Search

Objective: To determine the prognostic impact of the nuclear status at the two-cell stage on intracytoplasmic sperm injection (ICSI) outcomes., Design: Retrospective study., Setting: Hospital., Patient(s): Only ICSI cycles with time-lapse monitoring of transferred embryos with known implantation/delivery data from November 2012 to December 2014 were included. A total of 2,449 embryos were assessed for multinucleation rates at the two- and four-cell stage, and 608 transferred embryos were studied for ICSI outcomes., Intervention(s): None., Main Outcome Measure(s): Implantation rate (IR) and live birth rate (LBR) according to the number of multinucleated blastomeres at the two-cell stage: none (Without-MNB 2cell ), one (MNB 1/2cell ), and two (MNB 2/2cell ); morphokinetics of MNB 2cell embryos., Result(s): Embryos with MNB 1/2cell led to lower IR (27.7%) and LBR (22.7%) than embryos Without-MNB 2cell (33.4% and 29.8%, respectively). The MNB 2/2cell embryos led to significantly lower IR (18.3%) and LBR (13.4%) than embryos Without-MNB 2cell . This difference remained significant in multivariate analysis for implantation (odds ratio 0.57; 95% confidence interval 0.34-0.94) and birth (odds ratio 0.46; 95% confidence interval 0.26-0.80), independently of the other significant parameters (women's age, time of two-cell formation, and multinucleation at the four-cell stage). Among implanted MNB 2cell , if cleavage into four cells occurred later than 37 hours after insemination, embryos were significantly more likely to lead to birth., Conclusion(s): The presence of multinucleation at the two-cell stage and more specifically in both blastomeres had a significant negative impact on birth potential. Thus, embryo multinucleation at the two-cell stage should be used as an additional noninvasive criterion for embryo selection., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy., Design: Prospective case-control study., Setting: A private center for reproductive medicine., Patient(s): A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls., Intervention(s): Choice of embryos for transfer was based on the PGD NGS results., Main Outcome Measure(s): Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates., Result(s): The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%)., Conclusion(s): We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial., Clinical Trial Registration Number: ACTRN12614001035617., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the relationship between blastomere number and aneuploidy., Design: Historical cohort study., Setting: In vitro fertilization clinic., Patient(s): Two hundred fifty-nine patients undergoing in vitro fertilization (IVF) in combination with comprehensive chromosomal screening of embryos., Intervention(s): A total of 1,915 embryos were biopsied on day 3 and underwent comprehensive chromosomal screening with microarray-based comparative genomic hybridization., Main Outcome Measure(s): Relationship between day 3 blastomere number, aneuploidy rate, and progression to the blastocyst stage., Result(s): A number of day 3 blastomeres >9 was associated with significantly increased aneuploidy rates. Rapidly developing embryos were significantly more likely to blastulate regardless of their chromosomal status. Number of embryos per patient greater than 13 was independently associated with lower aneuploidy rates after controlling for maternal age. This trend was not significant with the use of a more clinically relevant threshold of greater than six embryos per patient., Conclusion(s): Embryos with 6-9 cells at the cleavage stage should be considered for transfer over embryos with >9 cells. Day 3 blastomere number may be used in conjunction with extended culture to improve selection of euploid embryos, especially when supernumerary embryos are available. Further studies are needed to show if these selection criteria improve clinical outcomes., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the prognostic impact of the embryo nuclear status at day 2 among other major morphologic parameters (first cleavage at day 1, number of blastomeres and anuclear fragmentation at day 2) on the birth rate., Design: Retrospective study., Setting: Hospital IVF department., Patient(s): Women undergoing 1,629 day 2 transfers of 2,732 embryos from May 2006 to November 2008., Intervention(s): Four groups according to the embryo nuclear status., Main Outcome Measure(s): Implantation, miscarriage, and birth rates., Result(s): Univariate analysis indicated significantly higher birth rates when all blastomeres were mononucleated (15.0%) compared with embryos with not all blastomeres mononucleated (9.2%), embryos without any visible nucleus (7.7%), and embryos where at least one blastomere was multinucleated (4.2%). Multivariate analysis found significant decreased birth rates when multinucleated blastomeres were observed., Conclusion(s): Blastomere nuclear status should be added to the kinetic and morphologic criteria traditionally used at day 2 to assess human embryo quality. The presence of multinucleated blastomeres has a negative impact on birth potential. The results argue for integrating the blastomere nuclear status at day 2 with the kinetic and morphologic criteria traditionally used to define the best embryo to transfer. Embryos with a single visible nucleus in all blastomeres should be given priority for transfer when available., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the distribution patterns of H3K4me3 and H3K27me3 in human oocytes and preimplantation embryos., Design: Experimental study., Setting: University reproductive medical center., Patient(s): Patients undergoing IVF cycles., Intervention(s): Oocytes and embryos were collected from patients undergoing IVF cycles., Main Outcome Measure(s): The distribution patterns of H3K4me3 and H3K27me3 in oocytes and embryos were analyzed by indirect immunofluorescent staining and scanning confocal microscopy., Result(s): H3K4me3 and H3K27me3 signals were detectable at each stage of oocyte and embryonic development. However, only one of the pronuclei showed signal for H3K27me3 in each of the zygotes, whereas H3K4me3 staining was always uniform in all zygotes. The level of H3K4me3 decreased steadily from germinal vesicle to metaphase II stage, obviously increased from zygote stage to four-cell stage, and reached the lowest at eight-cell stage. A sharp increase was then observed at blastocyst stage. The level of H3K27me3 slightly changed from germinal vesicle stage to zygote stage, then decreased steadily and reached the lowest at eight-cell stage, followed by a significant increase at blastocyst stage., Conclusion(s): The levels of H3K4me3 and H3K27me3 show dynamic changes during human oocyte maturation and preimplantation embryonic development. Asymmetric distribution of H3K27me3 exists in human zygote