Effect of arylamine acetyltransferase Nat3 gene knockout on N-acetylation in the mouse.

Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.

The impact of early testicular sperm extraction or cryopreservation on the outcome of intracytoplasmic sperm injection--a randomized controlled study.

PURPOSE: To compare the outcome of sperm extraction 24 h before ovum pickup and on the day of oocyte retrieval. METHODS: A controlled study was performed to compare the outcome of 90 sperm extractions and in vitro sperm injection cycles performed in 54 patients. RESULTS: Available fresh sperm for the sperm injection procedure and cryopreservation obtained on the day of ovum pickup were similar to sperm collected 1 day before (33.3% vs 39.4%, respectively). Fertilization rate obtained with fresh sperm was also similar (48.9% vs. 54%), respectively. Clinical pregnancy rate was 38% vs. 22% per embryo transfer, respectively (P = 0.235). When comparing an additional 24 cycles with cryopreservation of sperm retrieved on the day of ovum pickup, as well as a day previously, no significance was noted in the parameters. CONCLUSIONS: Sperm retrieved 24 h before oocyte retrieval and used as fresh or frozen-thawed for sperm injection are as effective as those used on the day of ovum pickup.

A prospective randomized controlled study of the effect of short coincubation of gametes during insemination on zona pellucida thickness.

A prospective, randomized study was conducted to evaluate the thickness, of zona pellucida (ZP) after brief or standard exposure of human oocytes to spermatozoa, and to determine the correlation between ZP thickness, fertilization rate and embryo quality. The mean ZP thickness 48 h after insemination was found to be significantly less in fertilized oocytes than in non-fertilized oocytes in all treated groups (13.72 +/- 3.0 microns and 15.08 +/- 2.5 microns, respectively; p < 0.007). Zona pellucida thickness correlated positively with embryo quality. Brief exposure of gametes was found to influence ZP thickness. The ZP was significantly thinner after brief and intracytoplasmic sperm injection (ICSI) exposure of oocytes to spermatozoa than after standard in vitro fertilization (IVF). The mean ZP thickness 24 and 48 h after fertilization was significantly greater in standard IVF (16.43 +/- 2.8 microns and 15.22 +/- 2.7 microns, respectively) than in either the brief exposure or ICSI groups (12.78 +/- 2.4 microns and 13.01 +/- 3.5 microns vs. 13.46 +/- 2.2 microns and 13.16 +/- 2.4 microns; p < 0.0001).

Endometrial ablation for the postmenopausal woman.

The postmenopausal woman with abnormal uterine bleeding is considered at risk for developing endometrial neoplasia or one of its precursors. She requires prompt evaluation of the endometrium followed by adequate treatment. In the subgroup of postmenopausal women taking hormone replacement therapy (HRT), the risk of abnormal bleeding is by far higher, is the main reason for discontinuing HRT, and deserves additional attention.

The classic approach to diagnosis of vulvovaginitis: a critical analysis.

OBJECTIVE: To correlate the symptoms, signs and clinical diagnosis in women with vaginal discharge, based on the combined weight of the character of the vaginal discharge and bedside tests, with the laboratory diagnosis. METHODS: Women presenting consecutively to the women's health center with vaginal discharge were interviewed and examined for assessment of the quantity and color of the discharge. One drop of the material was then examined for pH and the whiff test was done; a wet mount in saline and in 10% KOH was examined microscopically. The clinical diagnosis was based on the results of these assessments. Gram stain and cultures of the discharge were sent to the microbiology laboratory. RESULTS: One hundred and fifty-three women with vaginal discharge with a clinical diagnosis of vulvovaginitis participated in the study. Fifty-five (35.9%) had normal flora and the other 98 (64.1%) had true infectious vulvovaginitis (kappa agreement = 18%). According to the laboratory, the principal infectious micro-organism causing the vulvovaginitis was Candida species. Candida infection was associated with pH levels of less than 4.5 (p < 0.0001, odds ratio = 4.74, 95% confidence interval: 2.35-9.5, positive predictive value 68.4%). The whiff test was positive in only a small percentage of bacterial vaginosis (BV) (p = not significant (NS)). Clue cells were documented in 53.3% of patients with a laboratory diagnosis of BV (p < 0.02, positive predictive value 26.7%). CONCLUSIONS: The current approach to the diagnosis of vulvovaginitis should be further studied. The classical and time-consuming assessments were shown not to be reliable diagnostic measures.

"Primary" versus "secondary" vulvar vestibulitis: one disease, two variants.

