Advanced Methods for Dose and Regimen Finding During Drug Development: Summary of the EMA/EFPIA Workshop on Dose Finding (London 4-5 December 2014).

Inadequate dose selection for confirmatory trials is currently still one of the most challenging issues in drug development, as illustrated by high rates of late-stage attritions in clinical development and postmarketing commitments required by regulatory institutions. In an effort to shift the current paradigm in dose and regimen selection and highlight the availability and usefulness of well-established and regulatory-acceptable methods, the European Medicines Agency (EMA) in collaboration with the European Federation of Pharmaceutical Industries Association (EFPIA) hosted a multistakeholder workshop on dose finding (London 4-5 December 2014). Some methodologies that could constitute a toolkit for drug developers and regulators were presented. These methods are described in the present report: they include five advanced methods for data analysis (empirical regression models, pharmacometrics models, quantitative systems pharmacology models, MCP-Mod, and model averaging) and three methods for study design optimization (Fisher information matrix (FIM)-based methods, clinical trial simulations, and adaptive studies). Pairwise comparisons were also discussed during the workshop; however, mostly for historical reasons. This paper discusses the added value and limitations of these methods as well as challenges for their implementation. Some applications in different therapeutic areas are also summarized, in line with the discussions at the workshop. There was agreement at the workshop on the fact that selection of dose for phase III is an estimation problem and should not be addressed via hypothesis testing. Dose selection for phase III trials should be informed by well-designed dose-finding studies; however, the specific choice of method(s) will depend on several aspects and it is not possible to recommend a generalized decision tree. There are many valuable methods available, the methods are not mutually exclusive, and they should be used in conjunction to ensure a scientifically rigorous understanding of the dosing rationale.

The future is now: model-based clinical trial design for Alzheimer's disease.

Failures in trials for Alzheimer's disease (AD) may be attributable to inadequate dosing, population selection, drug inefficacy, or insufficient design optimization. The Coalition Against Major Diseases (CAMD) was formed in 2008 to develop drug development tools (DDT) to expedite drug development for AD and Parkinson's disease. CAMD led a process that successfully advanced a clinical trial simulation (CTS) tool for AD through the formal regulatory review process at the US Food and Drug Administration (FDA) and European Medicines Agency (EMA).

The Role of Modeling and Simulation in Development and Registration of Medicinal Products: Output From the EFPIA/EMA Modeling and Simulation Workshop.

The European Medicines Agency (EMA) and the Federation of Pharmaceutical Industries and Associations (EFPIA) hosted a workshop on modeling and simulation (M&S).(1) Representatives from industry, academia, and regulatory agencies from Europe and beyond discussed the role of M&S in the development and registration of medicinal products within plenary and breakout sessions (BOS). This manuscript summarizes the plenary discussion (Table 1) focusing on the European perspective. Deliverables from each BOS are included in separate papers.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e31; doi:10.1038/psp.2013.7; advance online publication 27 February 2013.

Gatekeepers and enablers: how drug regulators respond to a challenging and changing environment by moving toward a proactive attitude.

This article analyzes the role of regulatory authorities in facilitating innovation in the pharmaceutical sector. We describe how regulators are expanding their role to be not only gatekeepers but also enablers of development. They have already responded to the challenging and changing environment by moving toward a proactive attitude beyond evaluation of products, thereby more actively contributing to their development. Regulators have to continuously evolve their knowledge and standards alongside evolution in science. Creation of supportive regulatory frameworks and multistakeholder interaction will help address unmet regulatory needs.

Antisepsis for abdominal hysterectomy: a randomised controlled trial of povidone-iodine gel.

