Using the Eeva Test™ adjunctively to traditional day 3 morphology is informative for consistent embryo assessment within a panel of embryologists with diverse experience.

PURPOSE: Since many transferred, good morphology embryos fail to implant, technologies to identify embryos with high developmental potential would be beneficial. The Eeva™ (Early Embryo Viability Assessment) Test, a prognostic test based on automated detection and analysis of time-lapse imaging information, has been shown to benefit embryo selection specificity for a panel of three highly experienced embryologists (Conaghan et al., 2013). Here we examined if adjunctive use of Eeva Test results following morphological assessment would allow embryologists with diverse clinical backgrounds to consistently improve the selection of embryos with high developmental potential. METHODS: Prospective, double-blinded multi-center study with 54 patients undergoing blastocyst transfer cycles consented to have embryos imaged using the Eeva System, which automatically measures key cell division timings and categorizes embryos into groups based on developmental potential. Five embryologists of diverse clinical practices, laboratory training, and geographical areas predicted blastocyst formation using day 3 morphology alone and day 3 morphology followed by Eeva Test results. Odds ratio (OR) and diagnostic performance measures were calculated by comparing prediction results to true blastocyst outcomes. RESULTS: When Eeva Test results were used adjunctively to traditional morphology to help predict blastocyst formation among embryos graded good or fair on day 3, the OR was 2.57 (95 % CI=1.88-3.51). The OR using morphology alone was 1.68 (95 % CI=1.29-2.19). Adjunct use of the Eeva Test reduced the variability in prediction performance across all five embryologists: the variability was reduced from a range of 1.06 (OR=1.14 to 2.20) to a range of 0.45 (OR=2.33 to 2.78). CONCLUSIONS: The Eeva Test, an automated, time-lapse enabled prognostic test, used adjunctively with morphology, is informative in helping embryologists with various levels of experience select embryos with high developmental potential.

Computer-automated time-lapse analysis results correlate with embryo implantation and clinical pregnancy: a blinded, multi-centre study.

Computer-automated time-lapse analysis has been shown to improve embryo selection by providing quantitative and objective information to supplement traditional morphology. In this multi-centre study, the relationship between such computer-derived outputs (High, Medium, Low scores), embryo implantation and clinical pregnancy were examined. Data were collected from six clinics, including 205 patients whose embryos were imaged by the Eeva(TM) System. The Eeva scores were blinded and not considered during embryo selection. Embryos with High and Medium scores had significantly higher implantation rates than those with Low scores (37% and 35% versus 15%; P < 0.0001; P = 0.0004). Similar trends in implantation rates were observed in different IVF centres each using their own protocols. Further analysis revealed that patients with at least one High embryo transferred had significantly higher clinical pregnancy rates than those with only Low embryos transferred (51% versus 34%; P = 0.02), although patients' clinical characteristics across groups were comparable. These data, together with previous research and clinical studies, confirm that computer-automated Eeva scores provide valuable information, which may improve the clinical outcome of IVF procedures and ultimately facilitate the trend of single embryo selection.

Atypical embryo phenotypes identified by time-lapse microscopy: high prevalence and association with embryo development.

OBJECTIVE: To characterize atypical dynamic embryo phenotypes identified by time-lapse microscopy, evaluate their prevalence, and determine their association with embryo development. DESIGN: Retrospective multicenter cohort study. SETTING: Five IVF clinics in the United States. PATIENT(S): Sixty-seven women undergoing IVF treatment with 651 embryos. INTERVENTION(S): Embryo videos were retrospectively analyzed for atypical phenotypes. MAIN OUTCOME MEASURE(S): Identification of four groups of atypical embryo phenotypes: abnormal syngamy (AS), abnormal first cytokinesis (A1(cyt)), abnormal cleavage (AC), and chaotic cleavage (CC). Prevalence and association with embryo morphology and development potential were evaluated. RESULT(S): A high prevalence of atypical phenotypes was observed among embryos: AS 25.1% (163/649), A1(cyt) 31.0% (195/639), AC 18% (115/639) and CC 15% (96/639). A high percentage of embryos with atypical phenotype(s) had good quality on day 3 (overall grade good or fair): AS 78.6% (70/89); A1(cyt) 79.7% (94/119), AC 86.4% (70/81), and CC 35.2% (19/54), but the blastocyst formation rates for these embryos were significantly lower compared with their respective control groups: AS 21.5% vs. 44.9%, A1(cyt) 21.7% vs. 44.6%, AC 11.7% vs. 43.1%, and CC 14.0% vs. 42.3%. CONCLUSION(S): Embryos exhibiting atypical phenotypes are highly prevalent in human embryos and show significantly lower developmental potential than control embryos. CLINICAL TRIAL REGISTRATION NUMBER: NCT01369446.

Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial.

OBJECTIVE: To assess the first computer-automated platform for time-lapse image analysis and blastocyst prediction and to determine how the screening information may assist embryologists in day 3 (D3) embryo selection. DESIGN: Prospective, multicenter, cohort study. SETTING: Five IVF clinics in the United States. PATIENT(S): One hundred sixty women ≥ 18 years of age undergoing fresh IVF treatment with basal antral follicle count ≥ 8, basal FSH <10 IU/mL, and ≥ 8 normally fertilized oocytes. INTERVENTION(S): A noninvasive test combining time-lapse image analysis with the cell-tracking software, Eeva (Early Embryo Viability Assessment), was used to measure early embryo development and generate usable blastocyst predictions by D3. MAIN OUTCOME MEASURE(S): Improvement in the ability of experienced embryologists to select which embryos are likely to develop to usable blastocysts using D3 morphology alone, compared with morphology plus Eeva. RESULT(S): Experienced embryologists using Eeva in combination with D3 morphology significantly improved their ability to identify embryos that would reach the usable blastocyst stage (specificity for each of three embryologists using morphology vs. morphology plus Eeva: 59.7% vs. 86.3%, 41.9% vs. 84.0%, 79.5% vs. 86.6%). Adjunctive use of morphology plus Eeva improved embryo selection by enabling embryologists to better discriminate which embryos would be unlikely to develop to blastocyst and was particularly beneficial for improving selection among good-morphology embryos. Adjunctive use of morphology plus Eeva also reduced interindividual variability in embryo selection. CONCLUSION(S): Previous studies have shown improved implantation rates for blastocyst transfer compared with cleavage-stage transfer. Addition of Eeva to the current embryo grading process may improve the success rates of cleavage-stage ETs.

Biomarkers identified with time-lapse imaging: discovery, validation, and practical application.

"Time-lapse markers," which are defined by time-lapse imaging and correlated with clinical outcomes, may provide embryologists with new opportunities for improving embryo selection. This article provides an overview of noninvasive biomarkers defined by time-lapse imaging studies. In addition to comprehensively reviewing the discovery of each time-lapse marker, it focuses on the criteria necessary for their successful integration into clinical practice, including [1] statistical and biological significance, [2] validation through prospective clinical studies, and [3] development of reliable technology to measure and quantify the time-lapse marker. Because manual analysis of time-lapse images is labor intensive and limits the practical use of the image data in the clinic, automated image analysis software platforms may contribute substantially to improvements in embryo selection accuracy. Ultimately, time-lapse markers that are based on a foundation of basic research, validated through prospective clinical studies, and enabled by a reliable quantification technology may improve IVF success rates, encourage broader adoption of single-embryo transfer, and reduce the risks associated with multiple gestation pregnancies.

(1)H NMR based profiling of spent culture media cannot predict success of implantation for day 3 human embryos.

BACKGROUND: Identification of a non-invasive technique to assess embryo implantation potential in assisted reproduction would greatly increase success rates and lead more efficiently to single embryo transfer. Early studies suggested metabonomic analysis of spent culture media could improve embryo selection. The goal of this study is to assess if embryo implantation can be predicted based on proton nuclear magnetic resonance ((1)H NMR) profiles of spent embryo culture media from patients undergoing transfer of multiple embryos on cycle day 3. METHOD: We conducted a retrospective study in an academic assisted reproduction technology (ART) program and analyzed the data in a university research center. Two hundred twenty-eight spent culture media samples originating from 108 patients were individually analyzed. Specifically, five distinct sets (1 to 5) of different types of spent media samples (volume ~14 µL) from embryos that resulted in clinical pregnancy (positive heart rate at 6 weeks gestation) (n (1) = 29; n (2) = 19; n (3) = 9; n (4) = 12; n (5) = 33; n (total) = 102) and from embryos that did not implant (n (1) = 28; n (2) = 29; n (3) = 18; n (4) = 15; n (5) = 36; n (total) = 126) were collected on day 3 of embryo growth. The media samples were profiled using (1)H NMR spectroscopy, and the NMR profiles of sets 1 to 5 were subject to standard uni- and multi-variate data analyses in order to evaluate potential correlation of profiles with implantation success. RESULTS: For set 1 of the media samples, a borderline class separation of NMR profiles was obtained by use of principal component analysis (PCA) and logistic regression. This tentative class separation could not be repeated and validated in any of the other media sets 2 to 5. CONCLUSIONS: Despite the rigorous technical approach, (1)H NMR based profiling of spent culture media cannot predict success of implantation for day 3 human embryos.

