Impact of a single endometrial injury on assisted reproductive technology outcome: a preliminary observational study.

OBJECTIVE: To find a more acceptable strategy for our patients to improve endometrial receptivity in assisted reproductive technologies (ART) and to study the impact of a single endometrial sampling performed on day 21 of the spontaneous menstrual cycle. STUDY DESIGN: In this preliminary study, the possible beneficial effects on ART outcome of a single biopsy performed on the midluteal phase (day 21) of the spontaneous menstrual cycle preceding controlled ovarian stimulation was tested in a group of patients with 3 or more implantation failures. RESULTS: After endometrial biopsy 45.94% of patients with more than two failed ART cycles obtained clinical pregnancy. CONCLUSION: Although these preliminary results are encouraging, further investigation is needed to validate the study.

Effectiveness of a low gonadotrophin-releasing hormone antagonist dose in preventing premature luteinizing hormone rise during controlled ovarian stimulation.

The current prospective randomized study was designed to test the efficacy of a low dose (0.125 mg/day) of the gonadotrophin-releasing hormone antagonist cetrorelix in preventing premature luteinizing hormone (LH) rise during controlled ovarian stimulation in comparison with the standard dose of 0.25 mg/day. Ovarian stimulation was started with 225 IU of recombinant follicle stimulating hormone (FSH) on day 2 of the menstrual cycle. Cetrorelix was injected daily from day 6 of gonadotropin administration. Blood was sampled from each woman on day 3 of ovarian stimulation and then daily from day 5 onward up to human chorionic gonadotropin administration for analysis of FSH, LH, progesterone, and estradiol. LH rise was defined as serum LH ≥ 10 mIU/ml. There were 40 patients receiving cetrorelix at 0.25 mg/day and 36 patients receiving cetrorelix at 0.125 mg/day. Premature LH rise was recorded in 10% of patients injecting antagonist at 0.25 mg/day and in 14% of patients administered with antagonist at 0.125 mg/day. These frequencies did not differ statistically. In conclusion, our results suggest that a cetrorelix dose of 0.125 mg/day is effective as the standard dose (0.25 mg/day) in preventing premature LH rise during controlled ovarian stimulation.

Cytogenetic findings and reproductive outcome of infertile couples referred to an assisted reproduction program.

It is still undefined whether all the couples entering an assisted reproduction program should undergo to karyotype analysis. The present study was conducted to determine the prevalence of chromosomal abnormalities in a non-selected sample of 1,146 couples referred to assisted reproduction technologies (ART), and to analyze the outcome of pregnancies from couples in whom cytogenetic anomalies were detected. Irrespective of the infertility factor, fertilization was achieved by intracytoplasmic sperm injection (ICSI). A total number of 35 karyotype anomalies were diagnosed, corresponding to an abnormality frequency of 1.52% (1.83% for men and 1.22% for women). As could be expected, the majority of men presenting karyotype anomalies had a low sperm count. Among women, the majority of cytogenetic anomalies were detected in individual not presenting risk factors for aberrant karyotype. Around 41% of pregnancies achieved in couples presenting chromosomal anomalies ended in spontaneous abortion. Information on fetal karyotype was limited. No major malformations were observed among newborns from parents with abnormal karyotype. In consideration of the elevated frequency of pregnancy loss, it seems advisable to recommend that chromosomal analysis be performed in all couples undergoing ART. This with the aim of identifying patients that would possibly benefit from pre-implantation genetic diagnosis.

Proteome analysis of human follicular fluid.

We used proteomic approach to analyze the protein profile of human follicular fluid (HFF) obtained from 25 normo-ovulatory women undergoing assisted reproduction techniques due to a male infertility factor. In all HFF samples analyzed we found 695 common spots distributed in the 3 to 10 pH range and in the 10-200 kDa range. Only 625 of these spots were also present in the plasma. We used MALDI-TOF-MS analysis to unequivocally assign 183 HFF/plasma matched spots and 27 HFF/plasma unmatched spots. A large number of acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin and plasma amyloid protein, were identified in HFF in relatively high concentration supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. We also identified several important antioxidant enzymes; i.e., catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27 and protein disulfide isomerase. This indicates that during maturation the human follicle is well protected against toxic injury due to oxidative stress.

