Expression of the alphav integrin adhesion molecule during development of preimplantation human embryos.

OBJECTIVE: To investigate the expression of alphav integrin in developing human embryos. DESIGN: Observational study. SETTING: University medical center. PATIENT(S): Women undergoing IVF cycles. INTERVENTION(S): Immunofluorescent monoclonal antibodies against alphav integrin were used to stain human preimplantation embryos. MAIN OUTCOME MEASURE(S): Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative and quantitative expression of integrins in human embryos. RESULT(S): The alphav integrin subunit was found to be expressed in human embryos throughout their development (from two-cell stage up to blastocyst). The expression of alphav integrin subunit gradually increased throughout embryo development as measured quantitatively by image analysis. CONCLUSION(S): The expression of alphav integrin subunit throughout the development of preimplantation human embryos reinforces the concept of a role for integrins in the process of implantation.

Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures?

OBJECTIVE: To determine whether blastocyst transfer is of benefit to patients with multiple IVF failures. DESIGN: Retrospective cohort study. SETTING: The George Washington University Medical Center. PATIENT(S): Patients undergoing IVF between October 1, 1997, and November 30, 1998, who had previously undergone three or more unsuccessful IVF cycles. Patients who had at least three embryos at the 8- to 12-cell stage available on day 3 were eligible for the study. INTERVENTION(S): Patients were given the option of day 3 ET (group A) or blastocyst transfer (group B). MAIN OUTCOME MEASURE(S): Blastocyst-formation rate, clinical pregnancy rate (PR) per transfer, and implantation rate per transfer. RESULT(S): Groups A and B were similar in terms of age, the number of previous failed IVF cycles, fertilization rate, and the number of fertilized oocytes per cycle. The blastocyst-formation rate was 51.0%. Clinical pregnancy and implantation rates per transfer were statistically significantly higher in the blastocyst-transfer group. There were no multiple pregnancies after blastocyst transfer. CONCLUSION: Blastocyst transfer increases implantation rates and PRs in patients with multiple failed IVF cycles, without increasing the risk of multiple pregnancy.

Response to clomiphene citrate challenge test in normal women through perimenopause.

OBJECTIVE: To identify the physiological changes that discriminate subgroups of women along the reproductive continuum. METHODS: This prospective study was carried out at the outpatient facility of George Washington University Medical Center and the outpatient clinic of the National Institutes of Health Clinical Center. Twenty-five female subjects were divided into 4 biologically distinct groups: group 1 (n = 4) regular menstrual cycles and under age 35; group 2 (n = 11) regular cycles and over age 35; group 3(n = 6) irregular cycles and over age 35, and group 4(n = 4) menopausal. Measurements of basal and stimulated gonadotropins (immunoactive LH, LH-I; bioactive LH, LH-B; immunoactive FSH, FSH-I) and E2 were obtained before and after administration of clomiphene citrate. Basal and stimulated gonadotropins and E2 were analyzed to discriminate between subject groups 1-4. The relationship of menstrual cyclicity to hormone levels was evaluated. RESULTS: Basal levels of LH-I, LH-B, FSH-I could discriminate only group 4 vs. groups 1-3. Stimulated levels of FSH-I and E2 were significantly different for group 2 vs. 3 and group 2 vs. 4. Group 1 was similar to group 2. Both stimulated FSH-I and stimulated LH-I and LH-B differentiated group 4 vs. groups 1-3. The LH-B:LH-I (B:I) ratio was not discriminatory after the clomiphene citrate challenge test (CCCT). CONCLUSIONS: Baseline hormone values were useful in distinguishing only group 4. CCCT unmasked differences in FSH and E2 between irregularly and regularly cycling older women; these differences were not scen with LH-B or B:I ratio. Stimulated FSH was the most useful hormone parameter and paralleled menstrual cycle regularity as a useful discriminator in older women.

Increasing clinical pregnancy rates after IVF/ET. Can immunosuppression help?

