Accu-Beads as a quality control measure for manual and automated methods of measuring sperm concentration-an observational study.

PURPOSE: To determine the precision and accuracy of Accu-Beads and their utility as a quality control product for manual and automated measurements of sperm concentration. METHODS: This observational study was performed at an Assisted Reproductive Technology laboratory in a tertiary-care, university hospital. To simulate sperm concentration, bead concentrations were measured with the use of a manual and an automated method. RESULTS: The manual counts did not vary significantly from the automated counts regardless of the concentration. However, the counts did vary between lots of low concentration of Accu-Beads and between the two different types of fixed counting chambers. The two bead concentrations used in this study were below the 95 % confidence interval for the values listed by the manufacturer. CONCLUSION(S): In our laboratory, Accu-Beads met enough of the requirements of a good control material to be acceptable for daily quality control use, especially if we set our own ranges of acceptability for each vial of Accu-Beads. It is necessary to evaluate each new lot of Accu-Beads when they are received and again if they are used with a different counting chamber.

Temperature variations within and between incubators-a prospective, observational study.

PURPOSE: To determine if there is a temperature variation within and between incubators. METHODS: This prospective, experimental trial with external controls was performed at an Assisted Reproductive Technology laboratory in a tertiary-care, university hospital. Temperature values were taken at various locations within and between incubators. RESULTS: Even though they were both set to 37.0 °C, the same make and model incubators had significantly different internal temperatures. Temperatures differed significantly among top, middle and bottom shelves and between fronts and backs of shelves. CONCLUSION(S): We found temperatures differed within and between our front-loading incubators. Thus, laboratory personnel should evaluate their incubators for temperature variations within and between incubators and, if temperatures differ significantly, develop a plan to deal with discrepancies.

A randomized controlled trial of early versus delayed skin staple removal following caesarean section in the obese patient.

OBJECTIVE: To compare wound complications after Caesarean section in the obese patient, following early versus delayed skin staple removal. METHODS: We conducted a single-centre, non-inferiority, randomized controlled trial. Following Caesarean section, obese women (BMI ≥ 30 kg/m(2)) with subcutaneous wound depth ≥ 2.0 cm and skin staple closure of a transverse incision were randomized to staple removal on postoperative day 3 (early) or between postoperative day 7 and postoperative day 10 (delayed). The primary outcome was superficial wound dehiscence; a rate of 8% or higher in the early group was defined as inferior. Secondary outcomes were seroma/hematoma, surgical site infection, and visual analogue pain score. The planned sample size was 250 patients per group. RESULTS: The study was halted after 295 patients were randomized because of slow enrolment and exhaustion of funding. The rate of superficial wound dehiscence was 15.2% in the early group (n = 145) versus 11.5% in the delayed group (n = 148). The point estimate for this difference (3.7 %; 95% CI -4.4 to 12.4) favours delayed removal. However, because the 95% CI includes zero and the upper CI exceeds the predefined limit for non-inferiority (8%), non-inferiority was not demonstrated. The rates of all secondary outcomes were similar in the early group and the delayed group: seroma/hematoma (6.9% vs. 4.7%; RR 1.4, 95% CI 0.6 to 3.7, P = 0.4); surgical site infection (9.7% vs. 4.8%; RR 2.0, 95% CI 0.8 to 4.9, P = 0.1); and composite (superficial wound dehiscence, seroma/hematoma, and surgical site infection) wound complication (17.2% vs. 12.8%; RR 1.3, 95% CI 0.8 to 2.3, P = 0.3). CONCLUSION: The non-inferiority of early skin staple removal was not demonstrated. Delayed removal of staples should remain the accepted standard in the obese patient following Caesarean section.