Defining critical periods for itraconazole-induced cleft palate, limb defects and axial skeletal malformations in the mouse.

The main aim of the study was to identify the critical periods of susceptibility for itraconazole-mediated teratogenesis in the mouse. Pregnant ICR (CD-1) mice received a single oral administration of itraconazole at 50 mg/kg b.w., 150 mg/kg b.w. or 250 mg/kg b.w. on gestation day 8, 9, 10, 11 or 12. Control animals were administered with vehicle on gestation days 8-12. The gestational outcome was evaluated near term, on gestation day 18. Treatment-related morphological findings, as they can be evaluated by external, visceral and skeletal examination, mainly included cleft palate, limb defects and axial skeletal malformations. Cleft palate and limb defects resulted after single exposures between gestation days 9 and 11, with a tendency toward maximal response on gestation day 10. Significant incidences of axial skeletal defects were seen exclusively on gestation days 8 and 9. Exposure on gestation day 12 did not yield significant teratogenic responses. Cleft palate was the most sensitive teratogenic response, being the only developmental lesion associated to exposure to itraconazole at 150 mg/kg b.w.

Levels of apoptosis in human granulosa cells seem to be comparable after therapy with a gonadotropin-releasing hormone agonist or antagonist.

OBJECTIVE: To compare levels of apoptosis in granulosa cells from women treated with the gonadotropin-releasing hormone (GnRH) agonist triptorelin or the GnRH antagonist cetrorelix. DESIGN: Randomized, prospective study. SETTING: University hospital. PATIENT(S): Thirty-two women undergoing assisted reproduction techniques after ovulation induction with recombinant follicle-stimulating hormone (FSH) plus GnRH agonist or antagonist. INTERVENTION(S): Granulosa cells were isolated from follicular aspirates after oocyte removal. MAIN OUTCOME MEASURE(S): Apoptosis was assessed with Annexin V binding assay, terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) assay, flow cytometric analysis of DNA, and ultrastructural analysis of cell morphology in transmission electron microscopy. Serum and follicular hormonal levels were also determined. RESULT(S): Annexin V binding and TUNEL assays revealed comparable percentages of apoptosis in the two groups under investigation. Analysis of DNA histograms revealed a similar cell cycle distribution in the two groups. Ultrastructural analysis only occasionally displayed patterns of chromatin margination in apoptotic cells. The mean concentrations of all the follicular fluid steroid hormones evaluated (E2, T, and P) were significantly lower in the GnRH antagonist-treated group. CONCLUSION(S): Therapy with a GnRH agonist or antagonist is associated with comparable levels of apoptosis in granulosa cells.

Serological testing for celiac disease in women undergoing assisted reproduction techniques.

BACKGROUND: The assertion of a causal relationship between celiac disease and infertility is suggested by several lines of research. Nevertheless, robust evidence has not yet been provided. The present study evaluated, for the first time, the prevalence of celiac disease in women undergoing assisted reproduction techniques (ART). METHODS: Serum samples from 200 Italian women undergoing ART were evaluated for celiac disease by endomisium antibody (EMA) and transglutaminase antibody (t-TGA)-two highly sensitive and specific serological markers. Two hundred women not reporting reproductive problems and having delivered at least one child served as controls. In cases of positive serology, the diagnosis was confirmed by jejunal biopsy. RESULTS: Five (2.5%) women from the study group and two (1.0%) from the control group were found to have celiac disease (P = 0.44). The main indications for ART in women found to have celiac disease were tubal factor in two cases and male infertility in three cases. None of these women reported major gastrointestinal complaints. Extra intestinal signs linked to celiac disease were noted in four out of five patients. CONCLUSION: This study raises the issue of celiac disease screening in ART programmes. Given the available evidence in the literature combined with our observations from this study, the value of serological testing for celiac disease in infertile women remains uncertain. Further studies to address this issue are required.