pronuclei, whereas H3K4me3 is always uniform in all of the pronuclei., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To analyze whether the cleavage pattern redefined for all cleavage stages according to the relative blastomere size as a function of cell number has an additive value in predicting implantation potential of day-2 and day-3 embryos., Design: Retrospective analysis of standard embryo morphologic parameters (cleavage rate and degree of fragmentation) supplemented by cleavage pattern findings of 347 implanted embryos compared with those of a matched control group of 307 embryos that failed to implant., Setting: University-based tertiary medical center., Patient(s): Two hundred and nine women with successful implantation and 181 controls matched for age and demographic parameters with failed implantation., Intervention(s): In vitro fertilization (IVF), embryo assessment, and embryo transfer., Main Outcome Measure(s): Determination of cleavage patterns in synchronized and unsynchronized cleaving embryos and correlations with implantation outcomes., Result(s): Statistically significantly more embryos of the implanted group had good cleavage patterns compared with the failed implantation group (88% vs. 70%). A good cleavage pattern predicted implantation outcome even for nonsynchronized cleaving blastomeres at three, five, six, and seven cells (79% vs. 59%). Regression analysis demonstrated that adding cleavage pattern to the scoring system increased our ability to predict implantation in the training set; the area under the curve was the highest (0.707) as was the proportion of correct classification (>70%) when the cleavage pattern was assessed on both days 2 and 3., Conclusion(s): When combined with measurements of the cleavage rate and degree of fragmentation, the cleavage pattern refines our ability to predict the likelihood of implantation, representing a definitive tool in the selection of top-quality embryos., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos., Design: Experimental prospective controlled study., Setting: Research center., Patient(s): Human Blastomeres were obtained after biopsy., Intervention(s): Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing., Main Outcome Measure(s): Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres., Result(s): After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip., Conclusion(s): The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the validity of collecting day 3 embryo morphology variables into the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS)., Design: Retrospective., Setting: National database-SART CORS., Patient(s): Fresh autologous assisted reproductive technology (ART) cycles from 2006-2007 in which embryos were transferred singly (n=1,020) or in pairs (n=6,508) and embryo morphology was collected., Intervention(s): None., Main Outcome Measure(s): Relationship between live birth, maternal age, and morphology of transferred day 3 embryos as defined by cell number, fragmentation, and blastomere symmetry. Logistic multiple regressions and receiver operating characteristic curve analyses were applied to determine specificity and sensitivity for correctly classifying embryos as either failures or successes., Result(s): Live birth rate was positively associated with increasing cell number up to eight cells (<6 cells: 2.9%; 6 cells: 9.6%; 7 cells: 15.5%; 8 cells: 24.3%; and >8 cells: 16.2%), but was negatively associated with maternal age, increasing fragmentation, and asymmetry scores. An area under the receiver operating curve of 0.753 (95% confidence interval 0.740-0.766) was derived, with a sensitivity of 45.0%, a specificity of 83.2%, and 76.4% of embryos being correctly classified with a cutoff probability of 0.3., Conclusion(s): This analysis provides support for the validity of collecting morphology fields for day 3 embryos into SART CORS. Standardization of morphology collections will assist in controlling for embryo quality in future database analyses., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To report a clinical pregnancy after rebiopsy and vitrification of blastocysts following allele dropout (ADO) of biopsied day 3 embryos., Design: Case report., Setting: Private center., Patient(s): Thirty-year-old woman and her 33-year-old husband who carries the single-gene condition paraganglioma., Intervention(s): In vitro fertilization with day 3 embryo biopsy-ET-blastocyst biopsy and vitrification-subsequent frozen ET cycle., Main Outcome Measure(s): Results from preimplantation genetic diagnosis and pregnancy results after fresh and frozen ETs., Result(s): Nineteen oocytes were retrieved of which 13 were mature and 12 fertilized. Eleven embryos were biopsied on day 3: two were normal, five were affected, and four exhibited ADO. The two normal blastocysts were transferred, and three of the ADO blastocysts were biopsied and sent for reanalysis. The biopsied blastocysts were vitrified. No pregnancy resulted from the fresh ET. One of the biopsied blastocysts was normal, one received no result, and one exhibited ADO. A singleton clinical pregnancy resulted from a subsequent frozen ET of the thawed biopsied normal blastocyst., Conclusion(s): Rebiopsy and vitrification of blastocysts could be used in cases of ADO or lack of results after day 3 embryo biopsy., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To develop a simple method to purify blastomeres of inner cell mass (ICM) and trophectoderm (TE) lineage using magnetic activated cell sorting., Design: Prospective laboratory study., Setting: Embryology research laboratory., Patient(s): None., Intervention(s): Trophectoderm cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic cell sorting to separate cells into FITC-positive and -negative fractions., Main Outcome Measure(s): Purity and gene expression., Result(s): In the FITC-positive fraction, an average of 91.2% of cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an inner cell mass marker, was significantly higher in the FITC-negative fraction., Conclusion(s): Highly purified trophectoderm cells or inner cell mass cells can be collected using magnetic activated cell sorting. This method can be useful for understanding differentiation and function of cell lineages in the blastocyst., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study the implantation potential of unselected embryos derived from modified natural cycle IVF according to their morphological characteristics., Design: Cohort study., Setting: Academic department of reproductive medicine., Patient(s): A series of 449 single embryo transfers derived from modified natural cycle IVF., Intervention(s): None., Main Outcome Measure(s): Ongoing implantation rate according to embryo characteristics., Result(s): The best implantation was found in embryos with 4 and 8 cells on day 2 and 3 respectively,