Objectif : Comparer les effets du retrait précoce des agrafes cutanées aux effets de leur retrait différé, en ce qui concerne les complications de plaie à la suite d'une césarienne chez une patiente obèse. Méthodes : Nous avons mené un essai comparatif randomisé de non-infériorité au sein d'un seul centre. À la suite d'une césarienne, les femmes obèses (IMC ≥ 30 kg/m2) qui présentaient une profondeur de plaie sous-cutanée ≥ 2,0 cm et une incision transversale fermée au moyen d'agrafes cutanées ont été affectées, au hasard, à un groupe devant se faire retirer ces agrafes trois jours à la suite de l'opération (retrait précoce) ou à un groupe devant se faire retirer ces agrafes entre le septième jour et le dixième jour suivant l'opération (retrait différé). La déhiscence superficielle de la plaie constituait le critère d'évaluation principal; un taux de 8 % ou plus au sein du groupe « retrait précoce ¼ a été défini comme étant inférieur. Les critères d'évaluation secondaires étaient le sérome / hématome, l'infection du champ opératoire et le score sur l'échelle visuelle analogue. La taille planifiée de l'échantillon était de 250 patientes par groupe. Résultats : L'étude a été suspendue à la suite de la randomisation de 295 patientes, en raison de la lenteur du processus de sollicitation des patientes et de l'épuisement des fonds. Le taux de déhiscence superficielle de la plaie était de 15,2 % au sein du groupe « retrait précoce ¼ (n = 145) vs 11,5 % au sein du groupe « retrait différé ¼ (n = 148). L'estimation ponctuelle pour cette différence (3,7 %; IC à 95 %, −4,4 - 12,4) favorise le retrait différé. Toutefois, puisque l'IC à 95 % contient zéro et que l'IC supérieur dépasse la limite prédéfinie pour ce qui est de la non-infériorité (8 %), cette dernière n'a pas été démontrée. Les taux de tous les critères d'évaluation secondaires étaient semblables dans les deux groupes : sérome / hématome (6,9 % vs 4,7 %; RR, 1,4, IC à 95 %, 0,6 - 3,7, P = 0,4); infection du champ opératoire (9,7 % vs 4,8 %; RR, 2,0, IC à 95 %, 0,8 - 4,9, P = 0,1); et complication composite de la plaie (déhiscence superficielle de la plaie, sérome / hématome, infection du champ opératoire) (17,2 % vs 12,8 %; RR, 1,3, IC à 95 %, 0,8 - 2,3, P = 0,3). Conclusion : La non-infériorité du retrait précoce des agrafes cutanées n'a pas été démontrée. Le retrait différé des agrafes devrait demeurer la norme acceptée en ce qui concerne les patientes obèses à la suite d'une césarienne.

Endometrial receptivity defects during IVF cycles with and without letrozole.

BACKGROUND: Our aim was to study ways to improve IVF success rates in women with suspected endometrial receptivity defects. METHODS: We conducted a retrospective cohort study examining the effect of letrozole (aromatase inhibitor) on integrin expression as a marker of endometrial receptivity. We compared IVF outcomes in 97 infertile women who had undergone ανß3 integrin assessment by immunohistochemistry in mid-luteal endometrial biopsies. Of 79 women undergoing standard IVF, 29 (36.7%) lacked normal integrin expression. Eighteen other women with low integrin were studied after receiving letrozole during early IVF stimulation. An independent set of ανß3 integrin-negative patients (n = 15) who had undergone repeat endometrial biopsy for integrin testing while taking letrozole were re-evaluated. RESULTS: Clinical pregnancy and delivery rates were higher in women with normal ανß3 integrin expression compared with those who were integrin negative [20/50 (40%) versus 4/29 (13.8%); P = 0.02 and 19/50 (38%) versus 2/29 (7%); P < 0.01, respectively]. In 18 women who received letrozole early in IVF, 11 conceived (61.1%; P < 0.001) compared with integrin-negative patients who did not receive letrozole. In integrin-negative women who were rebiopsied on letrozole, 66.7% reverted to normal integrin expression. Positive endometrial aromatase immunostaining using a polyclonal antibody was a common finding in infertile patients compared with controls. CONCLUSIONS: Lack of endometrial ανß3 integrin expression is associated with a poor prognosis for IVF that might be improved with letrozole co-treatment. Prospective studies are needed to confirm and extend these findings but the data suggest that aromatase expression may contribute to implantation failure in some women.

