleeding patterns, lipid profile at baseline and 12 months of treatment were documented. RESULTS: There were no significant differences between the four populations on clinical measurements. Thirty-four women discontinued, 13 for bleeding problems. The five most common side-effects were mastalgia, bleeding problems, headache, pelvic pain and nausea. 44.8% of women experienced scanty vaginal bleeding during the first 3 months of therapy. Ninety seven percent of women had amenorrhea at the end of the study in MX, BR and AR, and 100% in CO. Body weight was constant during the study, and no correlation was found between body weight and total days with bleeding. The Kupperman index score was used to evaluate the climacteric complaints, and the score decreased from a mean of 25.4 to 5.1 at 12-months visit. Total cholesterol levels were significantly reduced in BR and CO (P < 0.05) between baseline and the final sample; serum triglycerides remained unchanged, HDL-cholesterol was significantly increased in MX (P < 0.05), and LDL-cholesterol was significantly reduced in CO (P < 0.05). The results of this 1-year study emphasize that a continuous combined HRT regimen with 2 mg E2/1 mg NETA is an attractive alternative for postmenopausal women who are at least 1 year after their menopause and optimally 2 years after their menopause. Although the combination of 2 mg E2 with 1 mg NETA in a continuous combined therapy scheme has been in use in the Nordic countries for over a decade and in Latin America for the last 6 years, there have been no previous published reports on its effectivity in Latin American women. This publication reports the experience in a group of 306 Latin American women, and it is the first Latin American publication with this formulation.

Spontaneous inflammatory pelvic disease in adult non-castrated female rats treated with estrogen.

The adaptive immune response of the genital tract is under the control of sexual steroids; however, the influence of sex hormones on innate immune mechanisms of the genital mucosa are only beginning to be understood. We found that long-term estrogen treatment increases the risk for inflammatory pelvic diseases in adult non-castrated female rats. Female rats (110 g to 130 g) received estrogen (10 rats; 17-beta estradiol, 50 mg pellet; 10 rats: subcutaneous weekly injection of estradiol valerate 0.166 mg/kg). Ten rats received a pellet of 17-beta estradiol and were treated with amoxicillin, 50 mg/kg after the 90th day of exposure to estrogen. Three control groups of ten rats were also used. The estrogen-treated rats developed an inflammatory pelvic disease, with abscess formation after the third month of hormonal treatment. All the surviving animals were killed after six months of hormonal exposure. Among 15 survivors of the two groups that received estrogen 13 animals presented tuboovarian abscesses. Among eight survivors of the group treated with amoxicillin, six had tuboovarian abscesses. None of the 30 control rats presented macro or microscopic signs of inflammatory disease in the uterus, tubes or ovaries. We conclude that estrogen impairs the defense mechanisms of the genital tract of non-castrated female rats, enhancing bacterial growth in the vagina and ascending infection to the uterus, tubes and ovaries.

Effect of the pretreatment with prolactin on the distribution of immunoreactive beta-endorphin through different ovarian compartments in immature, superovulated rats.

Beta-endorphin and prolactin (PRL) are natural inhibitors of ovulation via central and peripheral mechanisms, but their possible interactions within the ovary are still unknown. The aims of the present study were to determine the gene expression and the topographic distribution of beta-endorphin, and the possible changes evoked by the pretreatment with PRL on the ovarian beta-endorphin localization in immature, superovulated rats. Prepuberal female Wistar rats weighing 60-70 g were superovulated with 20 IU equine gonadotrophins and, 48 h later, 20 IU human chorionic gonalotropin (hCG). Four hours after the hCG injection, the rats received either 200 microg rat PRL .i.p. (n = 12) or saline vehicle (n = 10). In the following morning the rats were killed and their ovaries were quickly removed. Beta-endorphin localization was assessed by immunohistochemistry and proopiomelanocortin (POMC) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Beta-endorphin was expressed mostly in the corpora lutea and perivascular stroma, but a weak to moderate immunostaining was also present in the theca cells and some granulosa cells of tertiary/antral and preovulatory follicles. The main differences observed in the distribution of ovarian beta-endorphin between the two groups were a more intense immunostaining in the granulosa cells of antral follicles, corpus luteum and stroma of PRL-treated rats. POMC gene transcripts were detected in 2/5 samples from the control group and in 3/7 samples from the PRL-treated group. Thus, the expression of beta-endorphin in tertiary/antral follicles is enhanced by PRL treatment in immature, superovulated rats, providing a putative mechanism by which PRL could inhibit the ovarian response to induced ovulation.

Five-year follow-up of levonorgestrel-releasing intrauterine system versus thermal balloon ablation for the treatment of heavy menstrual bleeding: a randomized controlled trial.