Objective: To validate a novel system for embryonic DNA fingerprinting which can reliably distinguish sibling embryos from each other., Design: Prospective, randomized, and blinded study., Setting: Academic center for reproductive medicine., Patient(s): Blastomeres were obtained from discarded and transferred embryos from six patients undergoing IVF treatment., Intervention(s): None., Main Outcome Measure(s): Single lymphocytes from sibling cell lines and blastomeres from sibling day 3 human embryos were evaluated for accurate assignment of relationship using whole genome amplification and single-nucleotide polymorphism microarrays., Result(s): Assignment of single-cell relationships was accomplished with 100% accuracy. We also observed complete agreement between the molecular karyotype and DNA fingerprint-based identification of embryos implanted in three clinical IVF cases after multiple embryo transfer., Conclusion(s): These data demonstrate the first single-blastomere DNA fingerprinting technology capable of unequivocal discrimination of sibling human embryos., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

This study sought to validate a novel, minimally invasive system for embryo tracking by single nucleotide polymorphism microarray-based DNA fingerprinting of the first polar body. First polar body-based assignments of which embryos implanted and were delivered after multiple ET were 100% consistent with previously validated embryo DNA fingerprinting-based assignments., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR., Design: Techniques and instrumentation., Setting: Tertiary clinical and academic medical center., Patient(s): Thirty-six embryos from seven clinical PGD cycles., Intervention(s): Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5., Main Outcome Measure(s): PGD based on mutation identification or exclusion testing for at-risk HD carriers., Result(s): Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies., Conclusion(s): An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres., Design: Randomized study., Setting: Molecular genetics and animal reproduction laboratories., Patient(s): Cow ovaries obtained from slaughterhouses., Intervention(s): The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development., Main Outcome Measure(s): Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination., Result(s): A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR., Conclusion(s): Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages (splitting efficiency) and to determine the in vitro developmental capacity of twinned human embryos (developmental efficiency)., Design: Randomized comparative study., Setting: Private IVF centers., Patient(s): Couples undergoing IVF donating triploid embryos., Intervention(s): Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos. Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro., Main Outcome Measure(s): Development of twinned embryos to the blastocyst stage., Result(s): The number of developing embryos obtained after splitting could be increased in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency). Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts (developmental efficiency). Twinning success was related to the superior morphological quality of embryos used for splitting., Conclusion(s): This is the first report on twinned human embryos developing to blastocysts. This study exhibits the potential for novel applications in human assisted reproduction., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

This article describes a new methodology for preserving and banking isolated human blastomeres, whose originality is based on packing the blastomere into an emptied zona pellucida before vitrification. After warming, 75.7% of blastomeres survived and developed at a rate comparable to that in noncryopreserved blastomeres (62.5% cleavage, 26.6% compaction, and 20.3% cavitation)., (Copyright 2010 American Society for Reproductive Medicine. All rights reserved.)

We conducted a retrospective review of all frozen ETs from May 2002 to October 2007 using our new vitrification technique in order to determine its efficacy. We found high pregnancy (70.5%; 79/112) and implantation (40.6%; 110/271) rates with the use of this new method.

Objective: To report a simple and efficient fluorescence in situ hybridization (FISH) method for preimplantation genetic diagnosis (PGD)., Design: Technique and method., Setting: A hospital in vitro fertilization (IVF) laboratory., Patient(s): Women undergoing IVF or intracytoplasmic sperm injection (ICSI)., Intervention(s): None., Main Outcome Measure(s): The intensity and clarity of signals, technical difficulty, the percentage of successfully treated blastomeres, and blastomere integrity after FISH., Result(s): This paraformaldehyde fixation technique simplified the process of fixation of blastomeres for PGD without losing blastomeres during fixation. A total of 35 blastomeres derived from 10 arrested embryos or abnormally fertilized eggs (one pronucleus or three pronuclei) were used for three-dimensional (3D) FISH staining. Signals in all blastomeres were obtained successfully by this method. Approximately 0.1 microL of DNA probe was enough for the detection of the signals in each blastomere, less than the volume (1 microL) used in the conventional FISH., Conclusion(s): The 3D-FISH technique for PGD is easy to learn, less damaging to blastomeres, and loses no blastomeres during fixation. It is efficient, feasible, and economic, which allows more patients to benefit from this technique.

Significantly more embryos survived the vitrification procedure compared to slow freezing (85.5% vs. 61.8%) in cleavage-stage human embryos produced from in vitro maturation cycles, suggesting that vitrification is more efficient than slow freezing for cryopreservation.

Objective: To assess the potential benefit of preimplantation aneuploidy testing on the outcome of in vitro fertilization (IVF) for women of advanced maternal age (AMA)., Design: Prospective randomized clinical trial., Setting: Private IVF clinic., Patient(s): Sixty-two infertile AMA couples undergoing fertility treatment., Intervention(s): Fluorescent in situ hybridization (FISH) for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22., Main Outcome Measure(s): Preimplantation aneuploidy testing of biopsied blastomeres on day 3 of development., Result(s): Fertilization and blastocyst developmental rates were similar for the test and control groups: 80% versus 77.4% and 49% versus 48.2%, respectively. The average number of embryos transferred was comparable at 2.2 for the test group and 2.7 for the control group. Implantation rates were also equivalent across the two groups: 37.3% in the control group versus 36.5% in the test group. Nevertheless, the spontaneous abortion rate was observed to be lower for the test group: 25.9% versus 32.26% in the control group. This resulted in an observed increase in delivery rates for the test group: 78% versus 67.74% in the control group., Conclusion(s): Preimplantation aneuploidy testing does not appear to statistically significantly improve outcome parameters in infertile AMA patients; however, a trend toward a decrease in the spontaneous abortion rate with a subsequent higher delivery rate was observed.

