Severity of Diminished Ovarian Reserve and Chance of Success with Assisted Reproductive Technology.

Objective: To describe the relationship between severe diminished ovarian reserve (DOR) and assisted reproductive technology outcomes. Study Design: Retrospective cohort including all United States' fertility centers reporting to the CDC National ART Surveillance System, 2004­2012. Among women aged <41 (504,266 fresh autologous IVF cycles), we calculated cancellation rate/cycle and pregnancy rate/transfer, stratified by age, by maximum follicle-stimulating hormone (FSH). Cancellation rate per cycle and pregnancy, live birth, and miscarriage rates per transfer were compared among women with and without DOR. We used multivariable log binomial regression, stratified by age, to calculate adjusted relative risk (aRR) for the association between DOR and these outcomes and, within DOR groups, between stimulation type and outcomes. Results: Cancellation rate/cycle increased with increasing FSH and with DOR severity. For women aged <35 who underwent transfer, aRR for pregnancy and live birth indicated slightly reduced likelihood of these outcomes (severe vs. no DOR); confidence intervals approached the null. Among women with severe DOR, stimulation type was not associated with likelihood of pregnancy or live birth per transfer in any group except women ages 38­40. Conclusion: Women with severe DOR are at significantly increased risk of cancellation; however, those who undergo transfer have pregnancy and live birth chances similar to those of women without DOR after controlling for cycle characteristics.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Trends and outcomes for donor oocyte cycles in the United States, 2000-2010.

IMPORTANCE: The prevalence of oocyte donation for in vitro fertilization (IVF) has increased in the United States, but little information is available regarding maternal or infant outcomes to improve counseling and clinical decision making. OBJECTIVES: To quantify trends in donor oocyte cycles in the United States and to determine predictors of a good perinatal outcome among IVF cycles using fresh (noncryopreserved) embryos derived from donor oocytes. DESIGN, SETTING, AND PARTICIPANTS: Analysis of data from the Centers for Disease Control and Prevention's National ART Surveillance System, to which fertility centers are mandated to report and which includes data on more than 95% of all IVF cycles performed in the United States. Data from 2000 to 2010 described trends. Data from 2010 determined predictors. MAIN OUTCOMES AND MEASURES: Good perinatal outcome, defined as a singleton live-born infant delivered at 37 weeks or later and weighing 2500 g or more. RESULTS: From 2000 to 2010, data from 443 clinics (93% of all US fertility centers) were included. The annual number of donor oocyte cycles significantly increased, from 10,801 to 18,306. Among all donor oocyte cycles, an increasing trend was observed from 2000 to 2010 in the proportion of cycles using frozen (vs fresh) embryos (26.7% [95% CI, 25.8%-27.5%] to 40.3% [95% CI, 39.6%-41.1%]) and elective single-embryo transfers (vs transfer of multiple embryos) (0.8% [95% CI, 0.7%-1.0%] to 14.5% [95% CI, 14.0%-15.1%]). Good perinatal outcomes increased from 18.5% (95% CI, 17.7%-19.3%) to 24.4% (95% CI, 23.8%-25.1%) (P < .001 for all listed trends). Mean donor and recipient ages remained stable at 28 (SD, 2.8) years and 41 (SD, 5.3) years, respectively. In 2010, 396 clinics contributed data. For donor oocyte cycles using fresh embryos (n = 9865), 27.5% (95% CI, 26.6%-28.4%) resulted in good perinatal outcome. Transfer of an embryo at day 5 (adjusted odds ratio [OR], 1.17 [95% CI, 1.04-1.32]) and elective single-embryo transfers (adjusted OR, 2.32 [95% CI, 1.92-2.80]) were positively associated with good perinatal outcome; tubal (adjusted OR, 0.72 [95% CI, 0.60-0.86]) or uterine (adjusted OR, 0.74 [95% CI, 0.58-0.94]) factor infertility and non-Hispanic black recipient race/ethnicity (adjusted OR, 0.48 [95% CI, 0.35-0.67]) were associated with decreased odds of good outcome. Recipient age was not associated with likelihood of good perinatal outcome. CONCLUSIONS AND RELEVANCE: In the United States from 2000 to 2010, there was an increase in number of donor oocyte cycles, accompanied by an increase in good outcomes. Further studies are needed to understand the mechanisms underlying the factors associated with less successful outcomes.

Discontinuation of rLH two days before hCG may increase the number of oocytes retrieved in IVF.

