Faculty Bibliography

OBJECTIVE: Our purpose was to determine the feasibility of ascertaining aneuploidy for chromosomes X, Y, 18, and 16 by use of multiple-probe fluorescence in situ hybridization in blastomeres from preimplantation human embryos. STUDY DESIGN: A short fluorescence in situ hybridization procedure involving the simultaneous use of four deoxyribonucleic acid probes detected with red, green, blue, or a mixture of red and green fluorochromes was developed to determine numeric abnormalities of chromosomes X, Y, 18, and 16. Embryos underwent biopsy, and all or most cells were analyzed to distinguish true aneuploidy from mosaicism and to assess technique variations within the same embryo (n = 64). RESULTS: The analysis of all the blastomeres of an embryo was achieved in 91% of the embryos. Successful analyses including biopsy, fixation, and fluorescence in situ hybridization were achieved in 87.8% of the blastomeres. Of the four chromosomes tested, numeric aberrations were found in 23% and 42% of normally and abnormally developing embryos, respectively, including aneuploidy, polyploidy, haploidy, and mosaicism. When diploid embryos containing one or several tetraploid cells are counted as chromosomally abnormal, then 49% and 61% of normally and abnormally developing embryos, respectively, were chromosomally abnormal. Aneuploid embryos consisted of two monosomies for chromosome 16, one for chromosome 18, and a trisomy for chromosome 16. There was a tendency for aneuploidy to increase with maternal age. CONCLUSIONS: Fluorescence in situ hybridization is a more efficient method than cytogenetic analysis to study specific aneuploidies at preimplantation stages of development in human embryos. In addition, the preimplantation genetic diagnosis of two blastomeres per eight-cell embryo may be sufficient to ensure successful analysis of polyploidy, haploidy, and specific aneuploidies without endangering the survival of the embryo. The technique can be easily modified to consider other chromosomes, including 13 and 21. Because most chromosomally abnormal embryos do not develop to term, the use of this technique may increase the delivery rate per embryo by allowing only transfer of embryos normal for the tested chromosomes. This technique would be most useful for older women undergoing in vitro fertilization, because aneuploidy appears to increase with advancing maternal age

A large proportion of patients undergoing in-vitro fertilization (IVF) are aged > or = 35 years. It has been estimated that in this age group, 50% of embryos are chromosomally abnormal, with aneuploidy being the major contributing factor. Since the origin of most aneuploidies is maternal meiosis I non-disjunction, unfertilized oocytes could be safely screened for aneuploidy by analysing their first polar bodies. To determine the feasibility of first polar body aneuploidy analysis, polar bodies were analyzed by fluorescence in-situ hybridization (FISH) using probes simultaneously for chromosomes X, Y, 18, 13/21 or X, Y, 18 and 16. Within 6 h of retrieval, 88% showed a normal segregation involving a single chromosome of each kind, with double-dotted hybridization signals, corresponding to dyads (chromosomes in metaphase I composed of two chromatids). The rest showed non-disjunction of full dyads (6%), or an unbalanced pre-division of dyads (6%), which gives a segregation of one chromatid or one dyad and a chromatid with the first polar body. But only 34% of polar bodies analysed 24 h after retrieval or later showed a normal segregation, with most of the other polar bodies showing balanced pre-division, with two separated hybridization signals for all the chromosomes analysed. The rates of non-disjunction and unbalanced pre-division after > or = 24 h in culture were similar to the rates in fresh oocytes. When both types of aneuploidy were considered together, an increase of aneuploidy with maternal age was detected, which although slight, was significant (P = 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Patients who have a unicornuate uterus with a noncommunicating rudimentary horn that contains an endometrial cavity are at risk for endometriosis and obstetric complications. As in this case, resection of the rudimentary horn can be performed laparoscopically without increased risk to the patient and with some potential benefit

