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Objective: To provide full morphokinetic characterization of embryos ranked with different degrees of chromosomal mosaicism., Design: Retrospective cohort study., Setting: University-affiliated private in vitro fertilization clinic., Patient(s): We analyzed 1,511 embryos from 424 intracytoplasmic sperm injection cycles by culturing embryos in a time-lapse imaging system and performing next-generation sequencing. We assessed 106 mosaic embryos., Intervention(s): None., Main Outcome Measure(s): Comparison of chromosomal, morphological, and morphokinetic characteristics of blastocysts classified as euploid, aneuploid, low-degree mosaic (30% to <50% aneuploid cells in trophectoderm biopsy), and high-degree mosaic (50% to <70% aneuploid cells in trophectoderm biopsy). Statistical analysis was performed using χ 2 , Kruskal-Wallis, or analysis of variance tests according to data type and distribution. A two-way random effects model was used to calculate interoperator correlation of annotations, and a logistic mixed effects model was performed to evaluate the effect of confounders on morphokinetic timing., Result(s): The mosaicism rate was ∼7% regardless of parental age. Mosaicism and uniform aneuploidies were not evenly distributed across chromosomes. The percentage of high-quality blastocysts significantly decreased from euploid (66.9%) to mosaic (52.8%) and aneuploid (47.7%). Aneuploid blastocysts significantly delayed development compared with euploid blastocysts in start of compaction (median, 84.72 hours postmicroinjection [hpm], interquartile range [IQR], 13.2; vs. median, 82.10 hpm, IQR, 11.5), start of blastulation (median, 101 hpm; IQR, 11.7; vs. median, 98.29 hpm, IQR, 10.5), and timing of blastocyst (median, 108.04 hpm, IQR, 11.50; vs. median, 104.71 hpm, IQR, 11.35). However, embryo morphokinetics were not correlated to the degree of mosaicism or to a mosaicism configuration that was apt for embryo transfer., Conclusion(s): Morphokinetic timing of mosaic embryos overlaps with that of euploid and aneuploid embryos, which may reflect their unique genetic and developmental identity. Although this suggests mosaic embryos are not simply a misdiagnosis by-product, further studies are needed to reveal the true identity of this particular type of embryo., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Ovarian aging leads to a decrease in the quantity and quality of oocytes. Aged oocytes have significantly reduced amounts of mitochondria, the energy factories of cells, leading to lower fertilization rates and poor embryonic development. Various techniques have tried to use heterologous or autologous sources of mitochondria to reestablish oocyte health by providing more energy. However, heterologous sources are no longer used owing to the known risk of heteroplasmy. Although autologous methods have recently been tested in humans, they have not shown a clear improvement in embryo quality. In this review, we describe the techniques that have been tested in recent years to provide a state of the art on oocyte rejuvenation through extra injection of mitochondria., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study if autologous mitochondrial transfer (AUGMENT) improves outcome in patients with previously failed in vitro fertilization (IVF)., Design: Randomized, controlled, triple-blind, experimental study., Setting: Private infertility center, Valencian Institute of Infertility (IVI-RMA), Valencia, Spain., Patient(s): Infertile women ≤42 years of age, body mass index <30 kg/m 2 , antimüllerian hormone ≥4 pmol/L, >5 million/mL motile sperm, at least one previous IVF with at least five metaphase oocytes (MIIs) collected, and low embryo quality., Interventions(s): An ovarian cortex biopsy was performed to isolate egg precursor cells to obtain their mitochondria. Sibling MIIs were randomly allocated to AUGMENT (experimental) or intracytoplasmic sperm injection (Control). In AUGMENT, mitochondrial suspension was injected along with the sperm. Viable blastocysts from both groups were biopsied for preimplantation genetic testing for aneuploidy., Main Outcome Measure(s): Pregnancy, embryo quality., Result(s): An interim analysis was conducted. The patients' mean age was 36.3 ± 3.6 years, and they had an average of 2.5 ± 1.5 previous IVF cycles. Two of the 59 enrolled patients spontaneously conceived (one miscarried). Fifty-seven patients had ovarian biopsies and underwent stimulation. Oocyte retrieval was performed in 56 patients (premature ovulation; n = 1). A total of 253 MIIs were inseminated in AUGMENT