Objective: To report a simplified embryo biopsy method for preimplantation genetic diagnosis (PGD)., Design: Technique and method., Setting: A regional hospital in vitro fertilization (IVF) laboratory and private reproductive medicine clinic., Patient(s): Women undergoing IVF and PGD., Intervention(s): Blastomeres were successfully isolated from day-3 embryos at various stages., Main Outcome Measure(s): Blastomere integrity after biopsy, time of biopsy procedure, and subsequent blastocyst developmental rate., Result(s): Twenty embryos derived from abnormally fertilized oocytes (one pronucleus or three pronuclei) were used for biopsy at four-cell to 10-cell stages (day 3) by a laser zona drilling and assisted hatching micropipette delivery of culture medium inside the zona to push one blastomere out. Biopsies of all embryos using this method were successful. In two cases for PGD, fourteen 6-9-cell and four 3-4-cell stage embryos were successfully biopsied by this method. Ten out of 14 embryos from the 6-9-cell stage developed to hatching or hatched blastocysts. When two hatched blastocysts were vitrified, warmed, and cultured, both reexpanded, showing normal morphologic features., Conclusion(s): This technique is easy to learn, less damaging to the embryos, and less time consuming. It can be used for all stages of embryos without damage to either embryos or isolated blastomeres. It is an alternative method for embryo biopsy in PGD.

Objective: To develop a mouse model to investigate the possible causes for increased success rates when lysed cells are removed from thawed embryos., Design: Experimental study., Setting: Clinical IVF laboratory., Intervention(s): Assisted hatching, cell lysis, and removal of lysed cells., Main Outcome Measure(s): Embryonic growth rate and morphology., Result(s): The mouse embryos were divided into three groups; control (no cell lysis), group 1 (cell lysis and removal), and group 2 (cell lysis only). There was no significant difference in the initial number of blastomeres in each group or the number of cells lysed artificially in groups 1 and 2. The rate of embryonic development showed a significant delay in group 2 (7.97 +/- 4.92; control, 10.42 +/- 8.18; group 1, 5.74 +/-4.42; group 2). The embryo morphology on day 4 was significantly improved in group 1 and the control group when compared with group 2., Conclusion(s): Mouse embryos with artificially lysed cells after thawing had poorer developmental quality and growth rates compared with control embryos. However, removal of lysed cells restored the embryo's developmental potential to that of the control. Cell number and morphology was also significantly improved compared with embryos without lysed cell removal. These findings are consistent with human embryo development after thawing when lysed cells are present and thus mechanical lysis seems to be an appropriate method by which to further study frozen-thawed lysed cell removal.

The authors argue that a new method of deriving embryonic stem cell lines, which could be performed in conjunction with preimplantation genetic diagnosis, is unlikely to solve ethical concerns.

In patients with polycystic ovary syndrome (PCOS), the proportion of optimal embryos-blastomere number and fragmentation rate-derived from in vitro-matured oocytes after the hCG priming protocol was higher (43.3%) than the proportion of optimal embryos derived from oocyte in vitro maturation with the FSH priming protocol (11.1%). The embryos derived from in vitro-matured oocytes after the hCG priming protocol also showed a lower incidence of multinucleated blastomeres. Pregnancies were recorded only in the hCG primed patients.

Objective: To determine the extent to which embryo selection by blastulation and elective single blastocyst transfer, supported by efficient cryostorage of spare embryos, can reduce multiple pregnancies and maintain or improve on the live birth rate from IVF., Design: Prospective, nonrandomized cohort study., Setting: Sydney IVF, a private clinic in Australia., Patient(s): In vitro fertilization patients aged <38 years with three or more usable blastocyst, recruited from April 2000 through December 2001, with pregnancies followed up until August 2004., Intervention(s): Blastocysts were cultured and cryostored with stage-specific culture medium and low oxygen conditions., Main Outcome Measure(s): Fetal heart-positive twin pregnancy rate and accumulating "take-home baby" rate per retrieval leading to blastocyst transfer., Result(s): Among 121 women who elected single fresh blastocyst transfer (but who could elect to have two frozen blastocysts transferred at once), 79 (65.3%) took home a baby, with a twin pregnancy rate of 7%. Among 285 women who chose two blastocysts for fresh transfer, 184 (64.2%) took home at least one baby, with a twin pregnancy rate of 34% and five perinatal deaths., Conclusion(s): With technically appropriate blastocyst culture and freezing, and elective single blastocyst transfer in the fresh cycle, the overall multiple pregnancy rate can be reduced by >75%, permitting in this series a slight increase in the chance of taking home a baby.