OBJECTIVE: Our aim was to determine whether women with primary vestibulitis-since the first episode of sexual intercourse-differ in disease characteristics and outcome of operative treatment from women with secondary vestibulitis. STUDY DESIGN: A total of 111 patients with severe vulvar vestibulitis underwent perineoplasty from 1991 to 1995. Thirty-nine (35%) of them had primary vestibulitis (ie, dyspareunia from the first attempt at sexual intercourse). They were compared with 72 (65%) who had secondary vestibulitis with regard to demographic, social, and medical variables, the presence of human papillomavirus deoxyribonucleic acid, physical and histopathologic findings in the vestibule, and surgical outcome. RESULTS: Women with primary vestibulitis were 5 years younger than those who had secondary vestibulitis (22.9 +/- 2.9 years vs 27.7 +/- 8.6 years, respectively; P <.0001) and differed in their marital status (unmarried, 84% vs 56%, respectively; P <.008), parity (nulliparous, 97% vs 67%, respectively; P <.0002), and involvement of the whole vestibule (74% vs 93%, respectively; P <.006). The 2 groups were similar in all other variables, including use of oral contraception, smoking, presence of human papillomavirus, dysuria, success of perineoplasty (average, 83%), and histopathologic findings. CONCLUSIONS: Women with primary vestibulitis were younger than women with secondary vestibulitis. Most other differences were dependent on the different ages of the 2 groups. Primary and secondary vestibulitis may therefore be two presentations of the same disease.

Pure versus complicated vulvar vestibulitis: a randomized trial of fluconazole treatment.

OBJECTIVE: To examine the effectiveness of a 6-month treatment consisting of a weekly oral dose of 150 mg fluconazole for women with vestibulitis, and to explore the causes of treatment failure. METHODS: Forty women with vestibulitis were randomized to either of two treatment groups. One group received a 6-month low oxalate diet with calcium citrate complement, as a placebo, and the second group the same diet and calcium citrate with the addition of a weekly oral tablet of 150 mg fluconazole. The women were examined 3 months after completing treatment, for response to therapy. RESULTS: The addition of intensive 6-month fluconazole treatment did not lead to an outcome better than that attained by maintaining a low oxalate diet with calcium citrate supplementation. The satisfactory response rate was 15 and 30%, respectively. The presence of 'complicated vestibulitis', candidiasis concomitant with vestibulitis, decreases the satisfactory response rate regardless of the type of treatment administered (odds ratio 19.9, 95% CI 1.6, 250). CONCLUSION: Prolonged oral fluconazole is an ineffective treatment of vestibulitis, whether pure or complicated by concomitant vulvovaginal candidiasis. The coexistence of candidiasis and vestibulitis - complicated vestibulitis - might represent a subset of vestibulitis that is resistant to the currently available medical therapy.

Increased endometrial thickness in women with hypertension.

OBJECTIVE: We noticed an increase in endometrial thickness in women with hypertension who were treated with a combination of medications, including beta-blockers. The purpose of this study was to examine whether the endometrium of hypertensive women is thicker than that of healthy women and to determine whether endometrial thickening in hypertensive women is directly related to the antihypertensive beta-blocker treatment. STUDY DESIGN: We compared 3 groups of postmenopausal patients as follows: (1) women with a history of essential hypertension treated with a combination of medications, including beta-blockers; (2) women with a history of hypertension treated with a combination of medications that did not include beta-blockers; and (3) healthy women without hypertension. All patients were interviewed and examined, blood tests were performed, and endometrial thickness in the anterior-posterior diameter was measured by vaginal ultrasonography. Among the exclusion criteria were diabetes or an abnormal fasting blood glucose level, obesity, hormonal medication or replacement hormonal therapy during the previous 6 months, and a history of hormonal disturbances, infertility, or polycystic ovary syndrome. RESULTS: Of 45 hypertensive women enrolled in the study, 22 were treated with a beta-blocker combination medication and 23 were treated with other antihypertensive medications. They were compared with 25 healthy women. There was no statistically significant difference in endometrial thickness between women treated with medications, including beta-blockers, and those who were treated with other hypotensive agents. Twenty percent of women with hypertension and none of the healthy women had endometrium >5 mm thick (P <.017; odds ratio, 8.22; 95% confidence interval, 1.22-infinity). CONCLUSION: Twenty percent of hypertensive postmenopausal women were found to have increased endometrial thickness. However, we were unable to substantiate an association between the type of treatment administered, whether beta-blockers were included, and the increase in endometrial thickness.