Comparative transcriptome analysis of human trophectoderm and embryonic stem cell-derived trophoblasts reveal key participants in early implantation.

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC(troph)). The transcriptomes of hESC(troph) at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC(troph) are several molecules known to be involved in the implantation process, as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, and follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC(troph) is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

Follicle-stimulating hormone administered at the time of human chorionic gonadotropin trigger improves oocyte developmental competence in in vitro fertilization cycles: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To determine whether an additional follicle-stimulating hormone (FSH) bolus administered at the time of the human chorionic gonadotropin (hCG) trigger can improve the developmental competence of the oocyte. DESIGN: Randomized, double-blind, placebo-controlled, clinical trial. SETTING: Academic medical center. PATIENT(S): Women undergoing a long agonist suppression in vitro fertilization (IVF) protocol for treatment of infertility. INTERVENTION(S): FSH bolus at time of hCG trigger versus placebo. MAIN OUTCOME MEASURE(S): Primary outcome; fertilization; secondary outcomes: oocyte recovery, implantation rate, and clinical and ongoing pregnancy/live birth rates. RESULT(S): A total of 188 women (mean age: 36.2 years; range: 25 to 40 years) were randomized. Fertilization (2PN/#oocyte) was statistically significantly improved in the treatment arm (63% vs. 55%) as was the likelihood of oocyte recovery (70% vs. 57%). There was no statistically significant difference in clinical pregnancy rate (56.8% vs. 46.2%) or ongoing/live birth rate (51.6% vs. 43.0%). CONCLUSION(S): Improvements in IVF success rates have largely been due to optimization of embryo culture and stimulation protocols; less attention has been directed toward methods to improve induction of final oocyte maturation. This was the first randomized, double-blind, placebo-controlled trial to modify the ovulation trigger to improve oocyte competence, as demonstrated by the statistically significant improvement in fertilization.

Follicular fluid steroid hormone levels are associated with fertilization outcome after intracytoplasmic sperm injection.

OBJECTIVE: To investigate the association between hormone levels from individual follicles and fertilization outcome among patients undergoing intracytoplasmic sperm injection (ICSI). Differences in concentrations of selected sex steroids and pituitary hormones in individual follicular aspirates between oocytes that fertilize successfully, those that fail to fertilize, and those that degenerate with ICSI were examined. DESIGN: Prospective cohort study. SETTING: Academic medical center. PATIENT(S): Women undergoing ovarian stimulation and ICSI. INTERVENTION(S): Follicular fluid was sampled by transvaginal ultrasound-guided aspiration of the hyperstimulated ovary. Each follicle was individually aspirated and collected. Intracytoplasmic sperm injection and subsequent embryo culture were performed using standard laboratory technique. Follicular fluid gonadotropin and steroid hormone levels were measured by immunoassay. MAIN OUTCOME MEASURE(S): Oocyte fertilization outcome with ICSI. RESULT(S): Oocytes that fertilized normally came from follicles with higher estradiol (adjusted odds ratio [AOR]=1.28) and testosterone (AOR=1.35) concentrations compared with those that degenerated with ICSI. Oocytes that fertilized normally also came from follicles with higher estradiol (AOR=1.14) and progesterone (AOR=1.09) concentrations compared with those that failed to fertilize. CONCLUSION(S): The hormonal profile of the follicular fluid yielding a degenerative egg or an egg that fails to fertilize is different from that resulting in normal fertilization. Higher follicular fluid estradiol may be a marker for oocytes that will fertilize normally with ICSI.

Fertilization rate is an independent predictor of implantation rate.