Objective: Partially damaged frozen and thawed embryos are currently considered to have a lower viability than intact ones. This study was undertaken to compare the performance of intact frozen and thawed embryos with that of partially damaged embryos after removal of the necrotic blastomeres., Design: Observational clinical series., Setting: Private hospital., Patient(s): Three hundred twenty-six infertile couples undergoing frozen embryo transfer., Intervention(s): Removal of necrotic blastomeres from frozen-thawed human embryos., Main Outcome Measure(s): Pregnancy and implantations rates., Result(s): Outcomes of frozen embryo transfer cycles in which all embryos were fully intact (group 1) were compared with those in which all embryos have lost 1-2 blastomeres (group 2) or 3-4 blastomeres (group 3). Laser-assisted hatching was performed in all embryos, and necrotic blastomeres were removed from partially damaged embryos on this occasion. Only embryos that resumed mitotic activity after thawing were transferred. Comparable clinical pregnancy rates (PR) (38.7%, 39.6%, and 29.4%), delivery rates (34.4%, 34.0%, and 29.4%), and implantation rates (21.6%, 21.4%, and 17.2%) were obtained in groups 1, 2, and 3, respectively., Conclusion(s): The developmental potential of partially damaged frozen and thawed embryos can be equivalent to fully survived embryos if the necrotic blastomeres are removed from the partially damaged embryos and only those of them that show post-thaw cleavage are selected for transfer.

Objective: To test the hypothesis that pregnancy rates are low if grade Z1 pre-embryos are not available for transfer and to determine if pronuclear morphology is a better predictor of pregnancy than traditional embryo morphology., Design: Prospective clinical study., Setting: Academic human reproduction laboratory., Patient(s): One hundred couples undergoing IVF with conventional insemination or ICSI., Intervention(s): Embryo quality was assessed using both pre-embryo pronuclear morphology (zygote scoring or Z-scoring) at the time of fertilization evaluation and standard day 2 and day 3 embryo morphology (number of blastomeres and grading based on degree of fragmentation and blastomere size)., Main Outcome Measure(s): We tested two decision models, one based on Z-scores and another on morphology, to determine which grading system better predicted pregnancy outcomes in assisted reproductive technique. Zygote score and embryo morphology were measured for all embryos and the transferred embryo pool. Implantation and pregnancy rates resulting from the embryo transfers of all cycles were calculated., Result(s): The Z-score distribution of 552 embryos was 27% Z1, 8% Z2, 50% Z3, and 15% Z4. Z1 and Z3 embryos had significantly (P approximately .03) higher quality over Z2 and Z4 embryos. Using the Z-score decision model with Z1 embryos having highest priority for transfer, pregnancy rates were similar between Z1 and Z3 embryos. Using embryo morphology as a decision model, pregnancy rates were highest in transfers containing one or two "best"-quality embryos., Conclusion(s): Z1 and Z3 embryos had similar morphology and pregnancy rates. The decision model based on the Z-score model was not better than standard embryo morphology in predicting pregnancy outcome.

Objective: To quantify the mitochondrial DNA contents in human embryos with good or poor quality at the 8-cell stage., Design: Prospective study., Setting: Private infertility clinic., Patient(s): Five women aged 24 to 34 years., Intervention(s): Embryos obtained in standard superovulation and embryo culture procedures., Main Outcome Measure(s): Mitochondrial DNA (mtDNA) copy numbers in human embryos at cleavage stage were quantified by real-time polymerase chain reaction, in an effort to correlate with morphology., Result(s): The grade 8A embryos contained a mean mtDNA copy number at 1163937 (n = 8, from three patients); grade 8B embryos, at 939345 (n = 5, from two patients); grade 8C(+) embryos, at 637872 (n = 12, from 5 patients); and grade 8C(+) embryos derived from 3PN zygotes, at 300429 (n = 3, from a single patient)., Conclusion(s): Great variations were found among blastomeres from a single embryo and among embryos from a single patient. The native variations of mtDNA copy number may affect developmental ability irrespective of morphology.

Objective: To evaluate the effect of oocytes and spermatozoa on early embryonic development., Design: Retrospective study., Setting: Infertility Clinic, the Family Federation of Finland., Patient(s): Fifty-nine oocyte donation cycles with oocytes shared among 118 recipient couples., Intervention(s): Culture of all fertilized oocytes., Main Outcome Measure(s): Standard sperm (concentration, progressive motility, and morphology according to Tygerberg strict criteria) and embryo (morphology and cleavage stage) characteristics., Result(s): A marked effect of the oocyte on both embryo morphology and blastomere cleavage rate was demonstrated. In addition, a significant sperm effect on blastomere cleavage rate was found. Sperm morphology as determined according to strict criteria rather than sperm count or progressive motility was positively associated with the blastomere cleavage rate. None of the measured sperm characteristics influenced embryo morphology., Conclusion(s): Embryo morphology, i.e., fragmentation and blastomere uniformity, are predominantly determined by oocyte quality, whereas both the oocyte and spermatozoa influence the blastomere cleavage rate.

Objective: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected., Design: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH)., Setting: Clinical IVF laboratory., Patient(s): A 40-year-old IVF patient., Intervention(s): Polar body and blastomere biopsy., Main Outcome Measure(s): Detection of aneuploidy., Result(s): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued., Conclusion(s): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

Objective: To determine the predictive value of 72-hour blastomere cell number on blastocyst development and to compare success rates of subsequent transfer based on the degree of blastocyst development., Design: Retrospective clinical study., Setting: Private assisted reproductive technology center., Patient(s): Ninety-three women aged 32.0 +/- 5.1 years undergoing oocyte retrieval for IVF., Intervention(s): Bipronucleate oocytes obtained from IVF were grown for up to 168 hours after fertilization and subsequently transferred at the blastocyst stage., Main Outcome Measure(s): Percentages of embryos developing to blastocyst from 72-hour embryos by blastomere cell number and subsequent implantation and pregnancy rates of transferred blastocysts., Result(s): Rates of blastocyst formation and expansion increased as cell numbers at 72 hours increased. Implantation rates were 43% for embryos transferred to women receiving only expanded blastocysts and 17% for embryos transferred to women receiving one or more less developed blastocysts. Pregnancy rates were higher for women receiving only expanded blastocysts than for women receiving one or more less developed blastocysts, although the difference was not significant., Conclusion(s): More developed 72-hour embryos are more likely to become blastocysts and expand. Implantation rates are greater for the transfer of expanded rather than unexpanded blastocysts.

