Identification of rearranged sequences of HPV16 DNA in precancerous and cervical cancer cases.

Integration of HPV16 DNA into the host chromosome is considered to be a crucial step towards genomic instability and cervical cancer development. Aim of the present study was to investigate the presence of HPV16 rearranged intra-viral sequences in HPV16-positive normal, precancerous and cervical cancer samples using the method of Restriction Site-PCR (RS-PCR). Sequence analysis of HPV16 integrants revealed for the first time in clinical samples two distinct rearranged intra-viral sequences, concerning the conjunction of E2 and L1 genes and the conjunction of E1 and L1 genes with inverted orientation. Furthermore mapping analysis of the E1 and E2 genes in cervical samples with rearranged intra-viral sequences of HPV16 genome was conducted in order to determine the integrity of viral genes. The identification of intra-viral rearrangements provides valuable information regarding the HPV16 DNA integration, and may be a significant biomarker for the presence of chromosomal instability and DNA damages in clinical samples.

Evidence that termination of the estradiol-induced luteinizing hormone surge in women is regulated by ovarian factors.

BACKGROUND: The endogenous LH surge is the result of the estrogen-positive feedback effect. However, the factors that are responsible for the termination of LH surge are not known. OBJECTIVE: The objective of the study was to investigate the mechanism that terminates the LH surge in women. SUBJECTS AND METHODS: Eight normally cycling women (aged 42-48 yr) were investigated in two cycles, i.e. cycle 1 (control) and cycle 2. In cycle 2 total abdominal hysterectomy plus bilateral salpingooophorectomy was performed on d 3. In both cycles, estradiol was administered transdermally at the dose of 100 microg on d 3 and 150 microg on d 4 and 5. Blood samples were obtained every 12 h from d 3 to 5 and every 6 h thereafter until d 9. RESULTS: In both cycles, after suppression of gonadotropins, the women displayed an endogenous LH surge. The time intervals between the commencement of estradiol treatment and the LH surge onset (73.5 +/- 1.5 vs. 76.5 +/- 2.5 h) and peak LH values (11.4 +/- 1.9 vs. 12.4 +/- 3.1 IU/liter) were comparable in the two cycles (mean +/- sem). After peaking, LH values decreased gradually in cycle 1, whereas in cycle 2 they remained stable and were higher than the corresponding values in cycle 1 (P < 0.05). Before the LH surge onset, estradiol values showed in both cycles a preovulatory pattern of changes, but starting 24 h after the onset of the LH surge, they were lower in cycle 2 (P < 0.05). Progesterone levels were similar in both cycles until the day of the LH surge onset, but in cycle 2 they declined thereafter and were lower than in cycle 1 (P < 0.05). CONCLUSIONS: It is suggested that ovarian factors rather than exhaustion of pituitary reserves are important for termination of the endogenous LH surge during the normal menstrual cycle.

Alternate day and daily administration of GnRH antagonist may prevent premature luteinization to a similar extent during FSH treatment.

BACKGROUND: This randomized controlled trial was designed to evaluate whether a GnRH antagonist given every other day could prevent premature luteinization in women undergoing IVF/ICSI treatment. METHODS: A total of 73 women receiving ovulation stimulation IVF cycles with recombinant FSH were allocated randomly on cycle day 7 to GnRH antagonist ganirelix in multiple doses (0.25 mg each), either daily (n = 37 women, group 1) or every other day (n = 36 women, group 2) until the day of HCG administration. RESULTS: Serum FSH, LH, estradiol and progesterone values showed similar trends in the two groups. During FSH stimulation, 13 (35%) of the women in group 1 had premature LH rises (> or = 10 IU/l) of which eight (22%) were after the start of antagonist administration. In group 2 there were 14 (39%) LH rises during FSH stimulation of which 10 (28%) were after the start of antagonist administration. Luteinization (serum progesterone >2 ng/ml) occurred in only one woman in each group overall (3%). A significantly smaller total dose of the antagonist was used in group 2 than in group 1 (P < 0.001). The study did not have power to evaluate differences in total dose of FSH, number of oocytes recovered and clinical pregnancy rate, all of which appeared similar in the two groups. CONCLUSIONS: Whether alternate day is as effective as daily administration of ganirelix in preventing premature luteinization should be addressed in a non-inferiority trial powered to evaluate live birth rate.

Changes in pituitary sensitivity to GnRH in estrogen-treated post-menopausal women: evidence that gonadotrophin surge attenuating factor plays a physiological role.