OBJECTIVE: To evaluate the safety and efficacy of immunosuppression as an adjunct to improving the success of in vitro fertilization/embryo transfer (IVF-ET). STUDY DESIGN: A randomized, double-blind, placebo-controlled clinical trial. RESULTS: Seventy-five patients were randomized to receive either prednisone (39 patients, 51%) or placebo (36 patients, 49%). Patients in both groups had similar ages and numbers of preembryos transferred. CONCLUSION: Both the implantation and clinical pregnancy rates were higher in the prednisone group (16% vs. 11% and 43.5% vs. 32.3%, respectively). However, these differences did not achieve statistical significance. Evaluation of the ongoing pregnancy rate revealed little difference between the prednisone-treated patients (30.7%) and those receiving placebo (28.0%). There were no side effects reported by patients in either group.

Basal and stimulated gonadotropin levels in the perimenopause.

OBJECTIVE: We investigated whether perimenopausal menstrual cycle irregularity is associated with increased gonadotropin immunoactivity, bioactivity, or the bioactivity/immunoactivity ratio at baseline and after short-term stimulation with gonadotropin-releasing hormone. STUDY DESIGN: Subjects consisted of four groups: (1) young regular cycling women (< 35 years old), older women (> 35 years) with (2) regular or (3) irregular menstrual cycles, and (4) postmenopausal women. Gonadotropin-releasing hormone stimulation tests (100 micrograms intravenous gonadotropin-releasing hormone) were performed in the National Institute of Mental Health outpatient clinic during the follicular phase of the menstrual cycle or randomly in postmenopausal women. RESULTS: Perimenopausal women had baseline follicle-stimulating hormone and luteinizing hormone levels and stimulated follicle-stimulating hormone levels (area under the curve) that were similar to those of postmenopausal women and significantly greater than those of control (younger and older) women. Postmenopausal women had significantly greater baseline levels of luteinizing hormone bioactivity than did the other three groups. The bioactivity/immunoactivity ratio in the postmenopausal women was significantly greater than those in the perimenopausal and older cycling women, which were similar. No change in the bioactivity/immunoactivity ratio was seen after gonadotropin-releasing hormone stimulation in any group. CONCLUSIONS: Although the perimenopause is associated with increases in baseline and stimulated gonadotropin levels similar to those seen after the menopause, significantly increased baseline luteinizing hormone bioactivity and the bioactivity/immunoactivity ratio are seen only after the menopause.

Achieving multiple-order embryo transfer identifies women over 40 years of age with improved in vitro fertilization outcome.

OBJECTIVE: To define factors in patients > or = 40 years that may improve outcome and provide prognosis for success in IVF-ET. DESIGN: Retrospective. SETTING: University infertility center. PATIENTS: Patients (n = 501) undergoing IVF-ET from 1987 to 1994. INTERVENTIONS: IVF-ET (n = 713 cycles) with GnRH-analogue suppression and hMG stimulation. MAIN OUTCOME MEASURES: We evaluated data including age, diagnosis, prestimulation (day 3) FSH and E2, hMG ampules used, days of monitoring, follicle number and size, maximum E2, ova retrieved, cancellation rate, clinical pregnancy, nidation, and miscarriage rates. RESULTS: Overall, patients > or = 40 years had significantly decreased pregnancy rates (PRs), response to stimulation, and increased miscarriage rates. However, if these patients had four or more embryos transferred, their response and PRs (34.4% per ET) were not significantly different from younger women (47.7% per ET). The majority (77.8%) of pregnancies in women > or = 40 years occurred when four or more embryos were transferred. CONCLUSION: A subset (49%) of women > or = 40 years undergoing IVF-ET will respond to ovarian stimulation well enough to result in four or more embryos available for transfer with a resultant PR similar to that observed in younger patients. We recommend consideration of an attempt at IVF-ET before recommending oocyte donation.

Immunocytochemical localization of growth factors and their receptors in human pre-embryos and Fallopian tubes.