BACKGROUND: Administration of recombinant luteinizing hormone (rLH) in controlled ovarian hyperstimulation may benefit a subpopulation of patients. However, late follicular phase administration of high doses of rLH may also reduce the size of the follicular cohort and promote monofollicular development. METHODS: To determine if rLH in late follicular development had a negative impact on follicular growth and oocyte yield, IVF patients in our practice who received rFSH and rLH for the entire stimulation were retrospectively compared with those that had the rLH discontinued at least two days prior to hCG trigger. RESULTS: The two groups had similar baseline characteristics before stimulation with respect to age, FSH level and antral follicle count. However, the group which had the rLH discontinued at least two days prior to their hCG shot, had a significantly higher number of oocytes retrieved, including a higher number of MII oocytes and number of 2PN embryos. CONCLUSIONS: When using rLH for controlled ovarian hyperstimulation, administering it from the start of stimulation and stopping it in the late follicular phase, at least two days prior to hCG trigger, may increase oocyte and embryo yield.

Anti-Müllerian Hormone in Peritoneal Fluid and Plasma From Women With and Without Endometriosis.

Anti-Müllerian hormone (AMH) has potential local effects on ovarian function and endometrial tissue, including endometriosis, but its presence in peritoneal fluid is not fully understood. This is a cross-sectional study evaluating AMH in peritoneal fluid and plasma from women with endometriosis (N = 61) and from control women without endometriosis (N = 36). There was a significant correlation between AMH in plasma and peritoneal fluid from both patients with endometriosis (r(2) = .767 [P < .001]) and control participants (r(2) = .647 [P < .001]) less than 45 years of age. Anti-Müllerian hormone declined with women's increasing age in both plasma and peritoneal fluid in women with and without endometriosis. There were no differences in the plasma or peritoneal fluid AMH in women with endometriosis versus control women. The strong relationship between plasma and peritoneal fluid may allow plasma AMH to be a marker for peritoneal AMH in studies evaluating the local effects of AMH.

Endometriosis and assisted reproductive technology: United States trends and outcomes 2000-2011.

OBJECTIVE: To assess endometriosis-associated infertility trends among assisted reproductive technology (ART) cycles, and to compare cancellation and hyperstimulation risks and pregnancy and live birth rates among women using ART for endometriosis-associated vs. male factor infertility. DESIGN: Descriptive and multivariable analyses of Centers for Disease Control and Prevention (CDC) National ART Surveillance System data. SETTING: Fertility centers. PATIENT(S): All reported fresh autologous ART cycles in the United States between 2000 and 2011 (n = 1,589,079). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Oocyte yield, hyperstimulation, cancellation, implantation, pregnancy, live birth. RESULT(S): The absolute number of ART cycles with an endometriosis diagnosis fell in recent years, from 16,751 (2000) to 15,311 (2011); the percentage fell over time, from 17.0% (2000) to 9.6% (2011) of all cycles. Compared with male factor (n = 375,557), endometriosis-associated cycles (n = 112,475) yielded fewer oocytes (50.5% vs. 42.5% of cycles with only 0-10 oocytes retrieved), lower risk of hyperstimulation (1.1% vs. 1.3%, adjusted risk ratio [aRR] 0.82, 95% confidence interval [CI] 0.74-0.91), and an increased risk of cancellation (12.9% vs. 10.1%, aRR 1.30, 95% CI 1.25-1.35). Endometriosis was associated with a statistically decreased but likely clinically insignificant difference in the following outcomes: chance of pregnancy per transfer (43.7% vs. 44.8%, aRR 0.96, 95% CI 0.95-0.98) among couples who did not also have tubal factor infertility and live birth per transfer (37.2% vs. 37.6%, aRR 0.96, 95% CI 0.94-0.98). CONCLUSION(S): The percentage of endometriosis-associated ART cycles has decreased over time. As compared with male factor infertility, endometriosis is associated with increased cancellation and decreased hyperstimulation risks. Despite decreased oocyte yield and higher medication dose, the difference in pregnancy and live birth rates may be of limited clinical significance, suggesting comparable pregnancy outcomes per transfer.

Insulin and messenger ribonucleic acid expression of insulin receptor isoforms in ovarian follicles from nonhirsute ovulatory women and polycystic ovary syndrome patients.