BACKGROUND: The study was conducted to compare 5-year follow-up of levonorgestrel-releasing intrauterine system (LNG-IUS) or thermal balloon ablation (TBA) for the treatment of heavy menstrual bleeding (HMB). STUDY DESIGN: A prospective, randomized controlled trial comparing LNG-IUS (n=30) and TBA (n=28) was performed. Hysterectomy rates, hemoglobin level, bleeding pattern, well-being status and satisfaction rates were assessed. Comparisons between groups were performed by χ(2) test and by unpaired and paired t tests. RESULTS: After 5 years of follow-up, women treated with a TBA had higher rates of hysterectomy (24%) compared to the LNG-IUS group (3.7%) due to treatment failure (p=.039). Use of LNG-IUS resulted in higher mean hemoglobin (±SD) levels in comparison to the TBA group (14.1±0.3 vs 12.7±0.4 g/dL, p=.009). Menstrual blood loss was significantly higher in the TBA when compared to the LNG-IUS group (45.5% vs 0.0% p<.001). The psychological general well-being index scores were similar. Patient acceptability, perceived clinical improvement and overall satisfaction rates were significantly higher in women using LNG-IUS. CONCLUSION: Five-year follow-up of HMB treatment with LNG-IUS was associated with higher efficacy and satisfaction ratings compared to TBA.

Computer-assisted oocyte morphometry before ICSI: correlation of oocyte measurements with fertilization and embryo development.

The present study aimed to correlate morphometric parameters of the oocytes with the occurrence of fertilization following intracytoplasmic sperm injection (ICSI). In a prospective, controlled cohort design, women (n = 32) who were candidates for ICSI had oocytes (n = 258) collected and submitted to morphometric evaluation using the Cronus3 software program. The morphometric parameters obtained were oocyte diameter, perivitelline space width, zona pellucida thickness, and first polar body diameter. The median oocyte diameter was similar in cases in which fertilization occurred compared with those in which fertilization failed (75.2 and 75.9 µm, respectively; P = .218). The 2 groups also had similar measurements of perivitelline space, zona pellucida, and first polar body. However, the best quality zygotes identified by a morphological score resulted from oocytes with larger diameter (75.6 vs 74.0 µm; P < .01) and narrow perivitelline space (5.3 vs 7.1 µm; P < .01). Embryo development, as assessed by cleavage at second day of culture, was not significantly associated with oocyte morphometric parameters. These findings suggest that morphometric parameters of the oocytes do not correlate with the occurrence of fertilization following ICSI but may assist in selecting oocytes more likely to originate high-quality zygotes.

Angiotensin-(1-7), its receptor Mas, and the angiotensin-converting enzyme type 2 are expressed in the human ovary.

OBJECTIVE: To investigate whether angiotensin (Ang)-(1-7), its receptor Mas, and angiotensin-converting enzyme type 2 (ACE2) are present in human ovary. DESIGN: Cross-sectional study. SETTING: Academic hospital. PATIENT(S): Twelve reproductive-age women and five postmenopausal women undergoing oophorectomy for nonovarian diseases and seven women having controlled ovarian hyperstimulation for IVF. INTERVENTION(S): Ovarian tissue was obtained from the reproductive-age women and postmenopausal women undergoing oophorectomy for nonovarian diseases. Follicular fluid (FF) samples were obtained from the women having controlled ovarian hyperstimulation for IVF. MAIN OUTCOME MEASURE(S): Localization of Ang-(1-7) and Mas by immunohistochemistry; measurement of Ang-(1-7) in ovarian FF by RIA; detection of messenger RNAs encoding Mas and ACE2 with use of real-time polymerase chain reaction; assessment of 125I-labeled Ang-(1-7) binding to ovarian sections with use of autoradiographic binding assay. RESULT(S): Angiotensin-(1-7) and the receptor Mas were localized to primordial, primary, secondary, and antral follicles, stroma, and corpora lutea of reproductive-age ovaries. Postmenopausal women expressed both the peptide and its receptor in the ovarian stroma. Angiotensin-(1-7) was detectable in FF (mean±SE: 191±54 pg/mL). Both Mas and ACE2 messenger RNAs were expressed in ovarian tissue, as revealed by real-time polymerase chain reaction, and ovarian binding sites for 125I-labeled Ang-(1-7) were identified by autoradiography. CONCLUSION(S): Angiotensin-(1-7), its receptor Mas, and ACE2 are expressed in the human ovary. The peptide is present in several ovarian compartments and can be quantified in FF.

A randomized prospective trial comparing the levonorgestrel-releasing intrauterine system with thermal balloon ablation for the treatment of heavy menstrual bleeding.

BACKGROUND: Use of the levonorgestrel-releasing intrauterine system (LNG-IUS) was compared with thermal balloon ablation (TBA) for the treatment of heavy menstrual bleeding (HMB). STUDY DESIGN: A prospective randomized trial comparing the LNG-IUS (n=30 women) and TBA (n=28 women). RESULTS: Hemoglobin levels increased (p<.001) and blood loss was reduced (p<.001) in both groups after 1 year of treatment. Menstrual bleeding was less in the LNG-IUS group compared to the TBA group at 6 and 12 months of treatment (p=.035 and p=.048, respectively). Intermenstrual bleeding was significantly less in the TBA group at 6 months compared to the LNG-IUS group (p=.044); however, there was no significant difference at 12 months (p=.129). No difference was found in psychological aspects between pre- and posttreatment variables in either of the groups (p=.537). CONCLUSIONS: Both the LNG-IUS and TBA appear to be effective in controlling HMB; however, posttreatment uterine bleeding patterns are different.

c-fos gene and protein expression in pelvic endometriosis: a local marker of estrogen action.