OBJECTIVE: Chlamydia trachomatis infections of the female genital tract, although a major cause of infertility, are often asymptomatic and undetected. Since many infertile women now seek in vitro fertilization, a procedure whereby fertilization and embryo implantation are precisely timed, we sought to determine the relation between an unsuspected C. trachomatis infection and the ability of embryos to implant and develop after their transfer to the uterus. STUDY DESIGN: At the time of oocyte aspiration, endocervical samples were obtained from 216 women and assayed by enzyme-linked immunoassay for immunoglobulin A antibodies to C. trachomatis structural membrane components and to recombinant C. trachomatis heat shock protein. The presence of C. trachomatis in the cervices was assessed by the polymerase chain reaction. The outcome of each in vitro fertilization cycle was then ascertained. RESULTS: Oocytes from 198 (91.7%) of the women were fertilized in vitro and subsequently transferred to the uterus. Term deliveries of healthy infants occurred after 68 (34.3%) of these transfers. Cervical immunoglobulin A antibodies to chlamydial heat shock protein were detected in 5 (7.3%) of the women with term births, and 1 (1.5%) also had immunoglobulin A antibody to chlamydial structural components; 3 (4.4%) were positive by the polymerase chain reaction for C. trachomatis. In contrast, among the 130 women whose embryo transfers did not result in an ongoing pregnancy, 36 (27.7%) had cervical antiheat shock protein immunoglobulin A (p = 0.0007) and 24 (18.5%) had antichlamydial structural component immunoglobulin A (p = 0.0002); 15 (11.5%) of these women had positive results of polymerase chain reaction for C. trachomatis. The majority of women with cervical antibodies to chlamydial structural antigens were also positive for antibody to heat shock protein. However, only 35% of the women with antibodies to heat shock protein were also positive for the other chlamydial antibodies. C. trachomatis was detected by polymerase chain reaction in 29.2% of women with anti-C. trachomatis antibodies and 7.8% of women with anti-heat shock protein antibodies. Women positive for antichlamydial immunoglobulin A were more likely to be undergoing a repeat in vitro fertilization cycle than were women who were antibody negative (p = 0.007). CONCLUSION: Unsuspected C. trachomatis infection or reactivation of an immune response to the C. trachomatis heat shock protein may induce an inflammatory reaction in the uterus that impairs embryo implantation and/or facilitates immune rejection after uterine transfer of in vitro fertilized embryos

Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.

Preimplantation genetic diagnosis was performed in 122 embryos obtained by IVF from 11 patients carriers of haemophilia, Duchenne's muscular dystrophy, Barth's syndrome, cystic fibrosis, Pelizaeus-Merzbacher syndrome or Rett's syndrome. After multiplex polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) analysis with multiple probes, 28 embryos diagnosed as not affected were replaced. Of these, eight implanted (28%) and produced three ongoing pregnancies, three deliveries of four babies and a biochemical pregnancy. However, one case screened for cystic fibrosis was misdiagnosed and the pregnancy was terminated. In order to evaluate the efficiency of multiplex PCR, 55 non-replaced embryos were reassessed by PCR or by FISH. Identical results were obtained in all cases. However, one embryo which had only X-chromosome specific amplification by PCR was found to be XO in all its cells by FISH. Although multiplex PCR is demonstrated to be reliable for sexing of human embryos, FISH has the additional advantages of supplying ploidy assessment while not being affected by contamination

OBJECTIVE: Our objective was to analyze the risk factors, stimulation characteristics, and future fecundity of patients with ectopic pregnancies after in vitro fertilization (IVF). METHODS: We retrospectively evaluated all cases of ectopic pregnancy occurring between January 1989 and March 1993 (Cornell series 1 to 17). A case-control group of intrauterine pregnancies was used for comparison of the stimulation and transfer characteristics. RESULTS: Twenty-seven of 1123 pregnancies (2.4%) were ectopic, following 2812 fresh IVF embryo transfers, while 8 of 105 pregnancies (7.6%) were ectopic, following 405 frozen-thawed embryo transfers. Tubal factor was the cause of infertility in the majority (85.7%) of ectopic pregnancies. No difference was found between the ectopics and the matched controls in stimulation and transfer characteristics. Thirty ectopic pregnancies were ampullary, two were interstitial, two were cervical, and one was heterotopic. Twenty of the patients subsequently underwent 29 IVF attempts, with a pregnancy rate of 41.4% per transfer. CONCLUSIONS: Ectopic pregnancy after IVF appears to be related to preexisting tubal pathology; embryo transfer of cryopreserved thawed embryos in a natural cycle may result in a higher ectopic rate in these patients; in subsequent IVF cycles the intrauterine pregnancy rate of these patients is not decreased