and 250 in Control; fertilization rates were 62.7 ± 30.0% and 68.7 ± 29.1%, respectively. Statistical differences were observed in day 5 blastocyst formation rates (23.3 ± 32.0% vs. 41.1 ± 36.9%). Neither the euploid rate per biopsied blastocyst (43.8 ± 41.7% vs. 63.8 ± 44.1%) nor the euploid rate per MII (9.8 ± 20.5% vs. 11.9 ± 16.1%) between AUGMENT and Control achieved statistical significance. Moreover, no differences were seen regarding mitochondrial DNA content and relevant morphokinetic variables. Thirty patients were able to undergo embryo transfer. Cumulative live birth rates per transferred embryo were 41.6% in AUGMENT and 41.2% in Control., Conclusion(s): AUGMENT does not seem to improve prognosis in this population. Therefore, the study has been discontinued., Clinical Trial Registration Number: NCT02586298., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study the potential variables that affect the mitochondrial DNA (mtDNA) content of trophectoderm (TE) cells in blastocysts that have undergone TE biopsy., Design: Observational retrospective single-center analysis., Setting: University-affiliated private in vitro fertilization center., Patient(s): A total of 465 consecutive preimplantation genetic screening (PGS) cycles of 402 women undergoing preimplantation genetic testing., Intervention(s): Trophectoderm biopsy performed on blastocysts of women undergoing preimplantation genetic testing-aneuploidy (PGT-A)., Main Outcome Measure(s): The mtDNA content in trophectoderm cells., Result(s): We checked the possible influence of patient characteristics, ovarian stimulation variables, embryo morphology, and embryo culture conditions on mtDNA values. Of all the analyzed variables, some such as body mass index (BMI), serum progesterone (P4), aneuploidy, and trophectoderm quality had an effect on mtDNA content in blastocysts. Body mass index had a small but positive effect on the mtDNA copy number; as the BMI values increased, the probability of women producing blastocysts with an mtDNA content above the median increased by 6%. For P4 serum concentration, an increase in P4 lowered the probability of blastocysts having values above the median by 39%. Embryo-associated variables such as TE quality and aneuploidy status appeared to affect the mtDNA copy number. For the aneuploid blastocysts, the probability of being above the median increased by 42%. Finally, blastocysts with poor quality TE had more chances of carrying higher mtDNA values., Conclusion(s): Summarizing, larger quantities of mtDNA in blastocysts are associated with the condition of aneuploidy and low quality TE, as well as being from women with high BMI values. Understanding the biological meaning of mtDNA content in human blastocysts and what factors may interfere with their values is fundamental. Other key gaps, such as whether a correlation exists between mtDNA content and mitochondrial mass and biogenesis in human TE cells, and whether this correlation can be extended to the inner cell mass, need to be further addressed. These questions are currently being investigated., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To describe the events associated with the blastocyst formation and implantation that occur in embryos during preimplantation development based on the largest sample size ever described with time-lapse monitoring., Design: Observational, retrospective, single-center clinical study., Setting: University-affiliated private IVF center., Patient(s): A total of 7,483 zygotes from 990 first treatments of intracytoplasmic sperm injection (ICSI; 627 of oocyte donor vs. 363 autologous oocyte cycles), of which 832 blastocysts were transferred., Intervention(s): No patient intervention. Embryos were cultured in a time-lapse monitoring system, and the embryos were transferred on day 5 after ICSI. Embryo selection was based on the multivariable model previously developed and on blastocyst morphology., Main Outcome Measure(s): Using a time-lapse system, embryo images were acquired every 15 minutes for 120 hours. Embryos cleavage time points up to the 9-cell stage (t2-t9) as well as to the morula stage (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle and synchrony of the second and third cell cycles were defined. As a result, we have monitored the embryonic development of a total of 3,215 blastocysts, of which 832 were transferred. Finally, we analyzed the characteristics of embryonic development of blastocyst (phase 1) and of implanted and not implanted (phase 2) embryos as finally validated in an independent data set (phase 3)., Result(s): A detailed retrospective analysis