We utilized indirect immunocytochemistry to demonstrate the presence of growth factors and their receptors in human pre-embryos and Fallopian tubes. In pre-embryos, only transforming growth factor-alpha (TGF-alpha) and the intracellular domain of epidermal growth factor receptor (EGFR) were found at the 4-cell stage. In 8- to 14-cell pre-embryos, TGF-alpha, the intracellular and extracellular domains of EGFR, and insulin-like growth factor-I and its receptor were found. Antibodies against TGF-alpha stained all Fallopian tube specimens, while the extracellular domains of EGFR was only found in specimens from patients with either blood type A or AB. These results suggest a cross-reactivity between the extracellular domain of the EGFR and blood group antigens. Our novel demonstration of growth factor receptor staining in human pre-embryos shows that growth factor receptor localization is dependent on the developmental stage of human pre-embryos. We have also established a potentially important link between the Fallopian tube which secretes growth factors and the localization of growth factor receptors in pre-embryos. These findings are compatible with the hypothesis that tubal secretions are embryotrophic for the early development of the pre-embryo.

Prognostic value of day 3 estradiol on in vitro fertilization outcome.

OBJECTIVE: To evaluate the prognostic value of day 3 E2 levels, independent of day 3 FSH levels, on responses to ovulation induction and subsequent pregnancy rates (PRs) in IVF-ET patients. DESIGN: Prospective, observational. SETTING: University-based tertiary care and private reproductive endocrine-infertility units. PATIENTS AND INTERVENTIONS: A total of 225 patients underwent 292 IVF cycles with luteal phase GnRH agonist suppression and hMG stimulation. MAIN OUTCOME MEASURES: We evaluated response and outcome data including age, day 3 FSH and E2 levels from a menstrual cycle before IVF, ampules of hMG used, maximum E2 level, cancellation rates, and clinical PR. RESULTS: Despite similar age, number of ampules of hMG, and peak E2 levels, patients with an elevated E2 level (E2 > or = 80 pg/mL) (conversion factor to SI unit, 3.671) on day 3 of a cycle before IVF-ET achieved a lower PR per initiated cycle (14.8% versus 37.0%) and had a higher cancellation rate (18.5% versus 0.4%) compared with those with E2 levels < 80 pg/mL. Even when patients with elevated FSH levels (FSH > or = 15 mIU/mL) (conversion factor to SI unit, 1.00) were excluded (leaving 279 cycles), those with an elevated day 3 E2 still had a lower PR per initiated cycle (14.8% versus 38.9%) and maintained a higher cancellation rate (18.5% versus 0.4%). When the day 3 E2 was > or = 100 pg/mL there was a 33.3% cancellation rate and no pregnancies were achieved. CONCLUSION: Patients who presented with an elevated day 3 E2 (> or = 80 pg/mL) in a cycle before IVF-ET had a higher cancellation rate and achieved a lower PR independent of FSH level. A day 3 E2 level, in addition to a day 3 FSH level, appears very helpful in prospectively counseling patients regarding cancellation risk and ultimate IVF-ET success.

Lineage tracing demonstrates that blastomeres of early cleavage-stage human pre-embryos contribute to both trophectoderm and inner cell mass.

We injected a fluorescent lineage tracer (Texas Red-lysine-dextran) into individual blastomeres of donated human diploid 2- to 8-cell pre-embryos and cultured them to blastocysts. Once pre-embryos reached the expanded blastocyst stage, they were fixed and examined in a scanning confocal microscope to identify the location of fluorescent tracer. In successfully injected pre-embryos that developed to expanded blastocysts, we found that randomly injected blastomeres formed both trophectoderm (TE) and inner cell mass (ICM). More labelled progeny were found in TE than in ICM. Our results show that individual early blastomeres are not yet committed to form either TE or ICM but instead can form both rudiments.

Utility of in vitro fertilization at diagnostic laparoscopy.