Intraoperative detection of subtle endometriosis: a novel paradigm for detection and treatment of pelvic pain associated with the loss of peritoneal integrity.

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Changes in long luteal protocol affects the number of days of stimulation: evolution of an assisted reproductive technology practice.

OBJECTIVE: To determine if the types of drugs used in controlled ovarian hyperstimulation influence the number of days of stimulation. STUDY DESIGN: This retrospective study of couples presenting at an assisted reproductive technology (ART) facility January 1997 through December 2007 included patients who were < 40 years of age, had a body mass index (BMI) of 20-35, used fresh sperm or oocytes, had > or = 3 embryos available on day 3, never had previous ART, and did not use a gestational carrier. Data analysis was limited to patients treated with long luteal stimulation. Data were separated into two groups: patients stimulated with urinary drugs and those stimulated with recombinant drugs. RESULTS: The groups were not different in age and day 3 follicle-stimulating hormone values, but were different in BMI, diagnoses, and fertilization methods. There were a significantly greater number of days of stimulation, total number of follicles produced, and peak estradiol level in the recombinant group. The recombinant group had less drug given per day, less endometrial thickness, and fewer embryos transferred. CONCLUSION: A change in our long luteal stimulation protocol appears to have lengthened the days of stimulation for our patients without altering pregnancy rate.

Incubator management in an assisted reproductive technology laboratory.

OBJECTIVE: To study the effect of incubator management on assisted reproductive technology (ART) outcomes. DESIGN: Series of retrospective and controlled, randomized studies. SETTING: Tertiary care infertility practice. PATIENT(S): Mammalian gametes/embryos. INTERVENTION(S): Evaluation of human and bovine oocytes/embryos cultured in various environmental conditions. MAIN OUTCOME MEASURE(S): Fertilization and embryo development rate as well as clinical pregnancy rate (PR). CONCLUSION(S): Here we review the general topic of incubator management as it pertains to ART. Discussed within the context of this article will be our experiences as they relate to incubator management. Details as they apply to incubator environment also will include gamete/embryo positions within incubator, air quality, and quality control.

Quantitative and qualitative analysis of microorganisms in an assisted reproductive technology facility.

OBJECTIVE: To perform a quantitative and qualitative analysis of the environmental microorganisms associated with two assisted reproductive technology (ART) laboratories. DESIGN: Prospective descriptive study. SETTING: Two ART laboratories at Greenville Hospital System (GHS) University Medical Center in Greenville, South Carolina. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative and qualitative analysis of colony-forming units (CFU; bacteria and fungi) and particles from an embryology clean room and an andrology laboratory. RESULT(S): Within these laboratories, three of the sites sampled differed significantly in the mean number of CFU per plate. These sites included external laboratory surfaces, laboratory room air, and air within cell-culture incubators. The mean number of viable CFU per cubic meter of laboratory room air differed significantly between the two laboratories. A total of 23 unique microbial species were isolated from the andrology laboratory, whereas 10 unique species were isolated from the embryology clean room. CONCLUSION(S): The data herein suggest that the increased positive control measures used in the embryology clean room are effective in minimizing environmental biologic contamination.

Effect of human granulosa cell co-culture using standard culture media on the maturation and fertilization potential of immature human oocytes.

OBJECTIVE: To determine whether culture of immature human oocytes with and without autologous cumulus cells (CC) in standard culture medium would provide additional oocytes for use in IVF procedures. DESIGN: Prospective study. SETTING: Hospital-based IVF practice. PATIENT(S): Sixty-one women undergoing 62 IVF-embryo transfer procedures between August 2004 and June 2005. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Oocyte maturation after culture with and without CC, oocyte fertilization, and zygote cleavage. RESULT(S): Culture with CC did not alter the maturation rate for metaphase I (M I) oocytes to the metaphase II (M II) stage. Germinal vesicle (GV) stage oocytes cultured with CC matured at a significantly higher rate than did GV oocytes cultured without CC. Although acceptable fertilization rates were obtained (62.1%), "usable" embryo yield was very low (13.0%). CONCLUSION(S): This study demonstrated good maturation of M I oocytes but poor maturation of GV oocytes in standard culture medium. Of the immature oocytes that did mature, were fertilized, and cleaved to embryos, only a very small number were actually suitable for patient use. Therefore, these extended culturing techniques were inefficient in maturing and providing additional oocytes/embryos for patient use. The use of specialized in vitro maturation medium may improve the utility of this process.