OBJECTIVE: To assess whether infectious morbidity after total abdominal hysterectomy is decreased by the addition of 20 cc povidone-iodine gel at the vaginal apex after the usual vaginal preparation with povidone-iodine solution. STUDY DESIGN: Randomised controlled trial. SETTING: Fifteen secondary and tertiary hospitals in Canada. SAMPLE: A total of 1570 women undergoing planned total abdominal hysterectomy. METHODS: Computer-generated randomisation using a centralised telephone service was stratified by study centre with variable block size. In the operating room, a swab for bacterial vaginosis was taken before vaginal antisepsis. Study group remained concealed until the standard surgical preparation in the operating room was complete. Then povidone-iodine gel 20 cc was placed at the vaginal apex in the intervention group only. Participants were followed for one month post-operative. MAIN OUTCOME MEASURES: The primary outcome was post-operative infectious morbidity during the 30 days after surgery, defined as: febrile morbidity with hospital stay greater than five days or antibiotic treatment, or infection requiring readmission to hospital or additional visit. Other outcomes included abdominal wound infection, pelvic abscess and other pelvic infections. RESULTS: Post-operative infectious morbidity within 30 days occurred in 128/780 (16%) women receiving povidone-iodine gel preparation and 142/790 (18%) women not receiving gel (RR 0.9, 95% CI 0.7 to 1.1). Pelvic abscess was diagnosed in 0 patients in the gel group and in seven patients in the control group (P < 0.01). No significant difference was found in pelvic cellulitis (eight in each group) or abdominal wound infection (51 in the gel group and 58 in the control group, P= 0.5). CONCLUSION: Povidone-iodine vaginal gel antisepsis led to a 9% relative decrease in overall infectious morbidity after abdominal hysterectomy, which was not statistically significant. Povidone-iodine vaginal gel decreased the risk of pelvic abscess after total abdominal hysterectomy.

Effects of functional ovarian cysts detected on the 7th day of gonadotropin-releasing hormone analog administration on the outcome of IVF treatment.

OBJECTIVE: To investigate the impact of functional ovarian cysts on the time required to achieve pituitary suppression, follicular development, embryo quality, and pregnancy rates during IVF treatment. DESIGN: Prospective observational study. INTERVENTION(S): Daily treatment with buserelin (sc 500 microg) was initiated on day 2 of menstruation. Ultrasound and hormonal tests were performed on days 1, 7, 11, 14, and weekly thereafter until pituitary suppression was achieved. RESULT(S): 48 patients underwent 51 cycles of IVF treatment. A functional cyst was detected in three cycles (5.8%) with baseline ultrasound scan and in 27 cycles (52.9%) on day 7 of buserelin administration. Patients who developed a cyst required a significantly longer time to achieve pituitary suppression (21 vs. 7 days), had a significantly lower FSH level at the time of initiation of gonadotropins, required more ampules of gonadotropin (45 vs. 41 ampules), developed less follicles (13 vs. 17.5), and had lower embryo quality. However, there were no differences in the implantation (23.5% vs. 17.2%) and pregnancy rates (37.2% vs. 29.2%) between two groups. CONCLUSION(S): Functional cysts prolong the period to achieving pituitary suppression, increase gonadotropin requirements, and decrease follicular recruitment and embryo quality. They have, however, no negative effect on pregnancy rates.

Effects of pretreatment with an oral contraceptive on the time required to achieve pituitary suppression with gonadotropin-releasing hormone analogues and on subsequent implantation and pregnancy rates.

OBJECTIVE: To assess the effect of pretreatment with an oral contraceptive (OC) on ovarian cyst formation during pituitary suppression with buserelin acetate. DESIGN: Prospective randomized trial. SETTING: Academic medical center. PATIENT(S): Eighty-three patients who were undergoing IVF-ET treatment. INTERVENTION(S): Patients in the study group were pretreated with an OC for 14 days starting on the first day of menstruation. The administration of SC buserelin acetate was initiated on the last day of OC administration. Patients in the control group began to receive buserelin acetate on day 2 of menstruation. Hormonal assays and ultrasound scans were performed on the first day of menstruation, and 7, 11, and 14 days after the commencement of buserelin acetate administration. Thereafter, these tests were performed weekly until pituitary suppression was achieved. MAIN OUTCOME MEASURE(S): Incidence of cyst formation. RESULT(S): A cyst developed in 27 patients in the control group (52.9%) and no patients in the study group (odds ratio [OR]=115; 95% confidence interval [CI]=10-617). Patients in the study group achieved pituitary suppression faster (median difference [MD]=7 days; 95% CI=4-14) and required fewer ampules of gonadotropin (MD=10; 95% CI=6-14). They recruited more follicles (MD=3; 95% CI=0-5) and had higher pregnancy rates (37.2% versus 33.3%). CONCLUSION(S): Pretreatment with an OC abolishes ovarian cyst formation, shortens the time required to achieve pituitary suppression, and decreases gonadotropin requirements without having a negative effect on pregnancy rates.