OBJECTIVE: To compare stimulation and outcome variables for IVF in stimulated cycles when ova are retrieved during diagnostic infertility laparoscopy versus transvaginal ultrasound (US) directed retrieval and to investigate the presence of unexpected failed fertilization in the diagnostic laparoscopy group, which allows an opportunity to diagnosis an etiology of infertility based on gamete interaction. DESIGN: Consecutive patients who needed infertility diagnostic laparoscopy and agreed to combination with IVF were compared with concurrent patients undergoing transvaginal US IVF. Male factor screening parameters (semen analysis, sperm penetrating assay) and resultant fertilization were analyzed for these patients. SETTING: The George Washington University Hospital, a tertiary referral center offering assisted reproductive technologies. Patients for diagnostic laparoscopy combined with IVF were enrolled in the Program of Oocyte Retrieval at Diagnostic Laparoscopy (PORDL). PARTICIPANTS: One hundred twenty-four women enrolled for diagnostic laparoscopy combined with IVF; 237 women were concurrently enrolled for transvaginal US IVF. RESULTS: Response variables (number of follicles, days of monitoring, ampules of hMG, maximum E2) between the two groups were similar. Outcome variables (ova retrieved, ova fertilized, ova cleaved, clinical pregnancy rate per embryo transferred) were similar despite a significantly higher number of embryos transferred for the transvaginal US group. The clinical pregnancy rate per cycle was similar, 26% versus 28% for the women in the transvaginal US versus those women in the PORDL group, as was the clinical pregnancy rate per ET, 31% versus 34%, respectively. The number of fertilized ova for each group was not significantly different: 5.5 +/- 0.3 for the transvaginal group versus 4.8 +/- 0.4 for the PORDL group. Patients in the PORDL group with a known male factor (group B) had a lower fertilization rate than those with no male factor (group A). Within the group A with no detectable male factor prospectively, 17.2% had unexpectedly poor fertilization (group A1), whereas the rest of the group A patients had higher fertilization rates as was expected (group A2). The clinical pregnancy rate per ET for group A1 was 0% compared with 43.4% for the group A2 patients. CONCLUSIONS: In vitro fertilization can be successfully performed during diagnostic laparoscopy yielding comparable results to transvaginal ultrasound IVF while gaining diagnostic information concerning sperm-ova interaction (i.e., fertilization).

Ovarian suppression with leuprolide acetate: comparison of luteal, follicular, and flare-up administration in controlled ovarian hyperstimulation for oocyte retrieval.

Adjunct use of leuprolide (LA) in patients undergoing controlled ovarian hyperstimulation with human menopausal gonadotropins (hMG) was evaluated by three protocols: Group F (n = 24) began LA on day 2 of the cycle and Group L (n = 38) began LA on day 23 of the cycle until ovarian suppression, at which time hMG was added. Group FL (n = 17) began LA on day 1 and hMG on day 3. Compared to FL, more ova were collected, more ova fertilized, and more pregnancies resulted per initiated cycle in groups achieving suppression before hMG stimulation. Fewer days were necessary to attain suppression for L vs F. After achieving suppression, patients were maintained on either 0.5 mg LA or 0.25 mg LA daily during hMG coadministration with similar results. Lower maintenance doses of LA during hMG did not decrease the amount of hMG needed but retained the benefits of LA. We recommend luteal initiation of LA to achieve suppression before hMG.

Efficacy of assisted reproductive technology during diagnostic and operative infertility laparoscopy.

Women being evaluated for infertility were offered assisted reproductive technology at the time of diagnostic laparoscopy. Oocyte retrieval was performed after ovulation induction in 33 women, of whom 19 had concurrent operative laparoscopy. Gamete intrafallopian transfer (GIFT) or in vitro fertilization (IVF) and embryo transfer were performed subsequently depending on laparoscopic assessment of pelvic architecture, oocyte maturity, and semen parameters. The clinical pregnancy rate was 24% per cycle and 28% per gamete or embryo transfer (four pregnancies after GIFT and four after IVF/embryo transfer). The clinical pregnancy rate per transfer did not differ significantly between the 19 women who had therapeutic operations in conjunction with laparoscopy (lysis of adhesions and/or fulguration of endometriosis) and the 13 who did not (25 versus 30%; P greater than .05). Assisted reproductive technology can be performed successfully during diagnostic infertility laparoscopy. Operative endoscopic manipulation did not adversely influence pregnancy outcome.

Dexamethasone treatment prevents the inhibitory effect of corticotropin-releasing hormone on gonadotropin release in the primate.

Corticotropin-releasing hormone (CRH) has been shown to inhibit gonadotropin secretion and this effect is mediated by endogenous opioid peptides, presumably stimulated by CRH. Since glucocorticoids are known to block the CRH-induced ACTH response, it can be hypothesized that by concurrently preventing endogenous opioid peptide release, they would also prevent the inhibitory action of CRH on gonadotropin secretion. We tested this hypothesis in 4 ovariectomized rhesus monkeys, pretreated with dexamethasone (DEX; 1.5 mg b.i.d. for 5 days). In experiment 1, the effects of a 5 h i.v. hCRH infusion with or without DEX pretreatment and of physiological saline were compared. Blood samples were taken at 15-min intervals during a 3 hour preinfusion control and throughout the infusion. Sera were assayed for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and cortisol by RIA. In the absence of DEX pretreatment, LH and FSH levels were progressively decreased during the CRH infusion: by hour 5, LH and FSH areas under the curve were 34.1 ( +/- 7.6) and 65.3% ( +/- 2.5) (mean % of preinfusion control values; + SE), respectively (p less than 0.01 vs. saline). In contrast, DEX pretreatment prevented the CRH-induced gonadotropin decrease: by hour 5, LH and FSH areas under the curve were 91.9 ( +/- 9.0) and 99.0% ( +/- 5.7) (n.s. vs. saline). In experiment 2, we tested whether DEX-treated monkeys would remain responsive to the gonadotropin inhibitory action of an opiate agonist. After a 3 hour preinfusion control baseline, morphine (9 mg i.v.) was given as a bolus injection to the same 4 animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained effects of opioid antagonism during the normal human luteal phase.

The luteal phase of the menstrual cycle is characterized by a progressive decrease in LH pulse frequency. Short term administration of opiate receptor antagonists during the luteal phase increases the release of both LH and PRL. However, the effects of prolonged opioid antagonism throughout the luteal phase are unknown and, hence, the precise role of endogenous opioid peptides in the reproductive cycle remains to be elucidated. In this study, we examine the ability of longer term opioid antagonism during the luteal phase to alter pulsatile LH and PRL release. Naltrexone (NTX), a long-acting oral opioid antagonist, at a dose of 50 mg, was administered daily for 7 days during the luteal phase in five women. Blood samples were obtained at intervals of 10 min starting at 0800 h for 11-12 h on matched days of the luteal phase of both a control and the experimental cycle. LH and PRL pulse frequencies were significantly increased at the end of the 7-day NTX administration period compared to those in the control cycle [LH, 0.22 +/- 0.04 (+/- SE) vs. 0.07 +/- 0.03 pulse/h (P less than 0.01); PRL, 0.20 +/- 0.02 vs. 0.13 +/- 0.02 pulse/h (P less than 0.05)]. The concordance between LH and PRL pulses increased from 50% in the control cycle to 70% in the NTX cycle, and there was a significant positive correlation between the amplitudes of the concomitant LH and PRL pulses (r = 0.72; P = 0.01). In conclusion, prolonged oral opioid antagonism increased pulsatile LH and PRL secretion during the luteal phase in normal women. The results underscore the important role of endogenous opioid peptides in controlling LH pulse frequency during the luteal phase of the cycle.

Induction of ovulation--past, present and future.

