Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. Methods: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. Results: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. Conclusions: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.     

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.     

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome-and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. Conclusions: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

采用第一极体进行植入前诊断的实验研究

目的 :建立采用第一极体植入前染色体非整倍体诊断的方法。方法 :取试管婴儿后的未受精卵细胞 ,激光打孔法行第一极体活检 ,固定后行多色荧光原位杂交 (FISH) ,分析极体中 13,16 ,18,2 1和 2 2号染色体的核型情况。结果 :活检成功率为 94 .7% ,FISH成功率为 86 .7% ,6 1.5 %的极体核型正常 ,38.5 %的极体为非整倍体。结论 :激光打孔结合FISH法是一种快速有效的极体植入前诊断方法     

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal

Purpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes. Methods: Oocytes that were at metaphase II about17–20 hr after insemination or intracytoplasmic sperm injection(ICSI) were treated with pronase to remove the zonapellucida and polar body (PB) and then spread on slides usingHCl and Tween 20. Two rounds of FISH were performedusing direct-labeled probes: chromosomes 1, 13, 21 (round1); chromosomes X, 7, 18 (round 2). Results: Of the 63 oocytes from 18 patients (mean age,32 years), 48 (76%) had one DNA complement as expected, 9(14%) had 2 DNA complements, 3 (5%) gave incomplete FISHsignals, and 3 (5%) were not analyzable. Of the 48 oocyteswith one set of DNA, 48% were haploid, 44% were aneuploidfor one or more chromosomes, and 8% were polyploid. Wealso found an increased frequency of predivision of chromatidbivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneousassessment of six chromosomes in human oocytes, and thereforecan be useful for accurately determining the incidence andcauses of genetic imbalances in human oocytes andapparently low fertilization rates.     

Cytogenetics of uncleaved oocytes and arrested zygotes in IVF programs

Purpose: Cytogenetic studies of arrested oocytes and zygotes were used to understand in vitro fertilization (IVF) failures. Methods: We investigated the cytogenetics (Giemsa banding and FISH) of 710 uncleaved oocytes and 94 arrested zygotes from 208 patients undergoing IVF procedures. Results and Conclusions: Of uncleaved oocytes without a polar body, 39% were judged cytogenetically abnormal (17% unbalanced predivision and 21.5% diploid). Of 575 oocytes with a polar body, 124 (21.5%) showed numerical or structural chromosome aberrations. In arrested zygotes, approximately equal cases were found with separate condensed haploid complements (no syngamy), nuclear asynchrony and pulverized DNA, and apparently cytogenetically normal zygotes arrested at mitosis. These data on chromosome abnormalities were also analyzed with respect to two ovarian stimulation protocols and to maternal age. Both ovarian stimulation protocols showed the same levels of chromosome abnormalities. Overall chromosome abnormalities and premature chromosome condensation were also unchanged with maternal age. These data illustrate the significance of chromosome aberrations in IVF failures.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes--implications for the use of primate parthenotes for stem cell production

Purpose: Spontaneous parthenogenetically activated noninseminated oocytes and failed fertilized oocytes after ART activated by puromycin were studied to assess cleavage ability and the cytogenetic constitution of the resulting embryos. Methods: Failed fertilized oocytes were exposed to puromycin, and whenever activation occurred, they were further cultured until arrest of development. FISH was used to assess the ploidy of spontaneous (group A) and induced parthenotes (group B). Results: The mean number of oocytes exposed to puromycin and the percentage and type of activation were identical in IVF and ICSI patients. The more frequent types of activation were one or two pronuclei and one polar body suggesting that retention of the second polar body is a common event after parthenogenetic activation. Conclusions: Retention of the second polar body and chromosome malsegregation were observed after parthenogenetic activation, either spontaneous or induced by puromycin. This means that using parthenogenetic embryos for stem cell research will require great care and attention.     