PIP: Administration of a gonadotropin-releasing hormone analog (GnRH-a) before ovarian stimulation with gonadotropins in women undergoing in vitro fertilization (IVF) treatment produces higher pregnancy and live birth rates, but also results in formation of ovarian cysts that must be treated before stimulation can commence. The effect of pretreatment with an oral contraceptive (OC) on ovarian cyst formation during pituitary suppression with the GnRH-a buserelin acetate was investigated in a prospective randomized trial of women undergoing IVF at Royal Victoria Hospital (Montreal, Quebec, Canada). 51 women were pretreated with an OC for 14 days, starting on the first day of menstruation, and began buserelin acetate (500 mcg/day) on the last day of OC administration. The 51 women in the control group were treated with the standard protocol of 500 mcg/day of buserelin acetate starting on the second day of menstruation. A cyst developed in 27 controls (52.9%) but in no women pretreated with OCs (odds ratio, 115; 95% confidence interval, 10.7-617.5). 49 pretreated women (96.1%) compared with 22 controls (43.1%) achieved pituitary suppression after 7 days of GnRH-a administration. Pretreated women also required a median of 10 fewer ampules of gonadotropin than controls, recruited a median of 3 more follicles than their non-pretreated counterparts, and had higher pregnancy rates (37.2% and 33.3%, respectively). OCs are assumed to prevent the formation of ovarian cysts during GnRH-a administration through a dual effect of pituitary suppression and ovarian protection. OC pretreatment enables a significant simplification of the long standard protocol of GnRH-a administration.

Pretreatment with an oral contraceptive is effective in reducing the incidence of functional ovarian cyst formation during pituitary suppression by gonadotropin-releasing hormone analogues.

PURPOSE: Our purpose was to assess the effect of pretreatment with oral contraceptives (OCs) on the formation of functional ovarian cysts during pituitary suppression with gonadotropin-releasing hormone (GnRH) agonists, subsequent follicular development, and pregnancy rates. METHODS: A retrospective case-controlled study of 31 in vitro fertilization (IVF) patients, all of whom in a previous cycle had commenced the long protocol of GnRH-agonist (Buserelin) in the early follicular phase and were pretreated in a subsequent cycle with 2 weeks of an OC containing 30 micrograms of ethinyl estradiol and 150 micrograms of desogestrel prior to GnRH-agonist administration, was undertaken. Follow-up visits were arranged after a minimum of 11 days of GnRH-agonist administration and weekly thereafter until pituitary suppression was achieved. RESULTS: Cysts were detected in 16 (51.6%) of the 31 patients not pretreated with OCs, and in 0 (0%) of the 31 patients pretreated with OCs (odds ratio = 67.1; 95% confidence interval = 5.6-350.7). Patients pretreated with OCs achieved pituitary suppression more rapidly (median difference = 4 days; 95% confidence interval = 2-7) and had comparable gonadotropin requirements and pregnancy rates. CONCLUSIONS: Pretreatment with OCs prior to pituitary suppression in the early follicular phase decreases ovarian cyst formation, without an apparent effect on subsequent follicular recruitment or pregnancy rates.

PIP: A retrospective case-controlled study was undertaken to assess the effects of pretreatment with oral contraceptive (OC) on the formation of functional ovarian cysts during pituitary suppression with gonadotropin-releasing hormone (GnRH) agonists, subsequently follicular development, and pregnancy rates. In the period between January 1997 and December 1997, 31 in vitro fertilizations, all of which in a previous cycle, had commenced the long protocol of GnRH agonists in the early follicular phase and were pretreated in a subsequent cycle with an OC containing 30 mcg ethinyl estradiol and 150 mcg desogestrel for 2 weeks prior GnRH agonist administration and then weekly until pituitary suppression was achieved. After data collection and analysis, findings revealed that functional ovarian cysts were detected in 16 (51.6%) of 31 patients not pretreated with an OC and in 0 (0%) of 31 patients pretreated with an OC. Satisfactory pituitary suppression was achieved more rapidly with patients pretreated with an OC. Further, comparable gonadotroph requirements and pregnancy rates were detected among patients pretreated with an OC. In conclusion, pretreating patients with an OC prior to pituitary suppression in the early follicular phase decreases ovarian cyst formation, without an apparent effect on subsequent follicular recruitment or pregnancy rates.

Results of laparoscopic treatments of ovarian endometriomas: laparoscopic ovarian fenestration and coagulation.