Murine teratology of fluconazole: evaluation of developmental phase specificity and dose dependence.

The potential of in utero exposure to fluconazole to initiate teratogenesis was analyzed in ICR (CD-1) mice. Developmental phase specificity was determined by treating mice with single oral doses of 700 mg/kg on gestational day 8, 9, 10, 11, or 12. Control animals received vehicle on gestational days 8-12. Gestational day 10 was identified as the phase of maximal sensitivity for induction of cleft palate, the predominant teratogenic effect induced by fluconazole, with 50% of fetuses exposed on this developmental phase being affected. After treatments on gestational day 8, 9, 11, or 12, cleft palate occurred with lower frequencies: 12, 21, 28.7, and 2.7%, respectively. Examination of skeletal morphology revealed anomalies of the middle ear apparatus in 15% of the fetuses that were exposed on gestational day 8. Dysmorphic tympanic ring and absence of the incus were the more common ear anomalies recorded. Reduced humeral length was noted in 22% of fetuses that were exposed on gestational day 10. Dose-response relationship was investigated by treating animals with 0 (vehicle), 87.5, 175, or 350 mg/kg on gestational day 10, coincident with the phase of peak teratogenic sensitivity. Besides showing that fluconazole operates under a strict dose-response mechanism, the study identified 175 mg/kg as the lowest observed adverse effect level for cleft palate induction, with 7.6% of the exposed fetuses being affected.

Additional investigation on the potentiation of phenytoin teratogenicity by fluconazole.

Fluconazole (FCZ) is a potent inhibitor of the cytochrome P450 (CYP)-mediated metabolism of the anti-epileptic agent phenytoin (PHT), a well-known human and animal teratogen. It has been postulated that phenytoin must be bioactivated via the CYP system to initiate teratogenesis. In contrast with this view, FCZ pretreatment has been previously shown to result in a potentiation of PHT teratogenesis. The current study was initiated to determine the impact of FCZ pretreatment on PHT exposure levels in maternal and embryonal compartments. HPLC analysis revealed that under a co-dosing FCZ-PHT regimen resulting in enhanced PHT teratogenesis, statistically significant higher PHT levels are detectable in maternal plasma and embryonic tissue in comparison to controls. These results further argue against a role for CYP system in teratogenic bioactivation of PHT.

The nitric oxide synthesis inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) causes limb defects in mouse fetuses: protective effect of acute hyperoxia.

In the present study the relationship between exposure to the nitric oxide synthesis inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME) and the induction of limb defects, with respect to stage specificity and dose dependency, was investigated in the mouse. ICR (CD-1) mice were dosed s.c with l-NAME at 50 or 90 mg/kg on gestation d 12, 13, 14, 15, or 16. A group of animals treated with vehicle on gestation d 14 served as control. Uterine contents were evaluated for teratogenesis on gestation d 18. A treatment-related disruption of limb development was noted. The effect was dose dependent and phase specific. l-NAME became teratogenically operational on gestation d 13 and elicited its maximum effect on gestation d 14, whereas no significant teratogenicity was observed when exposure occurred after gestation d 15. In utero exposure to l-NAME also reduced embryo viability relative to controls. When the higher dose was injected on gestation d 16, a significant number of dams delivered preterm. In a parallel study, the ability of hyperoxia to prevent limb teratogenesis was investigated. To this aim, a group of l-NAME-treated animals (90 mg/kg s.c. on gestation d 14) were exposed to 98 to 100% O(2) for 12 h. l-NAME-treated mice breathing room air served as positive controls. In response to hyperoxia, a significant decrement of l-NAME-induced limb defects was found. This study characterizes for the first time the teratogenic capacity of l-NAME in the mouse. Results obtained with hyperoxia fit the hypothesis that hypoxic tissue damage may play a contributory role in l-NAME-induced limb defects.