Use of PRINS for preconception screening of polar bodies for common aneuploidies

It has been shown that preconceptional screening for oocyte aneuploidies could help increase the pregnancy rate after in vitro fertilization (IVF), particularly in cases of advanced maternal age. The FISH (fluorescent in situ hybridization) technique is usually used to examine the first polar body (I-PB) for oocyte screening and so avoid fertilizing and transferring embryos from aneuploid oocytes. We have tested the feasibility of using another technique, the primed in situ (PRINS) reaction for this purpose. PRINS is a rapid, inexpensive method of labelling chromosomes. Chromosomes were labelled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labelled nucleotides using Taq DNA polymerase. A total of 183 PRINS reactions were performed with primers for chromosomes 13, 16, 18, 21 or X on 63 I-PBs removed from oocytes that failed to become fertilized during IVF. Each I-PB underwent three successive double-labelling reactions and intense signals were obtained in less than 40 min. Our data suggest that PRINS may be a useful alternative or a complement to FISH for detecting the main aneuploidies in all oocytes obtained after follicular puncture.     

Polar body biopsy and aneuploidy testing by simultaneous detection of six chromosomes

OBJECTIVES: To simultaneously detect six chromosomes in a single round of fluorescence in situ hybridization (FISH) during polar body diagnosis and aneuploidy testing in human in vitro fertilization (IVF) treatment. METHODS: A commercially available five-color FISH probe was modified by an additional chromosome probe. This kit was first tested on lymphocyte spreads and then used for polar body diagnosis (PBD) in patients with advanced maternal age and repeated implantation failure. The outcome of IVF treatment was compared with a control group. RESULTS: All six chromosomes could be simultaneously detected and easily distinguished by FISH analysis. PBD and aneuploidy testing were performed in 75 treatment cycles and compared with 126 controls. The biochemical pregnancy rate was significantly higher in the PBD group (37.1% vs 22.9%, p < 0.05) and a trend was observed for higher clinical pregnancy and implantation rates (24.22% and 14.4% vs 18.62% and 10.8%, respectively) and lower abortion rates (20% vs 31.8%) following PBD. CONCLUSIONS: The simultaneous detection of six chromosomes in a single FISH round is possible and can be applied to PBD. This approach may present another step towards increasing the number of chromosomes for aneuploidy testing.     

Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies

Most chromosomal abnormalities originate from female meiosis and contribute significantly to pregnancy failures, particularly in women of advanced maternal age. A total of 8,382 oocytes were obtained in 1,297 IVF cycles from patients of advanced maternal age (mean 38.5 years). Following a standard IVF protocol, oocytes were tested following removal and fluorescence in-situ hybridization (FISH) analysis of the first (PB1) and second polar bodies (PB2), using probes specific for chromosomes 13, 16, 18, 21 and 22 (Vysis). FISH results were available in 67,33 (80.3%) oocytes tested, 3,509 (52.1%) of which were aneuploid, with the remaining 3,224 (47.9%) normal oocytes available for transfer. In all, 41.7% of oocytes had meiosis I errors, compared to 35.1% with meiosis II errors. Abnormalities in meiosis I were represented by extra chromatids in 15.4%, missing chromatids in 48.1%, missing chromosomes in 5.9%, extra chromosomes in 0.5%, and complex abnormalities in 30.1%. The proportions of abnormal oocytes with missing or extra chromatids in meiosis II were 36.6 and 41.2% respectively, with the remaining oocytes having complex abnormalities, involving missing or extra chromatids of different chromosomes (22.1%) following meiosis II. Overall, 41.8% oocytes had meiosis I, 30.7% meiosis II, and 27.6% both meiotic division errors. A total of 45.1% of the abnormal oocytes had complex errors, involving the same chromosome in both meiotic divisions (21.5%), or different chromosomes (78.5%), of which 74.8% were with abnormalities of two, and 25.2% with abnormalities of three chromosomes studied. Of 3,224 detected aneuploidy-free zygotes, 2,587 were transferred in 1,100 treatment cycles (2.35 embryos per transfer), resulting in 241 (21.9%) clinical pregnancies and 176 healthy children born, suggesting a positive clinical outcome following aneuploidy testing of oocytes in a group of IVF patients of average age 38.5 years.     

Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection

OBJECTIVE: To verify whether a correlation exists between pronuclear zygote morphology and the chromosomal condition of preimplantation embryos. DESIGN: Prospective analysis of pronuclear zygote morphology and preimplantation genetic diagnosis (PGD) for aneuploidy of the resulting embryos. SETTING: Reproductive medicine unit, day surgery clinic. PATIENT(S): Seventy-seven patients undergoing 107 PGD cycles because of advanced maternal age (77 cycles) or previous IVF failures (30 cycles). INTERVENTION(S):Evaluation of pronuclear zygote morphology and chromosomal condition of the resulting embryos. MAIN OUTCOME MEASURE(S): Rate of embryo development, proportion of euploid embryos, and distribution of chromosomal abnormalities.The position of pronuclei within the ooplasm, the size and distribution of nucleoli, and the orientation of polar bodies with respect to pronuclei were highly predictive for the presence of complex chromosomal abnormalities in the developing embryos; zygotes with juxtaposed pronuclei, large-size nucleoli, and polar bodies with small angles subtended by pronuclei and polar bodies were the configurations associated with the highest rates of euploidy. CONCLUSION(S): The combination of the patterns related to pronuclear zygote morphology indicated four configurations where the proportion of chromosomally normal embryos was significantly higher compared with the other configurations, suggesting the validity of this scoring system for the selection of embryos generated by PGD patients.     

Accuracy of Preimplantation Diagnosis of Single-Gene Disorders by Polar Body Analysis of Oocytes

Purpose: A number of pitfalls in single-cell DNA analysis, including undetected DNA contamination, undetected allele drop out, and preferential amplification, may lead to misdiagnosis in preimplantation genetic diagnosis of single-gene disorders. Methods: Preimplantation genetic diagnosis was performed by sequential first and second polar body analysis of oocytes in 26 couples at risk for having children with various single-gene disorders. Mutant genes were amplified simultaneously with linked polymorphic markers, and only embryos resulting from the mutation-free oocytes predicted by polar body analysis with confirmation by polymorphic marker testing were transferred back to patients. Results: Overall 529 oocytes from 48 clinical cycles (26 patients) were tested, resulting in the transfer of 106 embryos in 44 clinical cycles. As many as 46 (9.6%) instances of allele dropout were observed, the majority (96%) of which were detected. Seventeen unaffected pregnancies were established, of which nine resulted in the birth of an unaffected child, and the rest are ongoing. Conclusions: A high accuracy of preimplantation genetic diagnosis of single-gene disorders is achieved by application of sequential analysis of the first and second polar body and multiplex polymerase chain reaction.     

Optimized criteria for using fluorescence in situ hybridization in the prenatal diagnosis of common aneuploidies

OBJECTIVE: To evaluate the medical and economic performance of three strategies for selecting patients eligible for interphase FISH in the prenatal diagnosis of common aneuploidies. METHODS: We evaluated three protocols on the same population that was referred for prenatal diagnosis between June 2001 and December 2006. The number of aneuploidies detected by FISH and the relative cost (reagent and technical staff cost) are reported for each strategy. RESULTS: 2707 women were referred for prenatal diagnosis either because of advanced maternal age over 38 (48%), abnormal maternal serum screening (35%) or prenatal ultrasound anomalies (17%). A total of 4.8% chromosomal anomalies (balanced and unbalanced) were diagnosed after karyotyping. Theoretically, interphase FISH should have detected 79.4% of the unbalanced anomalies. We observed a significant improvement in the trisomy 21 detection by selecting the probes according to the reason for referral. The last protocol adopted, which offers a rapid test to 57% of women undergoing amniocentesis, presents the best aneuploidy detection rate (68% of total aneuploidies, 87% of trisomy 21). CONCLUSION: Selecting probes according to medical criteria patients combined with a technical procedure modification allows medico-economic improvement of interphase FISH in routine diagnosis.     

Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential first and second polar body analysis

OBJECTIVE: The purpose of this study was to determine the accuracy and feasibility of sequential polar body removal and analysis for preimplantation genetic diagnosis of mendelian disorders. STUDY DESIGN: Three couples with risk factors for cystic fibrosis had preimplantation genetic diagnosis with the use of sequential polar body analysis. After stimulation, oocytes were harvested and the first polar bodies were removed and analyzed on the day of aspiration. The following day, after fertilization, the second polar bodies were aspirated. Only embryos known to have inherited the normal maternal allele were transferred. RESULTS: All three couples had successful pregnancies resulting in the births of unaffected infants. CONCLUSIONS: Preimplantation diagnosis with the use of sequential polar body removal is feasible and can prevent the establishment of genetically abnormal pregnancies for couples at risk. (Am J Obstet Gynecol 1998;178:1298-306.)     

Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients.

OBJECTIVES: To assess the rate of chromosome aneuploidy (e.g., extra or missing chromosomes) in oocytes remaining unfertilized in our in vitro fertilization (IVF) program. To determine whether two parameters of the IVF technique, advanced maternal age and hormonal follicle stimulation, affect this rate. DESIGN: Data on oocyte retrieval, fertilization, and aneuploidy rates are analyzed to test for possible relations with maternal age and two hormonal stimulation regimens. SETTING: Patients of our IVF program from 119 stimulated cycles over 8 months. PATIENTS, PARTICIPANTS: In vitro fertilization patients selected for having oocytes (1 to 18) remaining unfertilized after insemination in vitro. RESULTS: Advanced maternal age decreases both the number of retrieved oocytes and the fertilization rate, but hormonal treatments have no effect. Aneuploidy (rate 27%), involving group G most frequently, appears associated with advanced age. Patients who were previously parous produced significantly reduced numbers of aneuploid oocytes compared with the nonparous group. A significant excess (P = 0.01) of patients had multiple oocytes all alike (all haploid or all aneuploid), showing correlation among multiple oocytes of a patient in chromosome status. CONCLUSIONS: Maternal age affects reproductive performance and is related to specific chromosomal aneuploidy. Women who were previously parous are more likely to produce normal oocytes than nonparous women; oocyte normality therefore may improve the chance for a future successive pregnancy. Nonrandomness in chromosome abnormality of some patients' multiple oocytes is evidence for maternal predisposition to meiotic nondisjunction. Consequently, these patients are at risk for failed IVF cycles.     

Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies

OBJECTIVE: To present and evaluate the use of a new ultra-fast multicolor primed in situ (PRINS) procedure for karyotyping human oocytes and first polar bodies. DESIGN: In situ chromosomal identification on isolated cells, using combinations of specific primers for chromosomes 1, 7, 9, 16, and 18 and fluorescent nucleotides. SETTING: Sixteen unfertilized oocytes were obtained from women participating in an IVF program. PATIENT(S): Five patients undergoing an IVF-ET. INTERVENTION(S): In vitro unfertilized oocytes were fixed on slides, and sequential PRINS reactions were performed on each preparation. MAIN OUTCOME MEASURE(S): Ultrarapid in situ identification of three or four chromosomes on oocyte and polar body chromosome spreads. RESULT(S): On the basis of the direct in situ mixing of the colors of fluorochromes (FITC, TRITC, Cascade Blue) that were incorporated in sequential PRINS reactions, this method allows rapid and efficient labeling of three or four individual chromosomes. Each PRINS reaction consists of a unique 4- to 6-minute step for both in situ annealing and elongation. The procedure can be combined with fluorescence in situ hybridization (FISH) reactions. CONCLUSION(S): By simplifying the multicolor PRINS procedure, this new protocol should facilitate the use and adaptation of PRINS to chromosome screening. This approach could be used in parallel or in combination with FISH for efficient aneuploidy assessment on isolated cells.     

Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review

Embryonic aneuploidies may be responsible for pregnancy failure in many IVF patients. In recent years, fluorescent in situ hybridisation (FISH) for multiple chromosomes has been used to document a high frequency of chromosomal errors and aneuploidy in human preimplantation embryos and, after embryo biopsy, to select embryos that are more likely to implant. Such studies suggest that women with recurrent miscarriage and advanced maternal age may benefit most from preimplantation genetic diagnosis with aneuploidy screening (PGD-AS). The success of PGD-AS is likely to be enhanced by new technologies, such as comparative genomic hybridisation, which enable full karyotyping of single cells.     

Chromosome 21 Detection in Human Oocyte Fluorescence In Situ Hybridization: Possible Effect of Maternal Age

Purpose: The purpose of this study was to evaluate, among 100 uncleaved oocytes, the incidence of numerical and structural chromosome 21 and X abnormalities and to analyze the influence of various factors, such as in vitro (IVF) indications, follicle stimulation protocols, and women's age. Methods: We investigated 150 uncleaved oocytes from 128 patients after an IVF attempt. After cytogenetic analysis (Giemsa) 100 oocytes (66%) were selected for fluorescence in situ hybridization (FISH). Fluorescent probes for human chromosomes X and 21 were used simultaneously according to standard procedures for their hybridization and detection. Results and Conclusions: We analyzed by the FISH protocol 100 metaphase II oocytes with 22 to 25 chromosomes. Our results demonstrate a high rate of disomy for chromosome 21 in human oocytes. Among them, eight were disomic (8%) and three were nullosomic (3%) for chromosome 21. Only one disomy of chromosome X was noted. The various indications of IVF and the different folliculogenesis stimulating protocols did not seem to influence the results but suggested a correlation between the maternal age and the aneuploidy rate of chromosome 21.     

Second polar body extrusion is highly predictive for oocyte fertilization as soon as 3 hr after intracytoplasmic sperm injection (ICSI)

Objective Our objective was to evaluate the time course and the predictive value of the extrusion of the second polar body after intracytoplasmic injection (ICSI) related to the fertilization rate, embryo cleavage and quality.Setting The setting was the in vitro fertilization program of a university hospital.Patients Twenty-one patients were treated with intracytoplasmic single sperm injection either for fertilization failure in IVF, low fertilization in IVF (<5%), or severe male factors.Design One hundred thirty-five of 205 metaphase 2 oocytes treated with intracytoplasmic single sperm injection were observed 1, 2, and 3 hr after the assisted fertilization procedure. Extrusion of the second polar body was recorded. For each of these oocytes, fertilization was noted 18 hr after ICSI and cleavage and embryo quality were assessed 24 hr later. The 70 remaining oocytes were used to assess a possible negative effect of repeated exposure to light microscopy.Results The extrusion of the second polar body 3 hr after injection was an observation with a sensitivity of 0.87, a specificity of 0.58, and a high positive predictive value (0.90) toward oocyte fertilization. Twenty-nine and four-tenths percent of the oocytes extruded a second polar body within the first hour, 56.6% within the first 2 hr, and 78.3% had a second polar body 3 hr after injection. This time course was related neither to the speed of embryo cleavage nor to the embryo quality. Fertilization, cleavage, and embryo quality were not affected by repeated observation as deduced from comparison with the control group and confirmed by a high pregnancy (62% per oocyte retrieval) and implantation rate (22% per replaced embryo).Conclusion Oocytes can be checked, in all safety, 3 hr after a single sperm injection for the presence of a second polar to predict oocyte fertilization with a high certainty.