Objective: To investigate the suitability of recycling single blastomeres to assess multiple genetic variables for preimplantation genetic diagnosis., Design: Prospective randomized study., Setting: An academic medical center., Patient(s): Patients undergoing IVF-ET., Intervention(s): Blastomeres were disaggregated from donated embryos obtained from patients., Main Outcome Measure(s): Polymerase chain reaction (PCR) amplification products., Result(s): Fifty-eight blastomeres individually fixed on slides were separated into four groups. Sequential PCRs (group I, n = 30), primed in situ labeling (PRINS) before five sequential PCRs (group II, n = 10), staining with hematoxylin before performing five sequential PCRs (group III, n = 11) and preamplification of whole DNAs by degenerate oligonucleotide primer (DOP) before performing PCR were executed. The amplification efficiencies of five sequential PCRs were 100%, 100%, 96.6%, 83.3%, 56.7% for group I; 100% 100%, 100%, 80%, 40% for group II; 54.5%, 36.4%, 18.2%, 9.1% for group III; and 100%, 100%, 100%, 100%, 100% for group IV., Conclusion(s): Blastomeres fixed for PRINS can be recycled for PCR to obtain more genetic information. Hematoxylin staining appears to increase the incidence of failed amplification. Preamplification of whole genomic DNAs by DOP-PCR appears to facilitate diagnosis with high efficiency.

Objectives: To demonstrate the effectiveness of ultrarapid cryopreservation in human embryos., Design: Retrospective study., Setting: An IVF unit of a medical center., Patient(s): All cases received ultrarapid freezing (URF) of embryos and frozen embryo transfer., Intervention(s): Embryos were placed in phosphate-buffered saline (PB1) + 20% maternal serum (MS) for 5 minutes. Embryos were loaded to the straws containing PB1 + MS + 0.25 M sucrose (SUC) + 3.5 M dimethyl sulfoxide for 3 minutes, and then were stored in a liquid nitrogen tank. Embryos were thawed in a 37 degrees C water for 6 seconds and then cultured in PB1 + MS + SUC for 10 minutes. Embryos were transferred into PB1 + MS for 5 minutes and were transferred into patients., Main Outcome Measure(s): The embryo grades before and after URF, the survival and transferred embryo numbers, and the pregnancy and abortion rates were analyzed., Result(s): A total of 1,582 embryos were thawed, of which 1,273 (80.5%) embryos were transferred and 1,032 (65.2%) embryos survived with > or = 50% intact blastomere. The embryo numbers with grade I or II before and after freezing and thawing were 1,110 (70.2%) and 790 (50.0%). The mean embryo numbers per transfer was 5.0. Twenty-eight pregnancies (11.4% per transfer) were established, which included 5 miscarriages, 1 ectopic pregnancy, 4 preterm and 18 term deliveries., Conclusion(s): Ultrarapid freezing is worthy of reconsideration for embryo cryopreservation.

Objective: To describe an investigation of the feasibility of carrying out polymerase chain reaction (PCR) and fluorescent in situ hybridization on the same single human cell for sex determination., Design: To develop protocol for a clinical diagnostic test in preclinical trials., Setting: Infertile human volunteers in a clinical environment., Patients: Polyploid embryos were obtained from patients treated by IVF at the Advanced Institute of Fertility., Interventions: Seventeen blastomeres biopsied from human polyploid embryos were analyzed for sex determination by simultaneous application of PCR and fluorescent in situ hybridization., Main Outcome Measure: Presence of an X- or X and Y-chromosome band after agarose gel electrophoresis of PCR products and presence of an X- or X and Y-chromosome fluorescent probe signal after fluorescent in situ hybridization following PCR analysis., Results: By PCR, all 17 blastomeres were amplified and, by fluorescent in situ hybridization, 12 (70.6%) blastomeres produced signals that were consistent with PCR results. Two blastomeres showed only X signals, although by PCR they had both X and Y-chromosome bands., Conclusions: The sequential use of PCR and fluorescent in situ hybridization on the same blastomere can be applied to improve the accuracy of sex determination before fresh ET.

Objective: To examine the effect of GnRH analogue (GnRH-a) on the quality of frozen-thawed embryos and the pregnancy rate (PR) resulting from transfer., Design: A retrospective study of two groups of women undergoing transfers of frozen-thawed embryos. In group 1 ovulation induction with hMG was begun after hypophyseal desensitization with GnRH-a for 2 weeks. In group 2 ovulation was induced with hMG only. The freezing and thawing techniques were identical for both groups., Setting: In vitro fertilization unit at a university hospital., Patients: The study group (group 1) included 108 women who underwent 137 transfer cycles of frozen-thawed embryos, and the control group (group 2) included 44 women in 51 cycles of thawed ETs., Interventions: Ovum pick-up and ET techniques were the same for both groups. Methods of embryo freezing and thawing were identical, as were the morphological criteria for grading the embryos., Results: The morphology of embryos was similar in both groups, as was the number of embryos that had at least 50% intact blastomeres (83% +/- 23% and 78% +/- 30% for group 1 and group 2, respectively). The PR (16 of 137 [11.7%] and 6 of 51 [11.8%], respectively) as well as the abortion rate (30%) were similar for both groups., Conclusions: The use of GnRH-a does not affect the quality of embryos nor the pregnancy outcome. Because the yield of frozen embryos per ovum pick-up is higher in cycles stimulated by GnRH-a/hMG, the PR per pick-up cycle is thus anticipated to be higher.