Attempts to induce ovulation have been made since the early 1920s, but the major breakthrough came in the early 1960s with the introduction of clomiphene citrate and the gonadotropins. Additional progress was made in the early 1970s with the introduction of bromocriptine and in the early 1980s with the introduction of pulsatile GnRH. At the present, 'pure' FSH and GnRH agonists are being evaluated as adjuncts to HMG for induction of ovulation. As more insight is gained in the neuroendocrine control of the ovulating cycle, we may soon be able to induce ovulation by direct manipulation of the central nervous system.

Polycystic ovarian disease.

Polycystic ovarian disease has a variety of biochemical and clinical features with great individual variation. In our clinical experience, oligo-ovulation, manifested as oligomenorrhea or frank amenorrhea, associated with an acyclic estrogen milieu, is a consistent finding. This may be associated with hyperandrogenemia, hirsutism, inappropriate gonadotropin levels, hyperprolactinemia, obesity, insulin resistance, and ultrasound evidence of multicystic enlarged ovaries. A common presentation is infertility or irregular menstruation secondary to oligo-ovulation and hirsutism secondary to altered androgen metabolism. A challenge in diagnosis is to differentiate polycystic ovarian disease from latent cases of congenital adrenal hyperplasia. Although the precise mechanism in the pathogenesis of polycystic ovarian disease remains undefined, altered function of the hypothalamic-pituitary-ovarian and adrenal axes is both involved and integrated. Results from clinical trials of ovulation induction using different agents have implicated one site or another as the major progenitor of the "vicious cycle" but with no definitive pathway established. Restoring fertility to these patients can be challenging in that not all patients with polycystic ovarian disease respond to clomiphene or do so satisfactorily. The use of glucocorticoid suppression, pituitary suppression with GnRH analogues, or the use of FSH alone may be of benefit in clomiphene treatment failures.

Endogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate.

Stress can induce endocrine abnormalities and menstrual dysfunction in the primate. Here, we examine the effects that CRF, the principal neurohormone in control of the hypothalamic-pituitary-adrenal axis, exerts on pulsatile gonadotropin secretion and the role that the endogenous opioid peptides may play in this phenomenon. Ovariectomized rhesus monkeys were given a 5-h continuous iv infusion of physiological saline (2 ml/h), human CRF (100 micrograms/2 ml . h), or hCRF plus the opiate receptor antagonist naloxone (2 mg/2 ml/h; 5 mg in two experiments; n = 7 experiments/group). LH and FSH concentrations were measured at 15-min intervals for a 3-h preinfusion baseline control, during the 5-h infusion, and during a 2-h postinfusion observation period, while cortisol concentrations were measured at frequent intervals during the entire experiment. CRF infusion produced a progressive and significant decrease in both LH and FSH. Mean areas (+/- SE) under the LH and FSH curves during the 5-h CRF infusion, expressed as a percentage of preinfusion baseline, were 59.9 +/- 4.6% and 83.0 +/- 3.1% (+/- SE), respectively (P less than 0.001 and P less than 0.01 vs. saline controls). Large amplitude LH pulses were abolished during the CRF infusion. However, after cessation of CRF infusion, there was a rapid resumption of LH pulsatile release in four of the seven experiments. Addition of naloxone to CRF prevented the CRF-mediated suppression of LH and FSH release. Mean areas for LH and FSH during the 5-h combined infusion were 100.3 +/- 6.6% and 99.6 +/- 4.3% of the preinfusion baseline, respectively (P less than 0.001 and P less than 0.05 vs. CRH alone; NS vs. saline), and pulsatile LH secretion was maintained. Regardless of whether naloxone was administered, CRF increased cortisol levels significantly. Mean cortisol levels at the end of the CRF and CRF plus naloxone infusions were 48.2 +/- 10.4 and 52.9 +/- 7.4 micrograms/dl (+/- SE), respectively, compared to 21.0 +/- 3.0 with saline (P less than 0.05). These results demonstrate that in the ovariectomized rhesus monkey, CRF suppresses the secretion of both LH and FSH, and this effect can be sustained. They also indicate that the CRF inhibitory action on gonadotropin is primarily mediated by endogenous opioid peptides, independent of glucocorticoid levels.