of cleavage times was made for 7,483 zygotes. We analyzed 17 parameters and found several significantly correlated with subsequent blastocyst formation and implantation. The most predictive parameters for blastocyst formation were time of morula formation, tM (81.28-96.0 hours after ICSI), and t8-t5 (≤8.78 hours) or time of transition of 5-blastomere embryos to 8-blastomere embryos with a receiver operating characteristic curve (ROC) value = 0.849 (95% confidence interval [CI], 0.835-0.854; phase 1). These parameters were less predictive of implantation, with a ROC value = 0.546 (95% CI, 0.507-0.585). We also observed that time for expansion blastocyst, tEB (107.9-112.9 hours after ICSI), and t8-t5 (≤5.67 hours after ICSI) predict blastocyst implantation, with a ROC value = 0.591 (95% CI, 0.552-0.630; phase 2). The model was validated on an independent data set and gave a ROC of 0.596 (0.526-0.666; phase 3)., Conclusion(s): The inclusion of kinetic parameters into score evaluation may improve blastocyst selection criteria and can predict blastocyst formation with high accuracy. We propose two multivariable models based on our findings to classify embryos according to their probabilities of blastocyst stage and implantation in the largest data set ever reported of human blastocysts., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether the global metabolomic profile of the spent culture media (SCM) of day-3 embryos is different in obese and normoweight women undergoing in vitro fertilization (IVF)., Design: Prospective cohort analysis., Setting: IVF clinic., Patient(s): Twenty-eight young, nonsmoking women with normoweight, nonsmoking male partners with mild/normal sperm factors undergoing a first IVF attempt for idiopathic infertility, tubal factor infertility, or failed ovulation induction: obese ovulatory women (n = 12); obese women with polycystic ovary syndrome (PCOS; n = 4); normoweight ovulatory women (n = 12)., Intervention(s): Fifty μl of SCM collected from two day-3 embryos of each cohort., Main Outcome Measure(s): Metabolomic profiling via ultrahigh performance liquid chromatography coupled to mass spectrometry of SCM from a total of 56 embryos., Result(s): The untargeted metabolomic profile was different in obese and normoweight women. Partial least squares discriminant analysis resulted in a clear separation of samples when a total of 551 differential metabolites were considered. A prediction model was generated using the most consistent metabolites. Most of the metabolites identified were saturated fatty acids, which were detected in lower concentrations in the SCM of embryos from obese women. The metabolomic profile was similar in obese women with or without PCOS., Conclusion(s): The metabolomic profile in the SCM of day-3 embryos is different in normoweight and obese women. Saturated fatty acids seem to be reduced when embryos from obese patients are present., Clinical Trial Registration Number: NCT01448863., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates., Design: Prospective cross-sectional study., Setting: Multicenter study., Patient(s): Seven hundred embryo transfers and 1,028 early-stage human embryos., Intervention(s): None., Main Outcome Measure(s): Implantation according to the presence of EC and embryo quality., Result(s): The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage., Conclusion(s): At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In view of the increasing emphasis being placed on patient safety and quality health care, it is extremely important to develop fail-safe mechanisms to prevent assisted reproductive technology (ART) mix-ups. Sample mismatch is the most undesirable event that can occur in an IVF laboratory as it may have catastrophic consequences for both patients and health care professionals. Many strategies can be adopted to reduce laboratory errors, such as improved quality control and quality assessment, certification, educational programs, and external quality assessment. Nevertheless, none suffices to absolutely prevent this error. Therefore, the implementation of specific policies, such as double-checking safety protocols, is receiving more and more interest. After some adverse events involving sample misidentification occurred in some countries, double-checking every step of the IVF clinical and laboratory procedure has become mandatory. However, double-checking protocols can also prove difficult to implement and a new generation of errors may occur. Other approaches, including electronic strategies for tracking, and even microlabeling embryos, are currently being evaluated.