Objective: To analyze different biopsy methods, embryo stages, and cellular masses that can be removed for preimplantation diagnosis of genetic diseases to find optimal biopsy conditions compatible with the subsequent development of the conceptus, the acquisition of intact viable blastomeres, and the reliability of the genetic analysis., Data Identification: The most important published studies have been identified through a computerized bibliographical search (MEDLINE; Dialog, Palo Alto, CA)., Study Selection: Studies reporting different embryo biopsy methods practiced at different stages have been selected., Results: The analysis carried out in the current review shows, at the present time, the following: [1] the displacement and push methods may be more suitable than the stitch and pull and aspiration (puncturing the zona pellucida) approaches at cleavages stages; [2] the aspiration and stitch and pull procedures may assure higher success rates than the herniation procedure at the blastocyst stage; [3] the mechanical division method and the use of acid Tyrode's solution would not be advisable before the eight-cell stage; [4] human embryos at the two-cell and blastocyst stages may not be suitable for preimplantation diagnosis because of an excessive reduction of cellular mass at the two-cell stage and a low or zero pregnancy rate after transfer at the blastocyst stage; and [5] biopsy of a quarter of the embryonic cellular mass on day 2 after insemination may increase biochemical pregnancies if the cleavage rate is not preserved., Conclusions: At the present time, biopsy of a quarter of the embryo on day 3 after insemination may be the most feasible approach for preimplantation diagnosis.

Objective: To determine if a culture medium, designated CZB after the authors who first described it, which is supplemented with 0.11 mM ethylenediaminetetraacetic acid, 1.0 mM glutamine, 31.30 mM lactate, and 0.27 mM pyruvate and is lacking glucose for the initial stages of culture that overcomes the in vitro two-cell block of mouse embryos, can improve the rate of blastocyst formation of human embryos in long-term cultures and increase the pregnancy rate (PR) when used in a clinical in vitro fertilization (IVF) program., Design: The study is in two parts. Initially, excess oocytes from IVF and gamete intrafallopian transfer patients were fertilized in vitro and then placed in long-term culture of either CZB plus 10% heat-inactivated human serum (32 zygotes) or Earle's balanced salt solution (EBSS) supplemented with 0.45 mM pyruvate plus 10% human serum (28 zygotes) to determine if CZB medium could enhance in vitro development and increase blastocyst formation when compared with EBSS. Subsequently, CZB or EBSS medium was used for short-term cultures of embryos in a clinical IVF program to determine if the use of CZB could increase the clinical IVF PR., Setting: Private practice of one author (M.D.)., Patients: The excess oocytes were donated by couples not wishing to have cryopreservation. In the clinical trial, 49 couples presenting with tubal or male factor infertility and who had three or more fertilized zygotes were randomly assigned to one of the culture media being used., Results: In long-term cultures, embryos were observed at 42, 66, 90, 114, and 138 hours after fertilization and scored for blastomere number, degree of fragmentation, and developmental arrest. When CZB- and EBSS-cultured embryos were evaluated over 138 hours, there was a significant increase in the number of CZB-cultured embryos reaching the blastocyst stage (56% versus 20%; P less than 0.009) and less fragmentation of CZB-cultured embryos (18.8% versus 50%; P less than 0.01). In short-term cultures, pronuclear stage embryos from patients undergoing IVF were cultured in CZB or EBSS for 24 hours, graded, and then used in embryo transfers. Of the 28 patients assigned to EBSS, 6 became pregnant (21.4%), and of the 21 assigned to CZB, 5 attained pregnancy (23.8%). These results were not significantly different., Conclusions: The use of CZB for the long-term culture of human embryos is highly beneficial and increases the rate of blastocyst formation, but its use in an IVF program does not increase the clinical PR.

Patients at low risk for producing abnormal gametes produce a wide range of embryo qualities that correlate well with PRs. These data may be useful in counseling patients and in reviewing treatment and laboratory protocols.

Delayed fertilization is common in cycles with immature oocytes. This phenomenon was observed in 42 of 423 with mature oocytes. Of the 42 cycles, 16 were excluded because of the presence of sperm autoantibodies. Sperm parameters of the remaining 26 (6.1%) cycles (group A) were compared with those in cycles with no fertilization at all (group B) and those in the control group (group C). The percentage of normal forms was 15% in group B and 24% in group A compared with 51% in group C. Fertilization rates were 32% in group A compared with 81% in group C. The incidence of poor embryonic morphology was 82% in group A compared with 29% in group C. Delayed fertilization and poor embryonic morphology associated with impaired sperm quality is of clinical and prognostic importance.

In order to determine optimal culture conditions for embryos, human fetal cord serum (HCS) small- and large-molecular-weight fractions of HCS, and human serum albumin were employed as media supplements. In the first phase, embryos were cultured with the various protein supplements for 96 hours and observed morphologically every 24 hours. In the second phase, embryos were cultured with the various protein supplements and 5-bromo-2-deoxyuridine for 29 hours. Blastomere number, sister chromatid exchange (SCE), micronuclei, and chromosomal aberrations were observed. Morphologically, any protein supplement was better than no media supplement. Whole HCS and large-molecular-weight fraction provided the best conditions for embryo growth. Blastomere number was increased with whole serum when compared with no supplementation, small-molecular-weight fraction, or human serum albumin. In the analysis of SCE, all supplements had a lower SCE number than no serum supplement, and HCS supplement demonstrated a significantly lower SCE number than the other supplements. No difference in micronuclei or chromosomal aberration was observed in any of the supplements. In conclusion, whole HCS provides optimal conditions for culturing embryos, compared with the various serum fractions, and the beneficial components are primarily found in the large-molecular-weight fraction.