Assisted Reproductive Technology (ART) in the upstate: effectiveness of surgical sperm removal techniques in conjunction with ICSI to promote the success of pregnancy.

With aspiration of sperm directly from the epididymis (MESA) or extracted from the testis (TESA) and intracytoplasmic sperm injection, it is possible to provide men who are incapable of producing viable sperm in an ejaculate the ability to overcome this severe sperm defect and produce a successful pregnancy. In our practice, 10 of 26 ART cycles have had successful pregnancies with a total of 17 children being delivered. Thus, MESA/TESE techniques coupled with ICSI provide this population of patients with a viable option for producing offspring.

Comparison of pregnancy rates for two embryo-transfer catheters.

OBJECTIVE: To compare two flexible catheters and determine whether pregnancy rates (PRs) differed. DESIGN: Prospective, randomized pilot study. SETTING: Tertiary-care infertility practice. PATIENT(S): The subjects for this study were 100 women undergoing assisted reproductive technology (ART) cycles between September 2003 and October 2005. INTERVENTION(S): None. MAIN OUTCOME MEASURE: Clinical PR for Edwards-Wallace (Cooper Surgical, Shelton, CT) and Cook World (Cook Urological, Spencer, IN) embryo-transfer (ET) catheters. RESULT(S): The outcomes of pregnancy between the Edwards-Wallace and Cook World ET catheters were not significantly different. Use of the flare stimulation protocol resulted in a significantly lower PR compared to long luteal and antagonist protocols. No other variable affected PR. CONCLUSION(S): A 5% difference in PR was demonstrated between ET catheters; however, this study does not have the power to detect a significant difference in clinical PR between the two catheter groups. The pilot study demonstrates good design due to the lack of significant differences between the variables when stratifed by cather as well as pregnancy status. In a larger study, the flare protocol should be excluded.

Factors affecting assisted reproductive technology (ART) pregnancy rates: a multivariate analysis.

PURPOSE: To determine factors instrumental in achieving a clinical pregnancy in assisted reproductive technology (ART) patients. METHODS: This study included 205 women undergoing their first ART cycle. Univariate and multivariate analyses were performed for patient demographics, in vitro production data, and factors associated with embryo transfer. Odds ratios (OR) were performed where appropriate. RESULTS: Our analyses indicated that age (OR: 0.879), specific year in which the cycle was performed (OR: 2.959), and use of intracytoplasmic sperm injection (OR: 2.867) altered potential pregnancy rate. In addition, percent fertilization (OR: 1.028), number of embryos transferred (OR: 1.842), type of catheter used to transfer the embryos (OR: 0.377), presence of blood on the catheter (OR .414), and embryologist (OR: 2.338) also altered pregnancy rate. CONCLUSIONS: Our data indicate patients' age, use of the Cook catheter, and presence of blood on the catheter reduce pregnancy rates. Performing ART in 1999, using ICSI, increasing fertilization rate, increasing number of embryos transferred (albeit less desirable when the chance of multiple gestation may occur), and transferring embryos via a particular embryologist, improve pregnancy rates.

Defining the typical work environment for assisted reproductive technology laboratories in the United States.