Insulin action is mediated by two insulin receptor (IR) isoforms, differing in mitogenic and metabolic function. IR isoform expression might occur in human granulosa cells and could be altered in polycystic ovary syndrome (PCOS) from hyperinsulinemia. To determine the relationship between granulosa cell IR isoform expression and follicular fluid insulin concentration in individual follicles, 18 normal women and seven PCOS patients receiving gonadotropins for in vitro fertilization were studied. Glucose tolerance testing was performed before pituitary desensitization, and fasting serum insulin was measured at oocyte retrieval. Granulosa cells and fluid aspirated from the first follicle were used to determine IR isoform mRNA expression and insulin concentration, respectively. IR isoform A mRNA expression was greater than that of IR isoform B expression in normal mural granulosa and cumulus cells, without a cell type effect. Intrafollicular insulin levels increased with adiposity and serum insulin levels at oocyte-retrieval but did not predict IR mRNA expression. Total IR mRNA expression, but not intrafollicular insulin levels, was elevated in PCOS patients, whereas intrafollicular insulin levels were increased in women with impaired glucose tolerance. Granulosa cell IR heterogeneity, together with adiposity-dependent intrafollicular insulin availability, introduces a novel mechanism by which insulin may affect granulosa cell function within the follicle.

Cumulative first live birth after elective cryopreservation of all embryos due to ovarian hyperresponsiveness.

OBJECTIVE: To estimate cumulative chance for first live birth after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. DESIGN: Retrospective analysis with longitudinal follow-up. SETTING: Academic hospital. PATIENT(S): Thirty subjects with elective cryopreservation of all embryos due to ovarian hyperresponsiveness. INTERVENTION(S): Elective cryopreservation of all embryos at the pronuclear stage (n = 30) and subsequent cryopreserved-thawed ET (n = 51). MAIN OUTCOME MEASURE(S): Cumulative chance for first live birth. RESULT(S): Cumulative chance for first live birth was 77% when analyzed by intention to treat and 82% by treatment with ET. Nearly 40% of live births were multiple. CONCLUSION(S): Cumulative first live birth increased with repetitive ET after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. Multiple births, however, were frequent. In the context of initial ET attempts in young women, transfer of no more than two cryopreserved-thawed embryos is advised.

Ultrasound-guided unification of noncommunicating uterine cavities.

BACKGROUND: The benefits of ultrasound-guided hysteroscopic metroplasty have been well described in the management of the partial septate uterus. The use of ultrasonography at the time of hysteroscopy provides visualization of the intrauterine instruments within the uterine cavity. This report describes a case where ultrasound-guidance was used to enter a noncommunicating uterine cavity hysteroscopically. METHODS: A 22-year-old female after her third miscarriage experienced worsening dysmenorrhea. The patient underwent a laparoscopy and hysteroscopy to further evaluate the cause of pelvic pain and to treat the Müllerian anomaly. Under ultrasound-guidance, the 2 uterine cavities were unified by hysteroscopic metroplasty. RESULTS: Postoperative sonohysterography demonstrated unification of the 2 cavities. CONCLUSIONS: This unique application of ultrasound-guidance in hysteroscopic surgery may aid the surgeon in entering a noncommunicating uterine cavity.

Assisted hatching: trends and pregnancy outcomes, United States, 2000-2010.

OBJECTIVE: To assess trends and outcomes of assisted hatching among assisted reproductive technology (ART) cycles. DESIGN: Retrospective cohort analysis using National ART Surveillance System (NASS) data. SETTING: U.S. fertility centers reporting to NASS. PATIENT(S): Fresh autologous noncanceled ART cycles conducted from 2000-2010. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation, clinical pregnancy, live-birth, miscarriage, multiple gestation. RESULT(S): Assisted hatching use statistically significantly increased in absolute number (from 25,724 to 35,518 cycles), percentages of day-3 (from 50.7% to 56.3%) and day-5 transfers (from 15.9% to 22.8%), and percentage of transfers among women ≥38 years (from 17.8% to 21.8%) or women with ≥2 prior ART cycles and no live birth(s) (from 4.3% to 7.4%). Both day-3 and day-5 cycles involving assisted hatching were associated with lower odds of implantation (adjusted odds ratios [aOR] 0.7 and 0.6, respectively), clinical pregnancy (aOR 0.8 and 0.7, respectively), live birth (aOR 0.8 and 0.7, respectively), and increased odds of miscarriage (aOR 1.4 and 1.4, respectively), as compared with cycles without assisted hatching. Assisted hatching was associated with lower odds of multiple gestation in day-5 cycles (aOR 0.8). In cycles for women with a "poor prognosis," the association of assisted hatching with pregnancy outcomes was not statistically significant. CONCLUSION(S): Assisted hatching use had an increasing trend but was not associated with improved pregnancy outcomes, even in poor-prognosis patients. Prospective studies are needed to identify the patients who may benefit from assisted hatching.

Tubal factor infertility and perinatal risk after assisted reproductive technology.