Objective: To describe the times associated with the morphological changes that occur in the embryo during preimplantation development based on the largest sample size described with time lapse., Design: Cohort study., Setting: University-affiliated private center., Patient(s): A total of 9,530 embryos from 1,806 intracytoplasmic sperm injection (ICSI) cycles., Intervention(s): None., Main Outcome Measure(s): Using a time-lapse system, embryo images were acquired for at least 68 hours, in some cases reaching 120-130 hours. Embryo cleavage time points up to 8-cell-stage (t2-t8) as well as morulae (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle (cc) and synchrony (s) of the second and third cell cycles were defined. Finally, four subgroups of embryos were considered: the "regular divisions" group excluded embryos with a direct cleavage from 1 to 3 or 2 to 5 cells, and the "viable 8-cell," the "viable blastocyst," and "implanted embryos" groups included only embryos viable to the 8-cell stage, blastocyst stage, or transferred and successfully implanted, respectively., Result(s): Averages of times in the general population were: t2 = 27.9 hours, t3 = 38.2 hours, t4 = 40.7 hours, t5 = 51.0 hours, t6 = 54.1 hours, t7 = 56.7 hours, t8 = 59.1 hours, tM = 86.6 hours, tB = 104.1 hours, cc2 = 10.3 hours, cc3 = 12.8 hours, s2 = 2.7 hours, and s3 = 9.9 hours. Comparison between groups showed significant differences between regular divisions and viable 8 cells for t2, t3, t5, cc2, cc3, s2, and s3; between 8 cells and blastocyst for t5, t8, tM, cc3, and s2; and between blastocyst and implanted embryos for t8, tM, tB, and s2. Differences in timing related to morphology of cleavage- and blastocyst-stage embryos were detected., Conclusion(s): A time-lapse monitoring system applied to embryology allows accuracy and objectivity when defining the basis of embryo development within a clinic. The sample size is the largest ever described that provides consistent information about the normal distribution of embryo developmental timings., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles., Design: Randomized trial., Setting: Private university-affiliated IVF center, university-based hospital., Patient(s): A total of 1,125 cycles of ovum donation., Intervention(s): Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air., Main Outcome Measure(s): Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients., Result(s): The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively)., Conclusion(s): In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the usefulness of preimplantation genetic screening (PGS) using fluorescence in situ hybridization (FISH) for two different indications: repetitive implantation failure (RIF) and advanced maternal age (AMA)., Design: Two prospective, randomized controlled trials with patients allocated in two arms: blastocyst transfer on day 5 (group A) or PGS with transfer on day 5 (group B)., Setting: University-affiliated private clinics., Patient(s): The RIF study included women <40 years with three or more failed IVF cycles without other known causal factors (91 patients). The AMA study included intracytoplasmic sperm injection patients aged between 41 and 44 (183 patients)., Intervention(s): In the PGS group, single-cell day 3 biopsy was performed with aneuploidy screening for chromosomes 13, 15, 16, 17, 18, 21, 22, X, and Y. In both the blastocyst transfer group and the PGS group, ET was performed on day 5., Main Outcome Measure(s): Live-birth rate per patient and per started cycle., Result(s): A significant increase in live-birth rates per patient was found in the PGS group compared with the blastocyst group for the AMA study (30/93 patients [32.3%] vs. 14/90 patients [15.5%]; odds ratio, 2.585; confidence interval, [1.262-5.295]). In the RIF study no significant differences were observed (23/48 patients [47.9%] vs. 12/43 patients [27.9%])., Conclusion(s): PGS with FISH was shown to be beneficial for the AMA group., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the dynamics of the nuclear maturation (NM) of in vitro-matured (IVM) oocytes and to determine the most favorable duration of meiosis II (MII) arrest in relation to the normal activation response., Design: Experimental., Setting: University-affiliated infertility clinic., Patient(s): Donated immature germinal vesicle oocytes (GV)., Intervention(s): The GV underwent spontaneous IVM and the dynamics of NM studied by real-time monitoring. The IVM oocytes were parthenogenetically activated at different MII arrest points and their response assessed., Main Outcome Measure(s): Moment of GV breakdown; extrusion of the first polar body; duration of MI and MII arrest; activation rate (AR) and type., Result(s): Two GV populations-early (E-IVM, 18.4 ± 2.7 hours) and late (L-IVM, 26.3 ± 3.8 hours) maturing-were defined according to the time required for extrusion of the first polar body. Significantly more E-IVM than L-IVM exhibited a normal activation response (61.3% vs. 34.6%), but AR were similar (average, 88.6%) in both groups. Duration of the GV stage differed between the two groups, but MI arrest (14.0 ± 0.3 hours) was constant. The E-IVM arrested at MII for at least 4.3 hours displayed significantly lower AR and similar normal activation rates (61.3%) to E-IVM arrested for a shorter time (83.9% vs. 100%). The L-IVM displayed a similar AR (80.8%), but lower normal activation rates than E-IVM (34.6%), regardless of when activation took place., Conclusion(s): The success of IVM depends on the NM timing rather than on the length of MII arrest., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome., Design: Observational study., Setting: Private university-affiliated IVF center., Patient(s): Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons., Intervention(s): Vitrification by the Cryotop open device., Main Outcome Measure(s): Delivery rate (DR) per warming cycle., Result(s): Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%-95.3%) for day 2 and 95% (95% CI 94.3%-95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%-34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%-96.8%) for day 2, 94.2% (95% CI 93.4%-94.9%) for day 3, 95.7% (95% CI 94.5%-96.9%) for day 5, and 97.6% (95% CI 96.9%-98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%-38.5%), 41.7% (95% CI 39.9%-43.4%), 32.6% (95% CI 31.0%-34.2%), and 38.1% (95% CI 36.4%-39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912-1.277) showed no impact on the DR., Conclusion(s): Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect on the survival and delivery rates., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the presence of viral RNA or DNA sequences in spent culture media used after ovum pickup (OPU) or embryo culture and in liquid nitrogen (LN) used for oocyte or embryo vitrification in patients seropositive for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) undergoing IVF cycles., Design: Descriptive study., Setting: Private university-affiliated IVF center., Patient(s): Twenty-four women who underwent controlled ovarian stimulation for oocyte vitrification or IVF/ET. A total of 6, 11, and 6 patients were seropositive for HIV, HCV, and HBV, respectively, whereas 1 patient showed a coinfection with HCV and HBV. Seven patients presented positive blood viral load (HIV, n = 1; HBV, n = 1; HCV, n = 5). Sixty-three samples were analyzed: follicular fluid, n = 3; spent culture media, n = 33 (20 after OPU and 13 after embryo culture); and LN, n = 27 (14 and 10 after oocyte and embryo vitrification; and 3 LN storage tank samples)., Intervention(s): Ovum pickup, oocyte and/or embryo culture, and/or vitrification by the Cryotop open device. Reverse transcription-polymerase chain reaction analysis was performed for viral screening., Main Outcome Measure(s): Detection of viral sequences of HIV, HCV, and HVB., Result(s): All the samples analyzed tested negative for the detection of viral RNA or DNA sequences., Conclusion(s): We have not detected viral sequences after culture and vitrification of oocytes/embryos from HIV-, HBV-, and HCV-seropositive patients. These findings represent good evidence of the lack of risk of cross-contamination among seropositive patients, even using an open device for vitrification., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effect of different ovarian stimulation protocols on oocyte respiration and to investigate the relationship between oocyte oxygen consumption and reproductive outcome., Design: Prospective observational cohort study., Setting: Infertility clinic in a university hospital., Patient(s): A total of 349 oocytes from 56 IVF treatment cycles in our oocyte donation program., Intervention(s): None., Main Outcome Measure(s): Average