Endometriosis is an estrogen-dependent disease, causing pelvic pain and infertility. c-fos is an early transcription factor that has been reported to be related to estradiol-dependent cell proliferation. The aim of the present study was to assess the c-fos gene and protein expression in pelvic endometriotic implants in comparison to normal endometrium from infertile women. An open, prospective and controlled study included 15 infertile women with endometriosis and 19 control infertile women. Endometrial and endometriotic biopsies were performed at the follicular phase and the samples were processed for RT-PCR and immunohistochemistry. ERalpha mRNA levels were similar in the endometriotic implants/eutopic endometrium from women with endometriosis and in normal tissue (P = 0.649). The aromatase gene, however, was not expressed in the eutopic endometrium from either control or endometriosis groups, and was only expressed in 50% of endometriotic implants (P = 0.044). c-fos gene expression was higher in endometriotic implants (1.32 +/- 0.13; P = 0.011) than in eutopic endometrium from patients with endometriosis (0.97 +/- 0.11) or from the control group (0.91 +/- 0.05). In addition, immunohistochemistry showed a more abundant distribution of c-Fos in the stroma of endometriotic tissue compared to eutopic endometrium. These data suggest that c-fos may play a role in the molecular mechanisms of estrogen action on the induction, promotion or progression of endometriosis.

Expression of 17beta-hydroxysteroid dehydrogenase type 2 in pelvic endometriosis.

Lack of expression or a deficiency of 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2), a key enzyme in estradiol inactivation, could be involved in the pathophysiology of endometriosis. The aim of the present study was to evaluate expression of the gene (17beta-Hsd2) encoding 17beta-HSD2 in eutopic and ectopic endometrial tissues of women with endometriosis. Thirty-four infertile women were divided into a control group, without any clinical or laparoscopic evidence of endometriosis (n = 19), and a group with pelvic endometriosis (n = 15). Diagnosis was confirmed by histological examination of the endometriotic lesions. 17beta-Hsd2 mRNA expression was detected by reverse transcription-polymerase chain reaction in the control group (54% of the samples), in the eutopic endometrium of patients with endometriosis (83% of the specimens analyzed) and in all endometriotic lesions. The semi-quantitative analysis of 17beta-Hsd2 mRNA showed a significantly higher gene expression in the endometriotic implants compared with the intrauterine endometrium of the control group (p < 0.05). 17beta-HSD2 protein was localized to the glandular epithelium of both eutopic endometrium and endometriotic implants. The present results refute the hypothesis of lower or absent 17beta-HSD2 expression in pelvic endometriosis; therefore further studies are needed to assess other potential mechanisms leading to increased estrogenic activity within endometriotic implants.

Rapid thawing human sperm does not affect basic parameters in normozoospermic men: a double-blind prospective study

PURPOSE: To compare sperm recovery from slow versus rapid thawing technique using thirty-eight normozoospermic human sperm samples, as follows. Twenty-one samples from men taking part in routine infertility screening exams (infertile group) and seventeen from proven fertile volunteer men with at least one child (fertile group). MATERIALS AND METHODS: After analysis of motility, concentration, strict morphology and functional integrity of membranes, sperm was divided into two aliquots of 0.5 mL each and frozen in TyB-G medium. Samples were thawed at room temperature (25 ± 2º C) for 25 minutes (slow thaw) or in a water bath at 75º C for 20 seconds followed by water bath at 37º C for 3 minutes (rapid thaw). After thawing, motility, strict morphology and functional integrity of membranes were evaluated by a blinded investigator. The results were expressed as mean ± standard deviation for parametric variables and analyzed using Student's t-test. Data with unpaired non-parametric variables were expressed as median (interquartile range) and analyzed by the Mann-Whitney test. Wilcoxon test was used to analyze non-parametric paired variables. RESULTS: There was no significant difference between techniques for total and progressive motility, percentage of normal morphological forms, hypoosmotic swelling test. CONCLUSIONS: Although the rapid thawing protocol was completed in a shorter time (three minutes and 20 seconds versus 25 minutes, respectively), it wasn't harmful since both techniques showed comparable spermatozoa recovery. Additional research is needed to confirm its safety in clinical research before introducing this methodology in routine assisted reproduction.

Histological evaluation of the effects of cryopreservation in bovine ovarian tissue.

The aim of this study was to show the effects of freezing and thawing in bovine ovarian tissue by histological analysis. Ten cortical slices (2-4 mm in diameter) were obtained from each ovary by tru-cut biopsy and randomly divided into two groups: five fragments were immediately processed as a fresh tissue control group, while the remaining 5 fragments were slowly frozen using DMSO plus sucrose as cryoprotectors, then stored for two weeks and quickly thawed. Histological examination of all cryopreserved ovarian fragments showed no damage in the structure of the organ. Furthermore, there was no difference in the average number of primordial and primary follicles between the two groups of ovarian tissue. These data suggest that the bovine ovary can be used as a suitable model to test new freezing and thawing procedures in search for a standard protocol of human ovary cryopreservation.