OBJECTIVE: To determine the long-term results of laparoscopic fenestration and coagulation of ovarian endometriomas and to compare them with the results of ovarian cystectomy performed by either laparotomy or laparoscopy. DESIGN: Case-control study. SETTING: Two university-affiliated hospitals. PATIENT(S): One hundred fifty-six premenopausal women with ovarian endometriomas of at least 3 cm in diameter (stage 3 and 4 endometriosis, revised American Fertility Society classification). INTERVENTION(S): Laparoscopic ovarian fenestration and coagulation (group 1, 80 patients); laparoscopic ovarian cystectomy (group 2, 23 patients); and ovarian cystectomy by laparotomy and microsurgical technique (group 3, 53 patients). MAIN OUTCOME MEASURE(S): Operative findings, recurrence rate, and cumulative clinical pregnancy rate (PR) over a 36-month follow-up period. RESULT(S): The mean (+/-SD) time to first pregnancy was significantly shorter in group 1 (1.4+/-0.2 years) than in group 2 (2.2+/-0.5 years) or group 3 (2.4+/-0.5 years). The difference between the cumulative clinical PR between the three groups was not statistically significant after 36 months of follow-up. The difference in the recurrence rate among groups 1, 2, and 3 was not statistically significant. CONCLUSION(S): Laparoscopic ovarian fenestration and coagulation of endometriomas leads to faster conception than ovarian cystectomy by laparotomy. Laparoscopic ovarian fenestration and coagulation of endometriomas is associated with cumulative clinical PRs and recurrence rates over 36 months that are similar to those associated with ovarian cystectomy.

Diagnostic accuracy of transvaginal ultrasound in detecting free pelvic fluid.

OBJECTIVE: To evaluate the diagnostic accuracy of transvaginal ultrasonography in detecting and measuring free pelvic fluid. STUDY DESIGN: Eighty-two patients undergoing diagnostic or therapeutic laparoscopy at a tertiary care center were prospectively assessed before surgery by transvaginal ultrasound. Free pelvic fluid was measured in two ultrasonographic planes. These measurements were compared to the volume of fluid aspirated during laparoscopy. RESULTS: The mean volumes reported for transvaginal ultrasound were significantly lower than those observed at laparoscopy (mean milliliters +/- SEM, 2.54 +/- 0.5 versus 9.42 +/- 1.3, P < .001). The smallest volume of free pelvic fluid that was consistently detected by ultrasound was 8 mL. Whenever no fluid or < 1 mL was detected by transvaginal ultrasound, a small volume of fluid was found at laparoscopy (mean milliliters +/- SEM, 1.6 +/- 0.47). The sensitivity of transvaginal ultrasound was 83% and specificity was 69%. CONCLUSION: Transvaginal ultrasound is a sensitive method of detecting the presence of > 8 mL of free pelvic fluid and therefore is an important diagnostic tool in the assessment of pelvic pathology associated with increased peritoneal fluid.

Combined treatment of endometriosis. GnRH agonists and laparoscopic surgery.

Use of preoperative gonadotropin-releasing hormone (GnRH) agonists may be beneficial in the case of laparoscopic surgery for large endometriomas. Further studies are necessary to confirm the claim that preoperative GnRH agonists facilitate laparoscopic surgery for endometriosis. Use of GnRH agonists after laparoscopic treatment of endometriosis has beneficial effects in preventing the recurrence of pelvic pain associated with endometriosis.

Use of nafarelin versus placebo after reductive laparoscopic surgery for endometriosis.

OBJECTIVE: To evaluate the efficacy of the GnRH agonist (GnRH-a) nafarelin compared with placebo administered for 6 months after reductive laparoscopic surgery for symptomatic endometriosis. DESIGN: Randomized, prospective, placebo-controlled, multicenter clinical trial. SETTING: Thirteen clinics including private practice and university centers. PATIENT(S): One hundred nine women aged 18-47 with laparoscopically proven endometriosis and pelvic pain who had undergone reductive laparoscopic surgery for endometriosis. INTERVENTION(S): Patients were randomized to receive either the GnRH-a nafarelin (200 micrograms twice daily) or placebo for 6 months. MAIN OUTCOME MEASURE(S): Time to initiation of alternative treatment (the length of time from beginning study medication to receiving alternative therapy or to deeming that the study drug was ineffective) and patient-reported and physician-assessed pelvic pain scores. RESULT(S): The median time to initiation of alternative treatment was > 24 months in the nafarelin group versus 11.7 months in the placebo group. Fifteen (31%) of 49 nafarelin-treated patients required alternative therapy, compared with 25 (57%) of 44 placebo-treated patients. The patients' pelvic pain scores dropped significantly in the nafarelin and placebo groups after 6 months of treatment. Physician summary ratings showed significant improvement in the nafarelin group and no significant changes in the placebo group after 6 months of treatment. CONCLUSION(S): Compared with placebo, nafarelin administered after reductive laparoscopic surgery for endometriosis significantly delays the return of endometriosis symptoms requiring further treatment.