oocyte oxygen consumption rate in fmol/s. We correlated oxygen consumption values with ovarian stimulation features, fertilization, embryo quality on days 2 and 3, and implantation., Result(s): Differences in the measured oxygen consumption rates were found depending on which type of gonadotropins were used in the stimulation protocol. Higher consumption rates were found for oocytes that underwent normal fertilization compared with rates from nonfertilized or abnormal oocytes (odds ratio = 1.340; 95% confidence intervals = 1.037-1.732). Furthermore, higher oxygen consumption was observed for those oocytes which generated embryos that implanted compared with those that did not implant (6.21 ± 0.849 fmol/s vs. 5.23 ± 0.345 fmol/s., Conclusion(s): Measurement of oxygen consumption rates for individual oocytes before fertilization provides a noninvasive marker of oocyte quality and hence a quantitative assessment of the reproductive potential for the oocyte., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effectiveness of long-term vapor-phase nitrogen storage of vitrified human oocytes as a strategy for preventing the risk of cross-contamination due to direct contact with the liquid nitrogen (LN)., Design: Prospective randomized study., Setting: Private infertility center, IVI, Valencia., Patient(s): Oocyte donors (n = 44) and recipients (n = 46)., Intervention(s): Vitrification by the Cryotop method. Storage of vitrified oocytes in a vapor-phase nitrogen storage freezer and a traditional LN storage tank. Donation of the surviving oocytes and evaluation of fertilization, embryo development, and clinical results., Main Outcome Measure(s): Survival, fertilization, and cleavage rates. Embryo quality and clinical outcome., Result(s): Survival was 95.3% (vapor-phase nitrogen) and 94.5% (LN). Fertilization rates (73.1% and 71.7%) or cleavage on day 2 (95.6% and 94.7%), day 3 (84.5% and 79.9%), and blastocyst formation (54.7% and 53.9%) were similar between vapor-phase nitrogen and LN. Implantation, clinical, and ongoing pregnancy rates were similar for vapor-phase nitrogen (40.5%, 58.1%, and 48.8%, respectively) and LN groups (33.7%, 53.3%, and 46.6%, respectively)., Conclusion(s): The vapor-phase nitrogen system permits the storage of oocytes vitrified, maintaining their potential to develop into competent embryos in a similar manner as those stored in a traditional LN freezer. This approach represents a practical alternative that prevents cross-contamination during the storage of vitrified samples., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the survival rate and clinical results of our vitrification procedure on preimplantation genetic diagnosis (PGD) blastocysts and to calculate its actual contribution to the reproductive outcome per cycle., Design: Retrospective clinical study., Setting: University Institute IVI, Valencia, Spain., Patient(s): Patients who requested cryotransfer of surplus PGD blastocysts after failed fresh elective transfer., Intervention(s): Retrospectively collected data during 2 years of experience with blastocyst vitrification., Main Outcome Measure(s): Primary outcome measures were the following: blastocyst recovery and survival; cryotransfer cancellation; and the implantation, pregnancy (PR), and ongoing-pregnancy rates. The secondary outcome measure was cumulative ongoing PR (COPR)., Result(s): Cocultured vitrified PGD blastocysts were recovered and progressed in development after overnight culture (survival rate) at rates comparable to those of non-PGD blastocysts (49% and 42%, respectively). After transfer to 64% of patients, no statistical differences were found between PGD and non-PGD blastocyst groups concerning the following: PR (44% vs. 37%), implantation rate (40% vs. 27%), and ongoing-pregnancy rate (32% vs. 37%). Moreover, blastocyst vitrification significantly increased the COPR in both PGD and non-PGD cycles, from 47% (62/133) to 53% (70/133) and from 45% (24/53) to 53% (28/53), respectively., Conclusion(s): A preimplantation genetic diagnosis blastocyst vitrification procedure showed survival rates and improvements on the COPR that were comparable to those in non-PGD blastocyst cycles. Moreover, vitrification of biopsied and diagnosed embryos at the more advanced stages instead of at earlier cleavage stages is presented as an attractive strategy to consider in PGD programs.