OBJECTIVE: To determine whether fertilization rate serves as a biological assay, reflects oocyte quality, and may be used to help predict patient implantation rate. DESIGN: Retrospective cohort study. SETTING: Academic center. PATIENT(S): Couples undergoing 3603 in vitro fertilization (IVF) cycles from 2001 to 2007. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We compared the implantation rate among cycles with high versus low fertilization rate. Univariate analyses were performed to determine the association of implantation rate with potential confounding variables: age, day-3 follicle-stimulating hormone level, day-3 estradiol level, antral follicle count, oocyte number, cycle attempts, embryo grading, and number of embryos transferred. Multivariate analysis was then performed to determine whether the fertilization rate remained an independent predictor. RESULT(S): Cutoffs for fertilization rate were 50% for intracytoplasmic sperm injection (ICSI) and 75% for conventional insemination. Higher ICSI fertilization was statistically significantly associated with the implantation rate (25.2% vs. 17.8 %). After adjusting for variables associated with implantation rate, fertilization rate for ICSI remained a strong independent predictor of implantation. Higher conventional insemination fertilization was statistically significantly associated with implantation (32.1% vs. 25.7%) and remained a statistically significant predictor after adjustment. CONCLUSION(S): Fertilization is a strong, independent predictor of implantation rate and may be useful in modeling to guide decision making for the number of embryos to transfer.

The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery.

BACKGROUND: Ovarian stimulation for assisted reproductive technology (ART) overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. METHODS: This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. RESULTS: Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR) = 1.21, 95%CI 1.03-1.42). The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93-1.07). Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96-0.99). After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45-2.34). CONCLUSION: The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

A quantitative assessment of follicle size on oocyte developmental competence.

OBJECTIVE: To quantitatively assess the impact of follicle size on oocyte maturation, fertilization, and embryo quality. DESIGN: Prospective study. SETTING: Academic medical center. PATIENT(S): Couples undergoing ovarian stimulation and in vitro fertilization (IVF). INTERVENTION(S): A total of 235 cycles were monitored prospectively, and 2934 oocytes were collected from five groups of follicle size. Repeated measures multivariate analyses were used to compare the smaller follicle sizes with the lead follicle. MAIN OUTCOME MEASURE(S): Oocyte maturation, fertilization, and embryo quality. RESULT(S): Compared with the lead follicular group (>18 mm), the odds of a mature oocyte from a 16 to 18 mm size follicle were 37% and declined progressively with each size. The odds of fertilization of oocytes from follicles 16 to 18 mm in size was 28% less than the lead group and decreased with each size. The rate of polyspermy with conventional insemination was increased for the smaller follicular groups (adjusted odds ratio = 2.37). Follicle size did not predict embryo cell number, but embryos from smaller follicles had a statistically significantly higher fragmentation compared with the lead group. CONCLUSION(S): The lead follicular group was most likely to have a mature oocyte that was capable of fertilization and best suited for development into a high-quality embryo. The smaller follicles were capable of producing metaphase II oocytes that could fertilize, but at rates approaching only 60% that of the lead follicular group.

Methylenetetrahydrofolate reductase (MTHFR) is associated with ovarian follicular activity.

OBJECTIVE: Polymorphisms in the MTHFR gene have been associated with decreased cell division and apoptosis. This finding led us to evaluate whether MTHFR polymorphisms were associated with follicular growth within the ovary. More specifically, we investigated the effect of the two common polymorphisms C677T and A1298C in our population of women undergoing ovarian stimulation. DESIGN: Prospective cohort study. SETTING: Academic medical center. PATIENT(S): Two hundred twenty-three women undergoing ovarian stimulation. INTERVENTION(S): The DNA from patients was genotyped at the MTHFR C677T and A1298C polymorphisms. MAIN OUTCOME MEASURE(S): Day 3 FSH, E(2), antral follicle count, amount of gonadotropin used, the number of follicles >13 mm, E(2) on the day of hCG administration, and oocyte number. RESULT(S): Women with the variant MTHFR 1298 C allele had significantly higher basal FSH levels, and after ovarian stimulation, produced fewer follicles >13 mm, had lower E(2) levels on the day of hCG administration, and required more ampules of gonadotropin hormone during treatment. Women with the variant MTHFR 677 T allele demonstrated no significant differences. CONCLUSION(S): The MTHFR A1298C polymorphism, but not the C677T polymorphism, is associated with higher basal FSH levels and may be a determinant of response to ovarian stimulation. These findings make a compelling case for the MTHFR A1298C polymorphism to modulate folliculogenesis.

Asian ethnicity is associated with reduced pregnancy outcomes after assisted reproductive technology.