OBJECTIVE: To assess trends of tubal factor infertility and to evaluate risk of miscarriage and delivery of preterm or low birth weight (LBW) neonates among women with tubal factor infertility using assisted reproductive technology (ART). METHODS: We assessed trends of tubal factor infertility among all fresh and frozen, donor, and nondonor ART cycles performed annually in the United States between 2000 and 2010 (N=1,418,774) using the National ART Surveillance System. The data set was then limited to fresh, nondonor in vitro fertilization cycles resulting in pregnancy to compare perinatal outcomes for cycles associated with tubal compared with male factor infertility. We performed bivariate and multivariable analyses controlling for maternal characteristics and calculated adjusted risk ratios (RRs) and 95% confidence intervals (CI). RESULTS: The percentage of ART cycles associated with tubal factor infertility diagnoses decreased from 2000 to 2010 (26.02-14.81%). Compared with male factor infertility, tubal factor portended an increased risk of miscarriage (14.0% compared with 12.7%, adjusted RR 1.08, 95% CI 1.04-1.12); risk was increased for both early and late miscarriage. Singleton neonates born to women with tubal factor infertility had an increased risk of preterm birth (15.8% compared with 11.6%, adjusted RR 1.27, 95% CI 1.20-1.34) and LBW (10.9% compared with 8.5%, adjusted RR 1.28, 95% CI 1.20-1.36). Significant increases in risk persisted for early and late preterm delivery and very low and moderately LBW delivery. A significantly elevated risk was also detected for twin, but not triplet, pregnancies. CONCLUSION: Tubal factor infertility, which is decreasing in prevalence in the United States, is associated with an increased risk of miscarriage, preterm birth, and LBW delivery as compared with couples with male factor infertility using ART.

Analysis of follicular fluid retinoids in women undergoing in vitro fertilization: retinoic acid influences embryo quality and is reduced in women with endometriosis.

Retinol (ROL) and its biologically active metabolite, all-trans retinoic acid (ATRA), are essential for a number of reproductive processes. However, there is a paucity of information regarding their roles in ovarian folliculogenesis, oocyte maturation, and early embryogenesis. The objectives of this study were to quantify and compare peripheral plasma (PP) and follicular fluid (FF) retinoid levels, including ATRA in women undergoing in vitro fertilization (IVF) and to investigate the relationship between retinoid levels and embryo quality. Retinoid levels were evaluated in PP and FF from 79 women undergoing IVF at the time of oocyte retrieval and corresponding embryo quality assessed on a daily basis after retrieval for 3 days until uterine transfer. Analysis compared the retinoid levels with day 3 embryo grades and between endometriosis versus control patients. Results demonstrated distinctive levels of retinoid metabolites and isomers in FF versus PP. There was a significantly larger percentage of high-quality grade I embryos derived from the largest versus smallest follicles. An increase in follicle size also correlated with a >50% increase in FF ROL and ATRA concentrations. Independent of follicle size, FF yielding grade I versus nongrade I embryos showed higher mean levels of ATRA but not ROL. In a nested case-control analysis, control participants had 50% higher mean levels of ATRA in their FF and PP than women with endometriosis. These findings strongly support the proposition that ATRA plays a fundamental role in oocyte development and quality, and that reduced ATRA synthesis may contribute to decreased fecundity of participants with endometriosis.

Follicular recruitment is reduced in minimal stimulation for controlled ovarian hyperstimulation using purified human menopausal gonadotropin compared with follicle-stimulating hormone.

Ovulation induction using a minimal stimulation protocol with clomiphene citrate and highly purified hMG was associated with a 20% reduction in the number of follicles recruited compared with clomiphene citrate and recombinant FSH. This reduction may reduce the risk of multiple gestation without reducing the pregnancy rate.

Current status of the approach to assisted reproduction.

Assisted reproductive technologies are important tools in the clinical armamentarium used to treat both female and male infertility disorders. Pre-implantation genetic diagnosis offers couples at risk of having children with inheritable disorders the ability to analyze the genetic make-up of embryos before transfer. For patients undergoing treatment of cancer with chemotherapy or radiation therapy, these technologies offer the potential for the preservation of future fertility. As technology evolves, it is likely the clinical applications of assisted reproduction will continue to develop and expand in the future to enhance fertility.

Ovulation induction in a poor responder with panhypopituitarism: a case report and review of the literature.