Pure follicle-stimulating hormone as an adjuvant therapy for selected cases in male infertility during in-vitro fertilization is beneficial.

OBJECTIVES: Since research has demonstrated the possibility of hormonal therapy for male infertility, we conducted a study to analyze the efficacy of pure follicle-stimulating hormone (pFSH) treatment in patients with idiopathic, severe oligoteratoastheno-spermia (OTA) syndrome, or failed fertilization before referral to an intracytoplasmic sperm injection (ICSI) in an in-vitro fertilization (IVF) program. STUDY DESIGN: A retrospective, clinical study was carried out on 178 men with OTA syndrome. Group I comprised 76 patients selected for treatment with pFSH. Group II comprised 102 men who served as the controls. Pure FSH was administered intramuscularly to the patients in group I. Upon cessation of therapy, an IVF treatment cycle was carried out. RESULTS: After treatment with FSH, sperm motility was the only parameter which significantly improved in Group I (34% vs. 23%, and 15% vs. 24% in the subgroups of Group 1, respectively; P<0.05). In-vitro fertilization pregnancy rates were similar in both groups. The highest spontaneous pregnancy rates were achieved in FSH-treated, severe OTA patients. CONCLUSIONS: Patients with severe male factor infertility may benefit from pFSH in terms of sperm motility, fertilization by IVF and spontaneous pregnancy rates. Selection criteria for FSH treatment are proposed.

Telomerase activity in HPV-associated vulvar vestibulitis.

OBJECTIVE: To find a possible correlation between telomerase activity, mean telomere length and human papillomavirus (HPV) presence and type in vulvar vestibulitis. STUDY DESIGN: Twenty-two tissues excised during surgery for the treatment of severe vulvar vestibulitis and nine control tissue samples were tested for telomerase activity, mean telomere length, and HPV presence and type. RESULTS: Thirty-six percent of the tissues from vestibulitis patients were infected with HPV, mainly type 16/18, and none of the control tissue samples showed presence of HPV DNA (P < .02). Telomerase activity was detected in all tissues harboring HPV DNA, whereas only 64% of tissues without HPV DNA exhibited telomerase activity (P < .02). The mean telomere length was unchanged as compared to control samples. CONCLUSION: Telomerase activity in vestibulitis may be increased as a result of HPV infection, suggesting that HPV infection may play a role in the etiology of some cases of vulvar vestibulitis.

Increased T-type Ca2+ channel activity as a determinant of cellular toxicity in neuronal cell lines expressing polyglutamine-expanded human androgen receptors.

We have analyzed Ca2+ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca2+ current that is not affected by L-type Ca2+ channel blocker (PN 200-110), N-type Ca2+ channel blocker (omega-conotoxin GVIA) or P-type Ca2+ channel blocker (Agatoxin IVA) but is blocked by either Cd2+ or Ni2+. This pharmacological profile most closely resembles that of T-type Ca2+ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca2+ channel window current. We suggest that Ca2+ overload due to abnormal voltage-dependence of transient Ca2+ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca2+ channel currents, Ni2+ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.

A repetitive DNA sequence that characterizes human papillomavirus integration site into the human genome is present in vulvar vestibulitis.

OBJECTIVE: To determine the DNA sequence of polymerase chain reaction (PCR) products obtained from surgical specimens of patients with severe vulvar vestibulitis, in order to identify and type the human papillomavirus (HPV)-DNA associated with vulvar vestibulitis. STUDY DESIGN: Fifty three women, referred for dyspareunia and diagnosed as having severe vestibulitis, underwent perineoplasty operation consisting of surgical removal of the sensitive vestibule. PCR analysis using L1 HPV primer was performed, and DNA sequencing of the samples that were found to contain HPV-DNA was undertaken, using the dideoxy chain termination method. RESULTS: Using PCR, HPV-DNA was detected in 31 of 53 tissue specimens (58%). DNA sequencing of 12 HPV-positive PCR products revealed extensive homology to human Alu consensus sequence, albeit not to any known HPV sequence. CONCLUSIONS: The presence of interspersed, repetitive-DNA sequence Alu, which is known to be the preferred site for HPV integration into human genome, in the PCR product reinforces previous observations, suggesting that HPV may have a role in the pathogenesis of vulvar vestibulitis. It further implies a possible integration of the HPV into human DNA in these cases.

Uterine curettage following second trimester abortion by extraovular instillation of prostaglandin E2. A prospective-randomized trial.