The purpose of this investigation was to determine the parental origin of the pronucleus furthest from the second polar body (the distal pronucleus) in dispermic human zygotes. Intact dispermic embryos (n = 53) and those from which the distal pronucleus (n = 50) was removed at the zygote stage were biopsied after cleavage. Blastomeres were sexed using either coamplification of X and Y probes using a duplex polymerase chain reaction (PCR), or simultaneous fluorescence in situ hybridisation (FISH) with directly fluorochrome-labelled probes for chromosomes X, Y and 18. The ratio X/Y was determined in both groups of embryos by assessing a minimum of two blastomeres. If the pronuclei in dispermic zygotes are topographically in a fixed position, the X/Y ratio should change from 1:3 in dispermic embryos to 1:1 in enucleated ones. The ratio of embryos containing only an X chromosome and those with X as well as Y chromosomes in the intact dispermic zygotes was 1.0:2.3 which is similar to the theoretical ratio of 1:3. This ratio was 1.0:1.3 in dispermic zygotes from which the distal pronuclei were removed. This ratio is not significantly different from the 1:1 ratio based on a statistical analysis with a sample size of 50. These sex ratios would have been considered different if more than 200 enucleations had been performed. Although the ratio X/Y was altered following removal of distal pronuclei, suggesting frequent targeting of male pronuclei, accidental removal of the female pronucleus could not be excluded. This indicates that enucleation of dispermic zygotes could produce high yields of gynogenetic and androgenetic embryos for research purposes. Clinical application aimed at producing biparental zygotes may be hazardous, since mosaicism was common among enucleated embryos

OBJECTIVE: To use fluorescence in situ hybridization to corroborate the polymerase chain reaction (PCR) preimplantation diagnosis of human embryos in three couples carrying a chromosome X-linked disease. SETTING: Clinical and research IVF laboratories. PATIENTS: Individuals undergoing preimplantation diagnosis. RESULTS: Four ETs were performed in couples undergoing preimplantation diagnosis by multiplex PCR or fluorescence in situ hybridization, resulting in the birth of two normal female twins. The result of another is pending. A total of 22 embryos were analyzed by PCR. Embryos that were diagnosed as being at risk of carrying the genetic abnormality (n = 8), embryos that failed diagnosis (n = 4), and genetically normal embryos that arrested development (n = 4) were further analyzed by fluorescence in situ hybridization. The sex of all 16 embryos was determined and confirmed the previous 12 preimplantation diagnoses by multiplex PCR. In addition, fluorescence in situ hybridization analysis allowed the detection of two aneuploid embryos, one XO and one XXY, previously diagnosed by PCR as a normal female and male. Two mosaics were also detected. CONCLUSION: Polymerase chain reaction and fluorescence in situ hybridization are possible for preimplantation sex determination in cases of genetic sex-linked disease. Fluorescence in situ hybridization, however, supplies additional information about sex chromosome aneuploidy and is not susceptible to contamination or misdiagnosis of monosomy X

A new technology called primer extension preamplification (PEP), which has been applied to single spermatozoa, increases the amount of polymerase chain reaction (PCR) templates by amplifying DNA of the whole genome. The current investigation was aimed at applying PEP to single human blastomeres. Two blastomeres with nuclei from arrested embryos were selected for this study. Using three different PEP protocols (experiments I, II and III), DNA from single blastomeres was amplified using 15-base oligonucleotide random primers. The efficiency of the procedure was determined by further amplifications of aliquots of the PEP products with two specific sequences. Three aliquots from each PEP product were used as PCR templates for the human X chromosome (X) or the exon 10 of the cystic fibrosis gene (CF). PCR amplified products were analysed by gel electrophoresis. In experiment I, when X primers were used, positive signals were detected in all 10 embryos (100%), 90.0% (18/20) of the blastomeres, and in 80.0% (96/120) of the replicates. When CF primers were amplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeres and 78.3% (47/60) of the replicates were positive. In experiment II, efficiency was significantly reduced when total time for the procedure was minimized from 8 h to 5 h and 45 min. Although the time was further reduced to 4 h and 40 min in experiment III, the efficiency remained the same as in experiment I when the volume of PEP was reduced from 60 microliters (experiments I and II) to 40 microliters. One out of 132 control replicates (0.8%) was contaminated.(ABSTRACT TRUNCATED AT 250 WORDS)