Povidone-iodine gel vaginal antisepsis for abdominal hysterectomy.

OBJECTIVE: Our goal was to assess the safety and effectiveness of vaginal povidone-iodine gel in reducing febrile morbidity after abdominal hysterectomy. STUDY DESIGN: This cohort study included 158 women treated with gel immediately before hysterectomy, after the usual surgical preparation, and 317 historic control subjects with the usual surgical preparation only, at two teaching hospitals. Febrile morbidity was assessed by a blinded review of temperature records and was analyzed by Fisher's exact test and multiple logistic regression. RESULTS: Febrile morbidity occurred in 17% of gel-treated patients and 26% of controls (adjusted odds ratio 0.52, 95% confidence interval 0.31 to 0.89). In patients receiving prophylactic antibiotics the adjusted odds ratio for febrile morbidity in gel-treated patients was 0.47 (95% confidence interval 0.27 to 0.83). Prolonged fever occurred in 17% of controls and 10% of gel-treated patients (adjusted odds ratio 0.52, 95% confidence interval 0.28 to 0.97). CONCLUSION: Preoperative vaginal povidone-iodine gel is a safe and promising technique for reducing febrile morbidity after hysterectomy.

Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization.

OBJECTIVE: To assess the value of heparinized saline as a flushing medium for oocyte recovery. DESIGN: Prospective randomized study. SETTING: Academic tertiary referral center for fertility treatment. PATIENT(S): Thirty-five patients, with both ovaries intact having IVF-ET. INTERVENTION(S): Patients were randomized either to have the follicles of the left or right ovary flushed with heparinized normal saline at the time of oocyte recovery for IVF-ET. The contralateral ovary was flushed with heparinized culture medium. Oocytes obtained from each side were cultured separately and assessed for fertilization 18-21 hours after insemination. MAIN OUTCOME MEASURE(S): Collection and fertilization rates. RESULT(S): A total of 481 follicles were aspirated yielding 366 oocytes. Of these, 240 fertilized. From the side flushed with saline 185 oocytes were collected from 237 follicles, which was not significantly different from 181 oocytes collected from 244 follicles on the side flushed with culture medium (odds ratio = 1.23; 95% confidence interval = 0.79-1.92). Similarly, there was no significant difference observed in fertilization rates between oocytes obtained after saline (median 71.4%) and culture medium flush (median 75.0%) (odds ratio = 1.08; 95% confidence interval = 0.68-1.72). CONCLUSION(S): Heparinized normal saline is an equally good but cheaper and more convenient medium than standard heparinized culture medium and could replace it for flushing follicles during oocyte recovery for IVF-ET procedures.

Flow cytometric evaluation of leukocyte subpopulations in the follicular fluids of infertile patients.

OBJECTIVES: To evaluate the leukocyte subpopulations present in follicular fluid (FF) of infertile patients undergoing IVF-ET for tubal factor, idiopathic infertility, and endometriosis. PATIENTS: Sixty patients undergoing IVF-ET with a tubal factor diagnosis (n = 35), idiopathic infertility (n = 13), and endometriosis (n = 12) had their subpopulations of FF leukocytes analyzed by flow cytometry. MAIN OUTCOME MEASURE: Nonblood-contaminated samples of FF were collected under sterile conditions and centrifuged. Cells were labeled with a panel of monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD14, -CD20, -CD45, and -CD56, and analyzed by cytofluorometry. RESULTS: Follicular fluid leukocytes from patients with idiopathic infertility had a significantly higher proportion of T lymphocytes than tubal factor and endometriosis patients. Endometriosis patients had significantly higher proportions of natural killer (NK) cells, B lymphocytes, and monocytes compared with groups of idiopathic infertility and tubal factor. CONCLUSIONS: The differences observed in the leukocyte subpopulations from FF of patients with idiopathic infertility and endometriosis may affect folliculogenesis and oocyte maturation. Moreover, these modifications could be one of the factors altering their fertility.

Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome.