OBJECTIVE: To evaluate the benefits associated with routine uterine curettage following complete second trimester termination of pregnancy by extraovular prostaglandin E2. STUDY DESIGN: Fifty-five patients between 15 and 24 weeks' gestation who had undergone complete termination of pregnancy by continuous extraovular instillation of prostaglandin E2 (PGE2), were randomly assigned into either no further intervention (n=25), or uterine curettage under general anesthesia (n=30). The need for late uterine curettage, clinical and ultrasonographic parameters at 1 and 42 days follow-up, as well as the incidence of the minor and major complications, were compared between groups. RESULTS: Baseline and post-abortion clinical and ultrasonographic characteristics were similar in both groups. Mean (+/- Standard error of the mean) number of post-abortion bleeding days in the curettage group was 8.9+/-1.8 versus 10.1+/-2.6 days in the non-curettage group (P=NS). No patient in the former group, compared to three patients in the latter group, needed late uterine curettage, (P=NS). Major and minor complications rates in the curettage and in the no-curettage groups were not significantly different. Considerably more patients in the curettage group needed analgesic agents following the abortion compared to the no-curettage group (60% vs. 3.3%, respectively; P<0.001). CONCLUSIONS: Routine uterine curettage in patients undergoing complete second trimester termination of pregnancy by extraovular instillation of PGE2, exerts no benefit.

Decision-making in the colposcopy clinic--a critical analysis.

OBJECTIVE: To consider the omission of several diagnostic steps from the management of patients with high-grade squamous intraepithelial lesion (SIL) by analyzing the role of each step on the choice of treatment. STUDY DESIGN: Each diagnostic procedure was correlated to the treatment and outcome in 87 women with high-grade SIL. Treatments considered were large loop excision of the transformation zone (LLETZ) cold knife conization, and CO2 laser vaporization. RESULTS: Unsatisfactory colposcopy (P< or =0.01) and positive endocervical curettage (ECC) specimen (P< or =0.01) were essential for choice of treatment. CIN2 diagnoses of the preoperative cervical biopsy were rediagnosed as CIN3 based on the surgical specimen in 57% of the cases. The margins of 33 and 23% of surgical specimens removed by LLETZ or knife conization, respectively, displayed CIN involvement. Forty and 47% of these patients, respectively, later developed recurrent CIN. CONCLUSIONS: Omission of colposcopy and ECC could have resulted in sub-optimal treatment in many cases. Excision by LLETZ or knife conization is recommended for cases of CIN2 and CIN3. Follow up is imperative for patients with involvement of the margins.

Pelvic actinomycosis. Is long-term antibiotic therapy necessary?

OBJECTIVE: To describe 11 cases of actinomycosis and analyze whether long-term antibiotic use in necessary. STUDY DESIGN: Analysis of 11 cases of pelvic actinomycosis diagnosed and treated during the last nine years. Four patients had an intrauterine device (IUD) for 6-20 years, three patients had an IUD for 3-5 years, and four patients had no known etiology. In most patients the symptoms lasted from several days to one month. The actinomycotic lesions involved one or both ovaries in all 11 cases. In five patients the lesion extended to other areas, such as the uterus, omentum, parametrium, pelvic walls, colon, bladder, cul-de-sac and gallbladder. RESULTS: All patients underwent surgery that included removal of the lesions with the ipsilateral or both adnexa and, in specific cases, with extension of the lesions, hysterectomy, omentectomy, hemicolectomy and cholecystectomy. Confirmation of the diagnosis of actinomycosis was done by histology in all cases, and antibiotic treatment usually began 1-14 days after surgery. The drug of choice was penicillin. The duration of treatment was 12 months in 6 patients, 6 months in 3 and < or = 3 months in two. All patients were alive and well after two to nine years of follow-up. CONCLUSION: In contrast to actinomycosis at other sites, where the literature recommends antibiotic therapy for 6-12 months, pelvic actinomycosis could be a limited disease. We propose that in cases of pelvic actinomycosis where the abscess can be completely removed surgically, a shorter period of antibiotic therapy can be effective.

Increased telomerase activity and decreased telomere length in genital condylomata acuminata.