BACKGROUND: Most women with panhypopituitarism will undergo successful ovulation induction with gonadotropin therapy. Few proven treatment options exist for those who respond poorly to such therapy. A poor response may indicate diminished ovarian reserve, or reflect a deficiency of other key components for ovarian function. CASE: A 31-year-old female with panhypopituitarism and a poor response to gonadotropin therapy took growth hormone (GH) replacement for 4 months prior to restarting gonadotropins. When the serum level of insulin-like growth factor-I normalized, she began ovulation induction with gonadotropins with transdermal estradiol. After 63 days of gonadotropin therapy, she had a leading follicle of 18 mm, followed by follicles of 16.5 mm and 15.5 mm. The serum estradiol was 796 pg/ml, and human chorionic gonadotropin was administered. The patient conceived with timed intercourse. A prior attempt at ovulation induction with gonadotropins alone failed to produce follicular development. CONCLUSION: Prolonged gonadotropin treatment may be necessary to achieve ovulation and avoid the misdiagnosis of ovarian failure. Co-treatment with GH and estrogen may improve the follicular response in a poor responder with panhypopituitarism.

Diagnosis of polycystic ovaries by three-dimensional transvaginal ultrasound.

OBJECTIVE: To study diagnostic thresholds for polycystic ovary (PCO). DESIGN: Retrospective cohort study. SETTING: Academic hospital. PATIENT(S): Normoandrogenic ovulatory women and patients with polycystic ovary syndrome (PCOS). INTERVENTION(S): Two-dimensional (2D) and three-dimensional (3D) transvaginal ultrasound. MAIN OUTCOME MEASURE(S): The mean follicle number per ovary (FNPO) of both ovaries and the maximum number follicles in a single sonographic plane (FSSP) of either ovary were determined using 3D transvaginal ultrasound. Ovarian volume was determined using 2D transvaginal ultrasound. RESULT(S): Twenty-nine normoandrogenic ovulatory women were compared with 10 patients with PCOS. Diagnostic thresholds for PCO with 100% specificity as determined by receiver operator characteristic (ROC) curves were > or =20 for mean FNPO, > or =10 for maximum FSSP, and > or =13 cm3 for ovarian volume. Both 2D and 3D transvaginal ultrasound were highly accurate in the diagnosis of PCO as determined by areas under the curve (AUC) that were >90% for all three measures. CONCLUSION(S): Mean FNPO and maximum FSSP by 3D transvaginal ultrasound have comparable high accuracy for diagnosis of PCO. The diagnostic threshold with 100% specificity for mean FNPO is > or =20, which is greater than suggested by the Rotterdam Consensus Workshop in 2003. Use of the consensus standard, consequently, may result in overdiagnosis of PCO. A threshold of > or =20 mean FNPO using 3D transvaginal ultrasound may be appropriate to minimize false-positive diagnoses of PCO.

Diminished intrafollicular estradiol levels in in vitro fertilization cycles from women with reduced ovarian response to recombinant human follicle-stimulating hormone.

OBJECTIVE: To correlate serum and intrafollicular E(2) levels in women receiving GnRH agonist and recombinant human (h)FSH for IVF. DESIGN: Prospective study. SETTING: Academic IVF center. PATIENT(S): Twenty-three nonhirsute ovulatory women. INTERVENTION(S): In vitro fertilization cycles were classified as high- or low-normal response, according to whether the amount of recombinant hFSH administered was below or above, respectively, the mean recombinant hFSH given to all women combined. Serum bioactive LH and immunoreactive FSH were measured during pituitary desensitization preceding recombinant hFSH and repeated at oocyte retrieval. The first mature follicle aspirated from either ovary had a mean diameter >15 mm. MAIN OUTCOME MEASURE(S): Serum and follicular fluid (FF) insulin, bioactive LH, immunoreactive FSH, and steroid levels. RESULT(S): Basal serum gonadotropin levels were comparable between female groups. Serum immunoreactive FSH levels, but not bioactive LH levels, at oocyte retrieval were greater in cycles of low-normal vs. high-normal response, whereas maximum serum E2 levels and total oocyte counts were similar between both groups. Despite elevated FF immunoreactive FSH levels, FF E2 was decreased in low-normal response cycles. Intrafollicular T and P also were decreased and increased, respectively, in cycles of low-normal response, although FF bioactive LH levels were comparable in both groups. CONCLUSION(S): Intrafollicular E2 is decreased in cycles of reduced ovarian responsiveness to GnRH agonist and recombinant hFSH and is not predicted by maximal serum E2 or number of oocytes retrieved.

Cryosystem assessment by glucose uptake of murine blastocysts.

Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control - not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 +/- 24.2 and 64.1 +/- 31.5) versus controls (85.7 +/- 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols.