On the use of post-transcriptional processing elements in transgenes.

RNA processing events modulate final productivity of a given transgene. We have evaluated a series of RNA elements for their ability to enhance alpha1-antitrypsin production in mammary cells. Our results indicate the need for a case-by-case assessment of each construct design and the occurrence of gene silencing events in vivo.

Physical separation of HLA-A alleles by denaturing high-performance liquid chromatography.

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.

Donor oocyte cytoplasmic transfer did not enhance implantation of embryos of women with poor ovarian reserve.

PURPOSE: To determine whether donor oocyte cytoplasm transferred into the oocytes of women < or = 40 years or with diminished ovarian reserve would enhance embryo quality, implantation, or pregnancy rates. METHODS: Study subjects included women > or = 40 years (15) or with abnormal FSH levels (3). Healthy volunteers (18) produced oocytes for cryopreservation. Donor oocytes were thawed and cytoplasm from surviving oocytes was injected with a single sperm into the cytoplasm of recipient oocytes. Outcome measures included embryo quality scores, implantation, and pregnancy rates. RESULTS: Eighteen donors produced 213 oocytes for cryopreservation and 39/171 (22.8%) survived thawing. Eighteen recipients initiated 25 IVF cycles with embryo transfer in 20 cycles after cytoplasmic transfer (CT). Four cycles resulted in three biochemical losses and one aneuploid clinical loss. Embryo quality did not improve with CT compared to pre-CT IVF cycles in six recipients. CONCLUSIONS: CT with cryopreserved donor oocyte cytoplasm did not enhance success in women with advanced reproductive age or low ovarian reserve.

The number of embryos available for transfer predicts successful pregnancy outcome in women over 39 years with normal ovarian hormonal reserve testing.

PURPOSE: The purpose was to determine whether the number of embryos available for transfer following IVF in women over age 39 predicted a successful pregnancy outcome. METHODS: Retrospective analysis of 455 consecutive IVF cycles in women > or = 40 years of age. RESULTS: Few cycles were canceled (29/455, 6.4%) or produced no embryos (5/455, 1.1%). Women 40-43 years of age with normal ovarian reserve had a significantly greater delivery rate when > or = 4 embryos were available for transfer than when < 4 embryos were available (17.8% versus 2.4%, P = 0.002). Subsequent IVF cycles, from women with normal FSH whose first cycle produced < 4 embryos, produced delivery rates of 13.0% when > or = 4 embryos were available. Women with abnormal ovarian reserve or age > or = 44 years had very low delivery rates (1.2% and 1.4% respectively). CONCLUSIONS: The number of embryos available for transfer significantly predicts delivery from IVF-ET among reproductively older women. Many women age 40-43 with normal ovarian reserve can achieve pregnancy through IVF.

Pregnancy rates in sequential in vitro fertilization cycles by oocyte donors(1).

OBJECTIVE: To evaluate the clinical outcome of in vitro fertilization (IVF) treatment cycles from individual oocyte donors who underwent multiple sequential donations. METHODS: We reviewed clinical outcome data from sequential anonymous oocyte donation cycles using donors who underwent multiple IVF stimulations. Donors were grouped by the interval between cycles and the cycle number (rank). The primary outcome measure was delivery rate by individual donor per retrieval from the combined derivative fresh and frozen embryo transfers. RESULTS: Duration and amount of gonadotropin therapy and the fertilization rates did not correlate significantly with the interval between cycles or cycle rank. Cumulative delivered pregnancy rates for cycles 1-6 were 51.5%, 54.6%, 50.5%, 51.5%, 51.1%, and 57.6%, respectively. Delivered pregnancy rates did not vary by interval between cycles. CONCLUSION: Young healthy presumed or proven fertile women can reliably donate oocytes for at least six cycles with the expectation of consistently high pregnancy rates.

Serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women.

OBJECTIVE: To determine the serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate treatment in anovulatory infertile women. DESIGN: Prospective cohort. SETTING: Tertiary institutional infertility clinic. PATIENT(S): Fourteen consenting anovulatory infertile women receiving standardized, cyclic, incremental treatment with clomiphene citrate for ovulation induction. INTERVENTION(S): Clomiphene citrate treatment (50-150 mg/d, cycle days 5-9), titrated to the minimum effective ovulation-inducing dose, was administered for three to six total cycles. Blood samples were obtained on cycle days 3 and 10 in each treatment cycle. MAIN OUTCOME MEASURE(S): Serum concentrations of enclomiphene and zuclomiphene. RESULT(S): Cycle day 3 zuclomiphene levels were below assay limits in all initial cycles, increased progressively across three consecutive cycles, and thereafter plateaued. Cycle day 3 enclomiphene concentrations were uniformly undetectable. Cycle day 10 enclomiphene levels increased with dose administered, but these observations were not statistically significant. CONCLUSION(S): Clomiphene citrate induction of ovulation results in an isomer-specific systemic accumulation of zuclomiphene across consecutive cycles of treatment. The combined maximum concentration of enclomiphene and zuclomiphene attained in practice approximates 100 nmol/L and is generally well below levels previously demonstrated to have adverse effects in vitro.

Preservation of reproductive function before therapy for cancer: new options involving sperm and ovary cryopreservation.

In summary, sperm cryopreservation for future ICSI and ovarian tissue cryopreservation for future autotransplantation are new opportunities to preserve reproductive options of great importance to patients with newly diagnosed cancer. Since patients must utilize these strategies before cancer therapy is initiated, and these patients will not have a future chance to benefit once therapy has damaged gonadal function, awareness of these technologies among oncologists, radiation therapists, and other colleagues who interface with the victims of cancer is a high priority.

Characteristics of gonadotropin response, follicular development, and endometrial growth and maturation across consecutive cycles of clomiphene citrate treatment.

OBJECTIVE: To examine the patterns of gonadotropin response, follicular development, and endometrial growth and maturation across consecutive cycles of clomiphene citrate (CC) treatment. DESIGN: Prospective analysis of cycle characteristics. SETTING: Academic tertiary medical center. PATIENTS: Nineteen consenting anovulatory infertile women receiving standardized, cyclic, incremental treatment with CC (50 to 150 mg/d, cycle days 5 to 9) for ovulation induction. INTERVENTIONS: In each of up to six consecutive treatment cycles, urinary LH was monitored twice daily from cycle day 10 until detection of the LH surge or day 21; blood samples and transvaginal ultrasound (US) examination were obtained on cycle days 3, 10, and every 1 to 3 days thereafter until collapse of the dominant follicle. Endometrial biopsy was performed 11 to 13 days after the LH surge in the first, third, and sixth ovulatory cycle. RESULTS: Follicular phase duration, peak follicular diameter, the number of preovulatory follicles, and peak endometrial thickness and echo pattern remained consistent across consecutive ovulatory (n = 55) and anovulatory (n = 23) treatment cycles. Endometrial dating was > or = 3 days out of phase in 2 of 31 (6%) cycles sampled. Peak serum E2 and P concentrations did not vary with cycle number or correlate with endometrial thickness or echo pattern. Cycle day 10 FSH levels were significantly higher in ovulatory subjects than in anovulatory subjects. CONCLUSIONS: Patterns of gonadotropin response, follicular development, and endometrial growth and maturation remain consistent across consecutive cycles of CC treatment.

Effects of enclomiphene and zuclomiphene on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of small and large ovine luteal cells.

We examined the effects of enclomiphene and zuclomiphene, alone and in combination with oestradiol, on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of both large (granulosa-lutein) and small (theca-lutein) ovine luteal cells. Isolated large and small luteal cells derived from intact, enucleated ovine corpora lutea were incubated for 48-120 h with or without 22R-hydroxycholesterol or pregnenolone (2.5 microM) and a range of enclomiphene, zuclomiphene, and/or oestradiol concentrations (3-100 microM), both with and without ovine luteinizing hormone (100 ng/ml). Spent media were assayed in duplicate for progesterone content by radioimmunoassay. Enclomiphene, zuclomiphene, and oestradiol exhibited equivalent dose-dependent inhibitory effects on basal and gonadotrophin-stimulated small and large ovine luteal cell progesterone secretion under all substrate conditions. Both cell types became more sensitive to clomiphene inhibition with increasing time in culture. In combined treatments, the effects of oestradiol and either enclomiphene or zuclomiphene became additive in longer-term cultures and were never antagonistic. In this model system, (i) clomiphene, like oestradiol, appears to inhibit 3beta-hydroxysteroid dehydrogenase activity, (ii) both stereoisomers act as oestrogen agonists, (iii) neither demonstrates any anti-oestrogenic properties, and (iv) both large and small luteal cells become more sensitive to clomiphene inhibition with increasing duration of exposure.

Comparisons of pregnancy loss patterns after intracytoplasmic sperm injection and other assisted reproductive technologies.

OBJECTIVE: To compare outcome of pregnancies after intracytoplasmic sperm injection (ICSI) with those of other assisted reproductive technologies. DESIGN: Pregnancy outcomes after ICSI were followed prospectively and compared with pregnancy outcomes after IVF with fresh and frozen ETs and donor oocyte cycles. SETTING: A private tertiary referral center for genetics and infertility in Fairfax, Virginia. PATIENTS: One hundred thirty-six couples achieving pregnancy after undergoing ICSI, 71 after IVF, 35 donor oocyte recipients, and 19 after transfer of frozen-thawed embryos. INTERVENTIONS: In vitro fertilization and/or ET for all couples. Dilatation and curettage to obtain products of conception for chromosome analysis in 28 women experiencing spontaneous abortion. MAIN OUTCOME MEASURES: Pregnancy outcomes were classified as preclinical loss, clinical loss, and ongoing pregnancy. RESULTS: The mean frequency of preclinical pregnancy loss was 26% after ICSI, 28% after IVF, 3% after ET using donor oocytes, and 11% after frozen ET. The rate of clinical loss after ICSI (21%) was compared with IVF (18%), donor oocyte cycles (11%), and frozen ETs (21%). CONCLUSIONS: Intracytoplasmic sperm injection is not associated with an increase in pregnancy losses, clinical or preclinical, compared with conventional IVF.

Single vs. multiple semen specimens in screening for male infertility factors. A comparison.

OBJECTIVE: To reevaluate the number of semen analyses necessary to establish whether further male infertility testing is necessary. STUDY DESIGN: The results of three consecutive semen analyses for infertility evaluations were retrospectively reviewed. A male factor was defined by an abnormal semen analysis if either the first specimen of three (single-sample screening) or two of the three specimens (multiple-sample screening) met World Health Organization criteria. Males considered abnormal by multiple-sample screening underwent sophisticated andrologic evaluation. RESULTS: A single-sample conventional semen analysis obtained from 209 males demonstrated a diagnostic accuracy of 10.4% false negatives and a sensitivity of 89.6% when compared to that of multiple-sample analysis. Andrologic evaluation of abnormal males by multiple-sample screening confirmed that 9 of the 11 men with normal first specimens were abnormal; all others were confirmed as abnormal. CONCLUSION: Analysis of multiple semen specimens provides a reliable screen in the evaluation of male factor infertility when the goal is to minimize the false negative rate of screening tests.

Ultrasonographic characteristics of first-trimester gestations in recurrent spontaneous aborters.

OBJECTIVE: To compile, for the first time, serial ultrasonographic findings during the first trimester of pregnancy in women with a history of primary recurrent spontaneous abortion so as to define the dynamics of early normal and abnormal gestations in this category of gravidas. STUDY DESIGN: Transvaginal ultrasonograms were obtained weekly from 5 to 12 weeks' gestational age in 40 women, 10 each of four groups: recurrent spontaneous aborters and primiparas (controls), with both successful and failed gestations. RESULTS: Embryonic heart motion was detected in 40-50% of successful pregnancies during the fifth week of gestation and in the balance by the sixth week, while heart motion was detected in no more than 50% of pregnancies that later failed. Of the failed pregnancies, all were evident by the eighth week of gestation, including those with previously documented viability. The gestational sac size and crown-rump length were smaller than expected in both failed groups, with the sac size difference evident as early as week 5 and the crown-rump length difference apparent by week 7. CONCLUSION: Appropriate timing of the initial ultrasonogram in recurrent aborters (i.e., 8 weeks' gestational age) can identify, by means of heart motion and gestational sac features, all pregnancies that will ultimately fail.

Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age.

This study evaluated the impact of age and ovarian reserve status on cumulative pregnancy rates. Approximately 1200 women from a general infertile population underwent ovarian reserve screening with the clomiphene citrate challenge test in the first few months of their initial evaluation. All patients then underwent a thorough infertility evaluation with therapy appropriate for their specific diagnoses. Patients with evidence of tubal disease, peritoneal adhesive disease, or male factor were eliminated. The 588 remaining patients were evaluated based on their age and ovarian reserve status, and their long-term pregnancy rates were compared using life table analyses. There was a dramatic decrease in pregnancy rates associated with an abnormal clomiphene citrate challenge test which was uniformly poor independent of age. Patients with normal ovarian reserve had much higher pregnancy rates, but a significant age-related decline in pregnancy rates was clearly identified. We conclude that women with evidence of diminished ovarian reserve have uniformly poor pregnancy rates independent of their age, but that age remains an important prognostic factor among those with a normal ovarian reserve. The combined use of maternal age and ovarian reserve screening should be used when counselling individual patients regarding their long-term prognoses for conception.