OBJECTIVE: To determine time and staffing requirements as they relate to laboratory personnel in the field of assisted reproductive technology (ART). DESIGN: Descriptive study. SETTING: Assisted reproductive technology clinics in the United States. PERSONNEL: All personnel working in ART laboratories, which included laboratory directors and technicians (those laboratory personnel trained in specific areas of embryology, andrology, and endocrinology). INTERVENTION(S): None. MAIN OUTCOME MEASURES(S): Frequencies were determined for time and staffing requirements as they pertained to individuals and procedures (e.g., oocyte retrieval, embryo transfer, semen analysis, hormone assay, etc.) performed in embryology, andrology, and endocrine laboratories. RESULT(S): Two different surveys of ART clinics in the United States were combined in this report. The average technician worked 40 hours a week, whereas the average laboratory director worked 49 hours per week. The average embryologist performed 181 procedures (not cases) per year. The average andrologist performed 648 procedures (not cases) per year, while the average endocrinologist completed 2,673 procedures (not cases) per year. CONCLUSION(S): This is the first report to describe time and staffing requirements as they relate to the laboratory personnel in an ART facility.

Developing techniques for determining sperm morphology in today's andrology laboratory.

PURPOSE: To investigate three areas: the staining of spermatozoa; the Computer Assisted Semen Analysis (CASA); and the variability of technicians. METHODS: Staining experiment: sperm from 15 beef bulls were randomized to one of three staining protocols. CASA experiment: slides were evaluated using the Integrated Visual Optical System and compared to technician results. Variability of technicians: five laboratorians analyzed the same set of 20 slides six different times. RESULTS: Staining experiment: the size of the sperm increased in proportion to increased time and heat associated with each successive protocol. CASA experiment: coefficient of variation ranged from 18.3 to 101.7% (12 slides). Variability of technicians: the mean sperm morphology results ranged from 7.3 to 15% normal forms. CONCLUSIONS: Until laboratories adhere to the universal standard set by the World Health Organization to evaluate sperm morphology, a laboratory must rely on its own quality control to insure repeatable results.

Flexible versus rigid intrauterine insemination catheters: a prospective, randomized, controlled study.

OBJECTIVE: To prospectively compare pregnancy rates for couples undergoing IUI by a single healthcare provider with random assignment to either rigid (Tomcat; Kendall Sovereign, Mansfield, MA) or flexible (Soft Pass; Cook, Spencer, IN) catheters. DESIGN: Randomized, controlled study. SETTING: Tertiary care infertility practice. PATIENT(S): One hundred infertile women enrolled from a single infertility practice who met the inclusion and exclusion criteria for IUI were enrolled. Of the 100 patients, 95 underwent ovarian stimulation before IUI: 35 with clomiphene citrate alone, 60 with clomiphene citrate combined with a single dose of gonadotropins. INTERVENTION(S): Timed IUI was performed with either rigid (Tomcat) or flexible (Cook) insemination catheters. MAIN OUTCOME MEASURE(S): Pregnancy rate per cycle. RESULT(S): No differences were noted between groups for the following: age, length of infertility, day-3 FSH level, number of prior IUI cycles, total motile sperm count, days of abstinence, infertility diagnoses, insemination ratings, and stimulation protocols. No difference in pregnancy rates was observed between rigid and flexible catheter groups. CONCLUSION(S): There is no statistically significant difference between flexible and rigid catheters for IUI.

Changes in the freezing protocol for human zygotes alter embryonic development and pregnancy rates.

OBJECTIVE: To determine the effect of various freezing protocols on postthaw development and pregnancy rates resulting from transfer of human zygotes. DESIGN: Prospective study. SETTING: Tertiary care center. PATIENT(S): Couples undergoing assisted reproductive technology (ART) procedures who wished to have their excess zygotes cryopreserved. INTERVENTION(S): We cryopreserved zygotes with one of three protocols. MAIN OUTCOME MEASURE(S): Post-thaw survival and development of the zygotes as well as pregnancy rate after transfer of these zygotes. RESULT(S): A 3-minute hold time after seeding, followed by a final preplunging temperature of -180 degrees C, resulted in a clinical pregnancy rate of 28.6%. In contrast, a 15-minute postseed hold time and a -30 degrees C final chamber temperature resulted in a 37.3% clinical pregnancy rate. When we combined the protocols to provide a 15-minute postseed holding time and a -180 degrees C before plunging into liquid nitrogen, we achieved a 69.6% clinical pregnancy rate. CONCLUSION(S): By increasing the postseeding hold time and decreasing the temperature of the freezing chamber before plunging the zygotes into liquid nitrogen, significant improvements can be made in postthaw development and pregnancy rates.