Objective: To prospectively determine the impact of concrete components of the sperm oxidative glutathione stress system in terms of enzymatic activity and mitochondrial RNA (mRNA) expression on embryo quality and reproductive outcome. Human spermatozoa use the glutathione system to inactivate reactive oxygen metabolites, and there is a close correlation between some components of the glutathione system and male fertility. However, very few data are published regarding this system in sperm cells and its effect on fertilization ability and embryo development in human beings., Design: An oocyte-donation model, used to homogenize the female factor., Setting: University-affiliated private IVF setting., Patient(s): Semen samples from infertile males (n = 43) of couples undergoing oocyte-donation cycles (n = 43)., Intervention(s): None., Main Outcome Measure(s): Gene expression and activity of glutathione peroxidases (GPXs) 1 and 4, glutathione reductase, and intracellular glutathione (GSH) by fluorescent quantitative polymerase chain reaction and spectrophotometry, respectively., Result(s): Fertilization rate, pronuclear number, asymmetry, and pronuclear body distribution were not correlated with any sperm glutathione parameters that were considered. When day 3 embryo parameters were evaluated, only GPX4 mRNA expression in sperm cells was statistically significantly lower when asymmetric embryos were observed. Also, worst embryo development and morphology on day 5 was statistically significantly correlated with lower sperm GPX1 activity (101.07 vs. 258.8 IU/mg protein). Glutathione system analysis in fresh sperm was not statistically significantly different in patients achieving pregnancy compared with those who not, and we did not find any correlation with implantation rate., Conclusion(s): We have been able to correlate embryo morphology on day 3 with the sperm expression of GPX family members. The results indicate that sperm-derived mRNA may condition human embryo quality and persist even to blastocyst stage. The correlation of the sperm GPX family mRNA expression with embryo health appears quite promising for discovery of molecular causes of male infertility.

Objective: To evaluate ultrastructural features of preimplantation genetic diagnosis (PGD) blastocysts before and after vitrification., Design: Descriptive study of both vitrified and fresh hatching blastocysts., Setting: PGD program at the Instituto Universitario, Instituto Valenciano de Infertilidad., Patient(s): Patients undergoing PGD donated their abnormal embryos for research (n = 26)., Intervention(s): Biopsied embryos were cultured in the presence of human endometrial cells until day 6. Sixteen blastocysts were vitrified. A total of 11 high-scored hatching blastocysts, 6 warmed and 5 fresh, were fixed for ultrastructure., Main Outcome Measure(s): The cytoskeleton structure, type of intercellular junctions, and basic intracellular organelles in trophoectoderm cells and the inner cell mass were analyzed., Result(s): Ten of 16 blastocysts (62%) survived the warming process. Six of these showed no signs of cell degeneration and light microscopy revealed similar ultrastructural characteristics to those of controls. However, in trophoectoderm cells from both fresh and cryopreserved blastocysts, a reduced number of tight junctions and the presence of degradation bodies were detected., Conclusion(s): The particular ultrastructural features observed in PGD-derived blastocysts could be related to embryo manipulation and culture conditions. Vitrification does not seem to alter blastocysts, as those that survive hatching do not display detectable cellular alterations when observed through electron microscopy.