Several lines of evidence indicate that immunologic effectors, particularly suppressor T cells and NK cells, may play a role in the pathogenesis of idiopathic repetitive abortions. To investigate the involvement of these immune cell populations, we determined the immunophenotypic characteristics of endometrial leukocytes from nonpregnant recurrent aborters. Habitual aborters with a negative investigation underwent an endometrial biopsy during their secretory phase and were followed prospectively to assess clinical outcome. Endometrial leukocytes were evaluated by two-color flow cytometric analysis. The percentage of endometrial CD8+ T lymphocytes was significantly decreased in recurrent aborters, and their CD4:CD8 ratio was increased. In contrast, the proportion of B lymphocytes (CD20+) was strikingly increased in these patients' endometria. The proportion of NK cells was identical in recurrent aborters and normal controls, but the CD16-CD56 bright NK cell subset, which is predominant in normal decidua and endometrium, was significantly decreased in favor of an important contingent of CD16+CD56 dim NK cells in all habitual aborters. Repetitive aborters who had normal CD8+ and CD20+ cell numbers and a normal CD4:CD8 ratio subsequently underwent successful pregnancies, while patients with continuing abortions presented lymphoid populations observed in the habitual aborters group. In conclusion, endometrial lymphocytes of recurrent spontaneous aborters harbor a distinct immunophenotypic profile that antedates implantation and suggests that endometrial immunologic conditions are intrinsically altered in recurrent aborters. Also, the prognostic impact of CD8 and CD20 expression supports their predominant role in the development of fetal tolerance. Finally, a role for NK cells in the abortion process is suggested by their altered subsets in all repetitive aborters.

Comparison of reoperation for moderate (stage III) and severe (stage IV) endometriosis-related infertility with in vitro fertilization-embryo transfer.

OBJECTIVE: To evaluate the efficacy of reoperation for stage III or IV endometriosis-related infertility versus IVF-ET. DESIGN: Retrospective analysis. SETTING: In vitro fertilization-embryo transfer unit and tertiary infertility clinic. PATIENTS: Twenty-three couples with stage III or IV endometriosis-related infertility undergoing IVF-ET and 18 women undergoing reoperation for stage III or IV disease, both groups undergoing treatment after failed initial surgery to restore fertility. RESULTS: The cumulative pregnancy rate (CPR) after reoperation for stage III or IV endometriosis-related infertility after 3, 7, and 9 months was 5.9 percent, 18.1 percent and 24.4 percent, respectively. The cumulative PR after one and two cycles of IVF-ET with stage III or IV endometriosis was 33.3 percent and 69.6 percent, respectively. The cumulative PR after one cycle of IVF-ET was higher than with reoperation 33.3 percent versus 24.4 percent. After two cycles the cumulative PR was significantly higher than reoperation 69.6 percent versus 24.4 percent. The mean number of oocytes retrieved was 8.5 +/- 4.6, the mean number of embryos was 4.8 +/- 2.9, and the fertilization rate was 64 percent +/- 21.8 percent. The PR per cycle, per oocyte retrieval and per ET was 38 percent, 42 percent, and 44 percent, respectively, with the implantation rate being 16 percent. The live birth rate per oocyte retrieval and per ET was 29.7 percent and 34.4 percent, respectively. No statistically significant difference could be demonstrated with regard to the fertilization, implantation, nor pregnancy or live birth rates, as compared with IVF-ET outcome with tubal infertility. CONCLUSION: If initial surgery fails to restore fertility in patients with moderate (stage III) or severe (stage IV) endometriosis-related infertility, IVF-ET is an effective alternative; reoperation for asymptomatic patients offers little added benefit.

Flow-cytometric characterization of hematopoietic cells in non-pregnant human endometrium.

PROBLEM: Immunologic evaluation and quantitation of hematopoietic cells in human endometrium has been difficult to perform, particularly in nonpregnant subjects. In this study, we describe a method for the flow-cytometric characterization of hematopoietic cells present in the endometrium of non-pregnant women. METHOD: Endometrial biopsy samples from normal donors were first mechanically disrupted and filtered to generate a single-cell suspension of leukocyte-enriched endometrial cells. Cells were labeled with a panel of monoclonal antibodies, stained with propidium iodide (PI), and one- or two-color flow-cytometric analysis performed on cells excluding PI. RESULTS: The methodology described in this study was highly reproducible in experiments evaluating the interrun and intrarun variability. We then determined the immunophenotypic profile of endometrial leukocytes from 12 normal females. The majority of leukocytes were T cells (CD3: 47%; CD4: 24%; CD8: 28%) with an important contingent of NK cells (CD56: 32%), the majority of which harbored the unusual CD16-CD56 bright phenotype, and a minority of B cells (CD20: 6%) and monocytes (CD14: 7%). CONCLUSIONS: Flow cytometry can be used to assess antigen expression on the surface of endometrial leukocytes from nonpregnant women. In future studies, it will be possible to use this approach to investigate the role of immune cell populations in the endometrium of patients experiencing reproductive failure.