OBJECTIVE: To determine whether success rates were similar in Asian and Caucasian women undergoing infertility treatment. DESIGN: Secondary data analysis and multivariate modeling. SETTING: Clinics reporting to the national Society for Assisted Reproductive Technology registry and a university-based clinic. PATIENT(S): Caucasian and self-identified Asian infertile women undergoing IVF. The study included 25,843 Caucasian and 1,429 Asian patients from the national registry; 370 Caucasian and 197 Asian patients were included from the site-specific clinic. INTERVENTION(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): Pregnancy rate and live-birth rate. RESULT(S): Infertile Asian women differed only minimally from their Caucasian counterparts in baseline characteristics and treatment response. Yet Asian women had a decreased clinical pregnancy rate (odds ratio, 0.71; 95% confidence interval 0.64-0.80) and a decreased live-birth rate (odds ratio, 0.69; 95% confidence interval 0.61-0.77). Subsequent multivariate analysis demonstrated that Asian ethnicity was an independent predictor of poor outcome. CONCLUSION(S): After treatment, infertile Asian women have significantly fewer pregnancies than do Caucasian women. Multivariate analysis indicates that this discrepancy cannot be accounted for by differences in baseline characteristics or by response to current therapeutic interventions.

Oocyte degeneration after intracytoplasmic sperm injection: a multivariate analysis to assess its importance as a laboratory or clinical marker.

OBJECTIVE: Oocyte degeneration has historically been associated with the intracytoplasmic (ICSI) technique. We sought to determine whether oocyte degeneration rates were associated with the technician performing the procedure, the baseline characteristics of the patient, and/or ovarian stimulation variables. We also evaluated whether the degeneration rate could serve as a surrogate marker for implantation potential. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Couples undergoing ICSI. INTERVENTION(S): Six thousand six hundred fifty-three injected oocytes were analyzed to determine whether the degeneration rate was technician dependent. Two hundred thirty first-entry down-regulated cycles were examined to identify predictors associated with oocyte degeneration. Multivariate analyses were performed using generalized linear model routines. MAIN OUTCOME MEASURE(S): Oocyte degeneration rates and implantation rates. RESULT(S): Neither the ICSI technician nor the stripping technician was associated with the oocyte degeneration rate. However, the day 3 FSH, number of mature oocytes retrieved, and E2 levels on the day of hCG were significant independent predictors of degeneration rate. Physician-adjustable ovarian stimulation variables were not associated with the degeneration rate. The degeneration rate did not appear to be associated with the implantation rate. CONCLUSION(S): These data suggest that oocyte degeneration is not technician or physician dependent. Degeneration is likely a function of the inherent oocyte quality in women who underwent ovarian stimulation. However, the remaining cohort of retrieved oocytes appears to be unaffected by virtue of an uncompromised implantation rate.

Day 2 transfer improves pregnancy outcome in in vitro fertilization cycles with few available embryos.

OBJECTIVE: Delaying ET to day 3 to optimize embryo selection is well accepted. However, in cases where there are not enough embryos to perform selection, it is not clear whether there is a difference in clinical outcomes with the day of ET. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Two hundred forty-two fresh IVF/intracytoplasmic sperm injection (ICSI) cycles from 2002-2004, where all generated embryos were transferred irrespective of quality because of an extremely low number of available embryos. INTERVENTION(S): In time period 1, ET was on day 3. In time period 2, ET was on day 2. MAIN OUTCOME MEASURE(S): Patient response to stimulation was analyzed along with pregnancy outcome and implantation rate. RESULT(S): Miscarriage rates were decreased, and ongoing pregnancy rates were increased with a day 2 ET in patients <40 years of age. CONCLUSION(S): In women <40 years of age, the day of transfer is a significant predictor of clinical outcome in cases in which a low number of embryos are available for transfer. The evidence suggests that limiting embryo culture to only 2 days reduces the incidence of miscarriage and increases ongoing pregnancy rates.

Triploidy formation after intracytoplasmic sperm injection may be a surrogate marker for implantation.