Activation of complement in humans with a first-trimester pregnancy loss.

Serum complement (C') activity in recurrent spontaneous aborters and primiparous controls with successful and unsuccessful pregnancies was quantified so as to define the dynamics of C' activation in early pregnancy loss. C' hemolytic activity was shown to be stable throughout the first trimester of pregnancy and did not differ from preconception levels in all of the successful pregnancies of recurrent aborters and controls and in the majority of pregnancy losses. However, 30% of recurrent aborters and 20% of controls with a pregnancy loss demonstrated activation of C' by the alternate pathway as early as the 7th week with a progressive decline in C' activity until abortion was clinically completed. Circulating levels of C3 dropped from 1.34 to 0.53 mg/dl, and factor B levels declined from 0.34 to 0.14 mg/dl in these hypocomplementemic women. Pregnancy loss is therefore associated with C' activation in a subset of both recurrent and nonrecurrent aborters and this occurs largely before loss of fetal viability.

The effect of a surgical lubricant on in vivo sperm penetration of cervical mucus.

The implications of these data is that coital lubricants may impair fertility of some couples. Unfortunately, conception was not an end point on this study, and our conclusions must be considered inferential. Nevertheless, the results from this study lead us to recommend avoidance of surgical lubricants by couples attempting pregnancy.

Outcome of pregnancy after laparoscopy and chromotubation during cycles of conception: a report of three cases.

BACKGROUND: Diagnostic tubal patency procedures may displace an early pregnancy. Hysterosalpingography performed in the presence of an unrecognized pregnancy has been reported to result in an ectopic pregnancy. No such information on laparoscopy performed during a cycle of conception has been reported. CASES: Three cases of laparoscopy were performed during the luteal phase of an unrecognized pregnancy cycle. The three patients were counseled to avoid conception before surgery. All had an undetectable serum hCG level within 3 days of surgery and an undetectable urine hCG level on the day of surgery. The three pregnancies had an uneventful prenatal course, with delivery at term. CONCLUSION: Although follicular phase scheduling of tubal patency tests would avoid potential pregnancy complications, the risk from luteal phase laparoscopy and chromotubation to an unrecognized pregnancy may be less than expected.

Characterization of peripheral blood and peritoneal fluid mononuclear cell subsets in fertile and infertile women.

Mononuclear cell subpopulations from the peripheral blood (PB) and peritoneal fluid (PF) of fertile and infertile women were quantified by flow cytometry using a double-staining monoclonal antibody technique. No differences in the percentage distribution of mononuclear cells between fertile and infertile women were demonstrated when either the PB constituents or the PF components were compared to one another. When the mononuclear cell composition in the PB was compared with that in the PF however, the percentage of PF-activated T cells and monocytes was increased in both fertile and infertile women as opposed to that in the PB, while there was a decrease in T helper cells (fertile and infertile women) and natural killer cells (fertile women and infertile women with endometriosis). Even though shifts do exist in the immunocytes of PF from fertile and infertile women, evidence is lacking that the PF is an immunologic mediator of infertility.

Natural killer cell activity in early human pregnancy.

Natural killer (NK) cell activity was determined in 41 women during the first trimester of pregnancy. NK cytotoxicity was similar between the study subjects and nonpregnant controls and was not a reflection of a change in the circulating number of cells or density in culture. Although NK cells may play a role in maternal immune status in advanced pregnancy, NK cells do not appear to be crucially involved in the first trimester, when reproductive wastage usually occurs.

First trimester sonographic characteristics of patients with recurrent spontaneous abortion.

First trimester ultrasonographic evidence of FHM infers a good prognosis for normal pregnancies, although similar data in RSA is scarce. Abortion before 20 weeks' gestation occurred in 12 of 167 control patients (7.2%) and five of 23 patients with RSA (21.7%) after detection of fetal heart motion (P = 0.0382). The abortion rate decreased with a MSS-CRL difference of > 8 mm in controls but not in patients with RSA. We conclude that the probability of pregnancy success is not improved with FHM identification in patients with RSA.