The effect of polyploidy on the early development of human embryos is unknown. This study compares the early development of 90 polyploid and 275 diploid human embryos conceived in vitro. Between May 1983 and January 1986, 3081 oocytes were recovered during 631 cycles of laparoscopy for in vitro fertilization (4.9 oocytes/cycle); 1924 oocytes (62.4%) fertilized. There were 90 oocytes with more than two pronuclei (4.7% of fertilized oocytes), identified in 72 cycles (11.4% of cycles). In these cycles, the proportion of diploid oocytes (n = 275) that cleaved (cleavage rate) (92.7%) was significantly greater than the proportion of polyploid oocytes (n = 90) that cleaved (65.5%) (P less than 0.001). The cleavage rate for all diploid oocytes (n = 1834) was 90.4%. There was no significant difference in the stage of development (number of blastomeres; mean +/- standard deviation [SD]) on the day of embryo transfer between diploid (4.3 +/- 2.1) and polyploid (4.1 +/- 2.1) embryos that cleaved, but a plot of the frequency distribution of cleavage stages revealed that significantly more polyploid than diploid embryos had an uneven number of blastomeres at that time (33% versus 8%, respectively; P less than 0.001). Polyploidy confers an immediate developmental disadvantage; one third of polyploid embryos fail to cleave, and those that do divide demonstrate more asynchronous divisions.

We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.3% and 64.3% of biopsied and control embryos, respectively, implanted into the uterine wall, and 52.6% and 52.4% of biopsied and control embryos, respectively, developed into fetuses. In a separate series of experiments, we have demonstrated that biopsied mouse embryos can be successfully cryopreserved by ultrarapid freezing even though they have a punctured zona pellucida. Biopsied (frozen-thawed), control (frozen-thawed), and nonfrozen embryos had an implantation rate of 81.1%, 74.3%, and 74.1%, respectively, and a fetal formation rate of 62.2%, 62.9%, and 66.7%, respectively.

Little research has been done on the in vitro and xenogenous fertilization of cryopreserved primate oocytes. This study reports the development of freezing and thawing methods for squirrel monkey oocytes with subsequent successful fertilization by these two methods. Preliminary results on techniques for blastomere separation using the hamster and squirrel monkey as models are also given. These studies have important implications relative to the long-term frozen storage of human oocytes, their subsequent thawing, in vitro fertilization and embryo transfer, and the use of the blastomere separation procedure, in conjunction with in vitro fertilization, in the diagnosis of embryonic normality and possible congenital defects prior to implantation.

An important factor influencing the pregnancy rate after in vitro fertilization-embryo transfer (IVF-ET) appears to be the number of embryos transferred to the uterus. In this study, the influence of oocyte maturity and embryo quality on pregnancy rate was assessed in patients undergoing IVF-ET. Ovarian hyperstimulation was performed by human menopausal gonadotropin (hMG [n = 29]), clomiphene citrate (CC)/hMG (n = 81), and hMG/follicle-stimulating hormone (FSH [n = 13]) protocols. Oocyte maturity was graded on a scale from 1 to 5 based on the morphology of the ooplasm, cumulus mass, corona radiata, and membrana granulosa cells. Embryos were graded according to the symmetry of the blastomeres and the presence or absence of fragmentation. Mature preovulatory oocytes yielded the highest fertilization rates. No differences were found among the protocols in terms of fertilization rate, embryo quality, or pregnancy rate. When all protocols were combined, patients who conceived had a significantly higher number of embryos transferred than those who did not conceive (3.6 +/- 0.1 [mean = SEM] versus 2.7 +/- 0.1). When embryo quality was compared, there was no difference in the number of "B" embryos transferred between patients who conceived and those who did not (1.2 +/- 0.2 versus 1.2 +/- 0.1), but the patients who conceived had significantly more "A" embryos transferred (1.6 +/- 0.3 versus 0.8 +/- 0.1). These data suggest that the treatment protocol did not determine embryo quality. Furthermore, the increase in pregnancy rates seen with an increase in embryos transferred is the result of the transfer of more "A" embryos.

A seven-cell human egg recovered from the proximal middle quarter of the oviduct is described. Whether or not it is a normal representative of this stage of human development cannot be established at the present time. The specimen was recovered 83 hours after intercourse and 77 hours after the first significant elevation of the luteinizing hormone level in the urine. According to these data and the results of the endometrial and corpus luteum biopsies, the age of the egg was estimated to be approximately 72 hours. An analysis of size and the reaction of the blastomeres to toluidine blue suggests that they already show some differentiation at this early stage of development. The addition of these findings to previous reports of eggs recovered from human oviducts and uteri gives support to the concept that human eggs are delivered to the endometrial cavity when they contain between 7 and 12 blastomeres.

A total of 100 mature oocytes from 13 consecutive patients were randomly assigned from each patient to one of two treatment groups (n = 53 for group 1, n = 47 for group 2). Group 1 oocytes were incubated throughout the culture periods in medium supplemented with 7.5% homologous patient serum. Group 2 oocytes were treated similarly, except the serum supplement was of fetal cord origin. End points for examination included fertilization frequency, normality of fertilization, stage of embryonic development at two time periods, and quality of embryonic development at two time periods. None of the end points examined revealed significant differences between patient serum and fetal cord serum.