Assisted reproductive technology (ART) in the upstate: reducing the risks of multiple births.

Multiple pregnancies from ART procedures are all too common. Efforts to reduce high-order multiple pregnancies will require both social and medical reforms. In lieu of a legislative agenda to reduce multiple pregnancies in the United States, efforts must come from the personnel within the individual ART clinics. The approach to the problem includes accepting a lower pregnancy rate than otherwise might be obtainable, if the observed rate of high-order multiple pregnancies is above a certain threshold. Expanding the opportunities for ART reimbursement will ultimately address this problem in the United States, but until then, it is a problem that will not go away without commitment and resolve.

Expectations for oocyte fertilization and embryo cleavage after whole sperm versus sperm head intracytoplasmic sperm injection.

OBJECTIVE: To compare oocyte fertilization and embryo development after intracytoplasmic sperm injection (ICSI) with a whole sperm vs. a sperm head. DESIGN: Retrospective study. SETTING: Hospital-based IVF practice. PATIENT(S): Fifty-three women undergoing 54 IVF-embryo transfer plus ICSI procedures between January 1999 and June 2002. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Oocyte fertilization, zygote cleavage, and embryo stage after 72 hours of culture. RESULT(S): A significantly higher fertilization rate was observed using whole sperm (72.2%) than when using sperm heads (56.4%). Zygote cleavage rates for whole sperm vs. sperm head ICSI were 96.4% and 92.7%, respectively. Embryo cell stage after 72 hours of culture for whole sperm vs. sperm head ICSI was 6.5 +/- 2.1 cells and 5.6 +/- 1.8 cells, respectively. Embryo grade at this same time point was not different (2.3 +/- 1.0 and 2.5 +/- 0.9, respectively). CONCLUSION(S): The ICSI using whole sperm produces superior fertilization rates compared to ICSI using sperm heads, but once oocytes were fertilized, zygote cleavage rates were not different between the two sperm sources. Oocytes injected with a whole sperm produced embryos of higher cell stage but equivalent quality compared to oocytes injected with sperm heads. Therefore, having only sperm heads for use in ICSI should not be a deterrent to using this procedure.

Conclusions about the effects on fertilization of time from aspiration to incubation and blood in the aspirate depend on the use of appropriate statistical techniques.

OBJECTIVE: To examine the effects of exposure to blood and time from aspiration to incubation on oocyte fertilization. DESIGN: Retrospective, cohort study. SETTING: Hospital-based IVF practice. PATIENT(S): Eighty-two women undergoing IVF procedures. INTERVENTION(S): Patients received a standard gonadotropin protocol or a microdose flare protocol. MAIN OUTCOMES MEASURE(S): Logistic regression was used to estimate odds of nonfertilization, adjusting for maternal age, time from aspiration to incubation, presence of blood in the aspirate, and a time-by-blood interaction. Two statistical methods were used: an analysis that assumed oocytes were independent and an analysis that adjusted for the lack of independence of oocytes within mothers (i.e., cluster-correlated data). RESULT(S): The 82 women contributed 1093 oocytes. In a model with blood dicotomized as present or absent, and with time elapsed and maternal age categorized into quartiles, both time and maternal age were statistically significant when conventional logistic regression was used. In analyses that accounted for clustering within mother, retrieval-to-incubation times more than 4 minutes were associated with an increased risk of nonfertilization, but age and blood were not; there was no evidence of an interaction between time elapsed and blood. CONCLUSION(S): If appropriate statistics are applied, retrieval-to-incubation times more than 4 minutes are associated with increased risk of nonfertilization.