Objective: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome., Design: Double-blind prospective study., Setting: University-affiliated private IVF setting., Patient(s): A total of 85 couples undergoing infertility treatment with IVF/ICSI., Intervention(s): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART)., Main Outcome Measure(s): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success., Result(s): Fertilization rate was inversely correlated with DNA fragmentation (r = -0.245 P = .045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P < .001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P = .044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r = -0.250 P = .042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7)., Conclusion(s): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies' pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study.

Objective: To investigate the changes induced by age in the function and secretory pattern of the human ovary. Immunoreactive alpha-inhibin, E2, and P secretion in vivo and in vitro have been compared in two different populations., Design: Prospective study. Women undergoing IVF-ET were divided into two groups according to age: group 1 (32.0 +/- 0.7 years; mean +/- SEM) and group 2 (40.3 +/- 0.3 years)., Setting: In vitro fertilization program at the Instituto Valenciano de Infertilidad., Patients: A total of 33 infertile women with regular menses, undergoing IVF-ET., Interventions: Follicle aspiration performed by transvaginal ultrasound. Four follicles per patient were aspirated in individual plastic tubes. Granulosa-luteal cells isolated with Percoll columns and cultured in vitro up to 4 days in the presence of hCG., Main Outcome Measures: In vitro fertilization parameters, serum levels of E2, immunoreactive alpha-inhibin, and P, as well as the secretion of immunoreactive alpha-inhibin and P by the cultured granulosa-luteal cells., Results: Serum immunoreactive alpha-inhibin levels the day of ovum pick-up were significantly lower in group 2 compared with group 1. Incubation of cells for 96 hours showed a significantly higher ability to accumulate immunoreactive alpha-inhibin in group 1 than 2. Human chorionic gonadotropin stimulated immunoreactive alpha-inhibin production after 96 hours. Cells from younger women displayed a significantly higher ability to secrete P than cells from older women. Human chorionic gonadotropin was able to significantly stimulate P production in group 1., Conclusions: These results confirm previous observations showing a reduced production of immunoreactive alpha-inhibin and steroids of ovaries from older women and suggest that a reduced cellular function, rather than a decrease in the follicular population, is the main mechanism by which these changes are produced.

Objective: To investigate the effect of short-term glucocorticoid administration on embryotoxicity of sera from infertile patients with mild to moderate endometriosis., Design: Prospective longitudinal study., Setting, Patients: Eight infertile patients with mild to moderate endometriosis and a control group of eight infertile patients with tubal infertility were selected on the basis of laparoscopic examination., Interventions: Basal (B) serum collection and day 1 (T1), day 3 (T2), day 6 (T3), and day 12 (T4) serum drawn after a 3-day glucocorticoid treatment in endometriosis patients., Main Outcome Measure: Embryotoxicity of endometriosis sera, before and after glucocorticoid treatment, was investigated using a bioassay performed on two-cell mouse embryos. Interleukin 1 alpha (IL-1 alpha) and antismooth muscle, antimitochondrial, and antinuclear autoantibodies were also tested in these sera., Results: At 50% concentration, endometriosis serum is embryotoxic in comparison with control; 0% versus 61% of the embryos reached the blastocyst stage at 72 hours, respectively (basal versus control, P less than 0.001). However, this embryotoxicity significantly decreases 12 days after glucocorticoid treatment in comparison with untreated sera; 32.4% versus 0% of the embryos reached blastocyst stage at 72 hours, respectively (T4 versus basal, P less than 0.001), although they did not reach nontoxic levels (greater than 50%). Interleukin 1 alpha was undetectable in all samples analyzed. In endometriosis sera, antismooth muscle antibody was detected., Conclusions: At 50% concentration, serum from infertile patients with minimal to moderate endometriosis appears to be embryotoxic to the in vitro development of two-cell mouse embryos. However, this embryotoxicity significantly decreases 12 days after a 3-day treatment with glucocorticoids.