Automatic time-lapse instrument is superior to single-point morphology observation for selecting viable embryos: retrospective study in oocyte donation.

OBJECTIVE: To correlate the different categories provided by a commercial diagnostic test with blastocyst formation, quality, implantation potential, and ongoing pregnancy (OPR) for the purpose of validating the automatic annotations and the classification algorithm. DESIGN: Observational, retrospective, multicenter cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): A total of 3,002 embryos, including 521 transferred embryos with known implantation, from 626 IVF cycles that were incubated in a conventional incubator and monitored with an automatic time-lapse test. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Embryo selection was based on morphology and the classification provided by a commercial diagnostic test. Implantation was the primary end point, and OPR, blastocyst formation (BR), and embryo morphology were secondary end points. RESULT(S): BR and number of optimal blastocysts were related to the classification test. This correlation was also observed when analyzing implantation rates (day 3 transfer: high 38.2%, medium 31.7% and low 26.1%; day 5 transfer: high 66.7%, medium 50%, low 31%). Patients where no high embryos were transferred (n = 75) had an OPR of 46.70%, and those patients where at least one high embryo was transferred (n = 109) significantly increased OPR to 67%. A logistic regression analysis studying other confounding factors (day of transfer, number of oocytes obtained, and embryo morphology classification) was included. In that model, if at least one of the embryos was labeled as high, OPR was 2.567 times higher than a cycle where no high embryos were transferred. CONCLUSION(S): Our study presents, to our knowledge, the largest set of transferred embryos after time-lapse analysis with the use of an automatic time-lapse test. The provided classification was related to reproductive outcome. Our results suggest that the automated embryo diagnostic test provided extra information to the embryologist to select the best embryos, independently from clinical features of the patient or day of transfer.

Collapse of blastocysts is strongly related to lower implantation success: a time-lapse study.

STUDY QUESTION: Is there an association between blastocyst collapse patterns and implantation potential? SUMMARY ANSWER: Embryos that exhibit collapse are as likely to hatch as those that do not, but are less likely to implant and should not be replaced if alternatives are available. WHAT IS KNOWN ALREADY: Studies of blastocyst collapse in different species of mammals have found that most blastocysts, that experience consecutive weak contractions, hatch successfully whereas those that exhibit strong contractions or collapse, fail to hatch. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study. Seven hundred and fifteen transferred blastocysts were analyzed from July 2012 to May 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility center. Participant patients were recipients of oocyte donation and autologous IVF cycles (n = 460). Embryo development was analyzed with a time-lapse imaging system. Variables studied included blastocyst collapse (defined as the separation of ≥50% of the surface of the trophectoderm of the blastocyst from the zona pellucida), kinetic variables, embryo morphology, implantation and clinical pregnancy rates. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 139 blastocysts presented collapse (19.4%), of these 8 presented 2 collapses and 2 presented 3 collapses. The timings of embryo cleavages and the time taken to reach the morula stage and blastulation were significantly shorter in embryos with collapse than in those without. Implantation rates were compared in cycles where either all or none of the embryos replaced, implanted so that implantation data were known for every embryo. Among 408 embryos without collapse and 94 embryos with at least one collapse (94) the implantation rates were 48.5% (95% confidence intervals (CI) 46.7-53.4%) and 35% (95% CI 25.3-44.9%), respectively. The percentage of embryos that hatched was similar in both groups 28.7 and 31%, respectively. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study limits its potential value. WIDER IMPLICATIONS OF THE FINDINGS: Study the collapse pattern of the blastocyst, may assist selection of the blastocysts most likely to implant and increase IVF/ICSI success rates.

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse.

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse.

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.

Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse.

This study aims to compare the effect of early and late onset administration of oral antioxidants on number and quality of oocytes retrieved from aged mice after exogenous ovarian stimulation. Control hybrid females were fed a standard diet supplemented or not supplemented with pharmacological doses of vitamins C and E either from the first day of weaning or from the age of 32 weeks until they were autopsied at the age 40-42, 50-52, or 57-62 weeks after exogenous ovarian stimulation. Analysis of chromosomal distribution, DNA organization and cellular morphology was performed in ovulated cumulus-enclosed and -free oocytes, ovarian non-germinal vesicle oocytes enclosed by or free of mucous cumulus cells and in vitro-matured ovarian germinal-vesicle oocytes. Both early and late onset administration of oral antioxidants counteracted the negative effects of female aging on number of ovarian oocytes and total percentage of oocytes retrieved from oviducts and ovaries exhibiting a normal distribution of chromosomes in the metaphase-II plate and/or morphological traits of apoptosis. Although both early and late onset administration of oral antioxidants can counteract the negative effects of female aging on number and quality of oocytes, transference of these results to human beings should be made with caution because of the potential side effects of high doses of vitamins on reproductive function as well as many other undesirable systemic disorders.

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects the quality of ovulated oocytes in the mouse.

The present study aims to analyze the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on number and quality of mouse oocytes retrieved from oviducts after exogenous ovarian stimulation. Cellular and morphological traits of ovulated oocytes from hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 12, 40-42, 50-52 or 57-62 weeks of age were analyzed. Superovulation was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Injection of PMSG at diestrus-1 was associated with: (1) increased percentage of cumulus-free oocytes; (2) raised total percentage of oocytes without polar body; (3) increased total percentage of oocytes with intracytoplasmic mitochondrial aggregates; (4) decreased percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate; and (5) raised percentage of oocytes with chromosome scattering when compared to injection at estrus, diestrus-2, and proestrus stage. On the contrary, estrus females displayed the highest percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate and the lowest percentage of oocytes denuded of cumulus cells, without polar body, with intracytoplasmic mitochondrial aggregates and/or with chromosome scattering. These data suggest that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females to optimize the quality of oocytes collected from oviducts.

Postovulatory aging of oocytes decreases reproductive fitness and longevity of offspring.

We analyzed the long-term effects of postovulatory aging of mouse oocytes on reproductive fitness and longevity of offspring. Hybrid (C57BL/6JIco x CBA/JIco) parental generation (F0) females were artificially inseminated at 13 h (approximately 1 h postovulation) or 22 h (approximately 10 h postovulation) after GnRH injection. Reproductive fitness of first generation (F1) females was tested from the age of 28 wk until the end of their reproductive life. In males, the testing period ranged from the age of 2 yr until their natural death. Experimental F1 females exhibited longer between-labor intervals, decreased frequency of litters, and lower total number of litters and offspring born. Experimental second generation (F2) pups displayed teratogenic defects, higher preweaning mortality, and decreased body weight at weaning. Incidence of infertility was higher in experimental F1 males, which translated into lower total number of offspring born when compared with the control group. Life expectancy of F1 offspring was decreased in the experimental group. These results clearly show that postovulatory aging of mouse oocytes decreases reproductive fitness and longevity of offspring.