BACKGROUND: The purpose of this study was to investigate changes in pituitary response to GnRH in post-menopausal women during substitution treatment with exogenous estrogen and progesterone. METHODS: Seven healthy post-menopausal women (group 1) were treated with various doses of E2 valerate for 43 days, so as the serum concentrations of E2 mimicked those of a follicular (FP-1), a luteal (LP) and a second follicular (FP-2) phase. During the LP, progesterone was also administered. The 30 min response of LH (DeltaLH) and FSH (DeltaFSH) to GnRH (10 microg i.v.) (pituitary sensitivity) was investigated every 24 h in group 1 and also in seven normally cycling women (group 2) during a spontaneous (control) follicular phase (FP). Based on the hormone profiles, day 32 in group 1 (FP-2) corresponded to day 2 in the spontaneous FP of group 2. RESULTS: Basal FSH concentrations were significantly higher in FP-2 than in the control FP (P < 0.05), while basal LH concentrations were similar in the two phases with higher values in FP-2 towards the end of the experiment (corresponding to days 10 and 11, P < 0.05). However, an LH surge was seen only in the control FP. DeltaFSH values remained stable in both phases and increased only in the control FP on days 12 and 13. DeltaLH values remained stable in the control FP and only increased on days 12 (P < 0.05) and 13 (P < 0.05), but in FP-2, DeltaLH values increased earlier (corresponding to day 7, P < 0.05). CONCLUSIONS: The present study demonstrates for the first time that in the absence of ovarian function, follicular phase E2 concentrations sensitize the pituitary to GnRH at an earlier stage (corresponding to the midfollicular phase) than in the normal menstrual cycle (late follicular phase). It is suggested that during the early to midfollicular phase the ovaries produce a gonadotrophin surge attenuating factor (GnSAF) that antagonizes the pituitary-sensitizing effect of E2 to GnRH.

FSH and LH responses to GnRH after ovariectomy in postmenopausal women.

OBJECTIVE: Whether the postmenopausal ovary is still playing a role in the control of gonadotrophin secretion in response to GnRH has not been investigated. The aim of the present study was to test this hypothesis by examining changes in basal and GnRH-induced gonadotrophin secretion in postmenopausal women after bilateral ovariectomy. DESIGN: The responses of LH and FSH to GnRH [10 microg intravenously (i.v.)] were investigated in postmenopausal women from 2 days before to 8 days after total abdominal hysterectomy plus bilateral ovariectomy. PATIENTS: Nine postmenopausal women aged 52-67 years and between 5 and 15 years after menopause. In all cases the ovaries were histologically normal. MEASUREMENTS: Pituitary responses to GnRH were calculated every 12-24 h as the net increases in LH (DeltaLH) and FSH (DeltaFSH) at 30 min above the basal values. Basal values of oestradiol (E2) and testosterone were also measured. RESULTS: Basal values of FSH showed a significant decrease on postoperative days 2 (P < 0.01) and 8 (P = 0.03) as compared to day 0, while at the same time points after the operation LH values were marginally lower than on day 2 (P = 0.05). Serum E2 values showed a gradual increase up to postoperative day 1 (P = 0.04) and a gradual decline thereafter. Basal testosterone concentrations decreased gradually and significantly after ovariectomy and were significantly lower on day 8 than on day 0 (P < 0.01). DeltaFSH and DeltaLH responses to GnRH did not change significantly with time. A temporary increase at 12 h after the operation was not significant. CONCLUSIONS: These results demonstrate for the first time that the removal of the ovaries in postmenopausal women does not affect GnRH-induced gonadotrophin secretion in the short term. It is suggested that the postmenopausal ovary is not a dominant regulator of hypothalamic-pituitary interactions.

Detection and typing of HSV-1, HSV-2, and VZV by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction method for the rapid and accurate detection and typing of HSV-1, HSV-2, and VZV from clinical specimens is described. A sensitive multiplex polymerase chain reaction was achieved by optimization of parameters such as the primers, magnesium, and dNTPs concentrations. False-negative results that sometimes arise due to inhibitors of DNA amplification or failure of DNA extraction procedure used may be avoided by assaying each specimen with alpha-tubulin primers. Multiplex PCR amplified viral sequences from all 55 specimens obtained from patients with clinical evidence of HSV or VZV infection indicated 100% sensitivity. From 55 patients who were investigated by multiplex PCR, HSV-1 was detected in 28, HSV-2 in 20, and VZV in 7 specimens. The reported results indicate that the present multiplex PCR assay has a potential application in clinical diagnosis when a rapid and accurate detection and typing of involved viruses HSV-1, HSV-2, or VZV is needed.