Our objective was to find a possible correlation between telomerase activity, mean telomere length and human papillomavirus (HPV) presence and type in genital condylomata acuminata. Fifteen biopsies from women with genital condylomata acuminata and nine control tissue samples were tested for telomerase activity, mean telomere length, and HPV presence and type. All condylomata exhibited telomerase activity, compared to 78% of the control samples. The mean telomere length of condylomata was significantly (P<0.002) shorter compared to telomere length in control tissue samples. All condylomata lesions were infected with HPV types 6/11, and more than half had additional infection with HPV 16/18. Mixed HPV 6/11 with 16/18 infection correlated with shorter telomeres than presence of HPV 6/11 alone in the lesions (4.68 +/- 0.44 kb vs 4.97 +/- 0.57 kb). None of the control tissue samples showed presence of HPV DNA. Telomerase activity may be a marker of proliferation rather than malignancy, whereas the mean telomere length could better serve as a marker for the progression of HPV lesions toward malignancy.

Cessation of gonadotropin-releasing hormone analogue (GnRH-a) upon down-regulation versus conventional long GnRH-a protocol in poor responders undergoing in vitro fertilization.

OBJECTIVE: To determine whether a controlled ovarian hyperstimulation (COH) regimen that involves GnRH agonist (GnRH-a) discontinuation before administration of gonadotropins would benefit poor responders. DESIGN: A prospective, randomized controlled trial. SETTING: Hospital-based IVF Unit. PATIENT(S): Sixty-three patients with previous poor response to COH and/or high basal FSH level (> or =9 mIU/mL) undergoing 78 IVF-ET cycles. INTERVENTION(S): In both groups, administration of GnRH-a was started in the midluteal phase. Whereas in the study group (40 cycles), it ended before administration of gonadotropins, in controls (38 cycles) GnRH-a treatment was continued throughout the follicular phase. MAIN OUTCOME MEASURE(S): Ovarian stimulation patterns and IVF outcome. RESULT(S): A significantly higher cancellation rate was noted in the study group than in the controls (22.5% versus 5%, respectively). The new and control regimens resulted in similar stimulation characteristics and clinical pregnancy rates (11% versus 10.3%, respectively). In 13 patients with a basal FSH level that was not persistently high, the new regimen resulted in a significantly higher number of retrieved oocytes compared with the standard protocol (7.6+/-1.03 versus 4.0+/-0.68, respectively). CONCLUSION(S): Whereas for most low responders, the new COH regimen offers no further advantage, future prospective studies may demonstrate whether it can confer a benefit for a subset of patients with a basal FSH level that is not persistently high.

Shortened exposure of oocytes to spermatozoa improves in-vitro fertilization outcome: a prospective, randomized, controlled study.

A prospective, randomized study of 158 patients undergoing in-vitro fertilization (IVF) and embryo transfer was conducted to evaluate whether a shortened exposure of oocytes to spermatozoa enhances oocyte development, and subsequently influences the IVF outcome. A comparison was made between conventional treatment time and shorter exposure of retrieved oocytes to spermatozoa. Fertilization and cleavage rates, embryo quality, implantation and pregnancy rates in the study group (short exposure) versus controls (standard IVF procedure) were evaluated. Fertilization (56 versus 61%) and cleavage rates (96 versus 92%) were similar in the two groups respectively. However, embryo quality was significantly higher in the study group (P < 0.05). Moreover, the pregnancy and implantation rates were significantly increased (42.4 versus 26% per embryo transfer, and 16 versus 10% respectively; P < 0.05). Our results demonstrated that shorter exposure of oocytes to spermatozoa is superior to the standard time in IVF and may have a favourable effect on implantation rates by improving embryo quality.

Polymorphism for mutation of cytosine to thymine at location 677 in the methylenetetrahydrofolate reductase gene is associated with recurrent early fetal loss.

OBJECTIVE: This study was undertaken to determine whether a cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase is associated with particular subtypes of recurrent unexplained spontaneous abortion. STUDY DESIGN: The prevalences of cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase among 41 patients with recurrent unexplained spontaneous abortions and among 18 healthy control subjects were determined with polymerase chain reaction. RESULTS: Homozygosity and heterozygosity for the cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase were observed at nonsignificantly different rates among patients and control subjects (9% and 48% versus 22% and 38%, respectively, P <.95). Among patients with recurrent unexplained spontaneous abortions both homozygosity and heterozygosity were associated with significantly increased prevalence of recurrent early fetal loss rather than with repeated anembryonic gestations (P <.0001). CONCLUSION: The observation that polymorphism for the cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase is associated with repeated early fetal losses rather than with anembryonic gestations strengthens the argument for the role of hypercoagulability and abnormal uteroplacental vasculature in recurrent spontaneous abortion.