OBJECTIVE: Triploidy after intracytoplasmic sperm injection (ICSI) is due mostly to retention of the second polar body. Our interest was to determine the predictors of triploidy and to determine whether the presence of triploidy can serve as a surrogate marker of implantation for the remaining cohort of zygotes. DESIGN: Cohort study. SETTING: Academic research center. PATIENT(S): Infertile couples undergoing IVF/ICSI. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Triploid zygote (3PN) rate, implantation rate. RESULT(S): The 3PN rate is a significant predictor of implantation rate for the remaining cohort of zygotes. The starting and total dose of gonadotropins administered and the total days of stimulation are independent predictors of the 3PN rate. CONCLUSION(S): In couples with a normal semen analysis undergoing IVF/ICSI, the 3PN rate may serve as a surrogate marker of oocyte quality and may be altered by adjusting the stimulation protocol.

The difficult MESA: findings from tubuli recti sperm aspiration.

PURPOSE: To investigate sperm quality aspirated from the tubuli recti compared to that obtained from microsurgical epididymal sperm aspiration (MESA). METHODS: Sixteen patients with congenital bilateral absence of the vas deferens (CBAVD) underwent MESA. Six MESA procedures were difficult, and therefore sperm were retrieved from the tubuli recti ductules. Intraoperative sperm parameters, recovery after freeze-thaw, and ICSI outcomes were analyzed and compared between tubuli recti and MESA sperm. RESULTS: Mean initial sperm concentration was similar in both groups (18 vs. 16 million sperm/mL). Initial sperm motility was significantly higher in the tubuli recti group (35%) than the MESA group (25%). However, post thaw motility was higher with MESA compared to tubuli recti sperm (8.7 vs. 1.5%). ICSI fertilization rates after sperm freeze-thaw were 66% for tubuli recti sperm and 71% for MESA sperm. CONCLUSIONS: Tubuli recti sperm may provide an attractive alternative to testis sperm extraction. Poor sperm recovery after freeze-thaw should be expected.

The unique transcriptome through day 3 of human preimplantation development.

Successful human development is dependent upon a cascade of events following fertilization. Unfortunately, knowledge of these critical events in humans is remarkably incomplete. Although hundreds of thousands of human embryos are cultured yearly at infertility centers worldwide, the vast majority fail to develop in culture or following transfer to the uterus. In this study, we sought to characterize global patterns of gene expression in individual, normal embryos during the first three days of embryonic life using microarrays; we then compared gene expression between normally growing and growth-arrested embryos using quantitative PCR. Our results documented several novel findings. First, we found that a complex pattern of gene expression exists; most genes that are transcriptionally modulated during the first three days following fertilization are not upregulated, as was previously thought, but are downregulated. Second, we observed that the majority of genes exhibiting differential expression during preimplantation development are of unknown identity and/or function. Third, we show that embryonic transcriptional programs are clearly established by day 3 following fertilization, even in embryos that arrested prematurely with 2-, 3- or 4-cells. This indicates that failure to activate transcription is not associated with the majority of human preimplantation embryo loss. Finally, taken together, these results provide the first global analysis of the human preimplantation embryo transcriptome, and demonstrate that RNA can be amplified from single oocytes and embryos for analysis by cDNA microarray technology, thus lending credence to additional studies of genetic regulation in these cell types, as well as in other small biological samples.

Statistical analysis of factors affecting fertilization rates and clinical outcome associated with intracytoplasmic sperm injection.

OBJECTIVE: To identify and evaluate the statistically significant predictors of intracytoplasmic sperm injection (ICSI) fertilization rates and clinical pregnancy in a single population using appropriate statistical techniques. DESIGN: Retrospective study. SETTING: Fertility and Endocrinology Center, University of Washington Medical Center, Seattle, Washington. PATIENT(S): Four hundred forty-one patients undergoing their first attempt at IVF-ICSI from January 1, 1999, to May 21, 2001. INTERVENTION(S): Each ICSI procedure for an individual patient was performed by a single operator. Sperm parameters, oocyte age, culture condition, ICSI technique, and ICSI operator were assessed as variables influencing the fertilization rate. We also assessed the impact of patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred on the probability of achieving a clinical pregnancy. MAIN OUTCOME MEASURE(S): Fertilization rate and clinical pregnancy. RESULT(S): The 2 pronuclei (2PN) rate was significantly correlated with sperm motility, and there were significant differences in the 2PN rates among the ICSI operators. There was no difference in the 2PN rate among different sperm types or among the eight laboratory incubators or whether the eggs were cultured individually or in groups. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy. CONCLUSION(S): In our program, sperm motility and ICSI operator are the two most important predictors for the ICSI fertilization rate in vitro. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy.