Faculty Bibliography

Proinflammatory cytokines and activated macrophages and T lymphocytes have been detected in peritoneal fluids of women with endometriosis and may impair fertility. Expression of the 60 kDa heat shock protein (hsp60) is one mechanism leading to a localized activation of macrophages and T lymphocytes and cytokine release. Peritoneal fluids, obtained from 68 women undergoing a diagnostic laparoscopy, were assayed for hsp60. As independent evidence of local immune activation, the fluids were analysed for interferon gamma (IFN gamma). Fluids were also tested for antibodies to Chlamydia trachomatis because a chronic asymptomatic infection by this organism may also release hsp60. At laparoscopy, 26 women were diagnosed with pelvic adhesions, 19 had endometriosis, 16 had a visibly normal pelvis, four had ovarian cysts while three had myomas. The prevalence of hsp60 was higher in peritoneal fluids from the women with endometriosis than in the other subjects (P = 0.005). Hsp60 was detected in seven (36.8%) of the endometriosis patients and in only one each of the women with adhesions, a normal pelvis or an ovarian cyst; all women with myomas were negative. Detection of IFN gamma in peritoneal fluids was highly correlated with the presence of hsp60 (P = 0.0003). IFN gamma was present in seven of nine (77.8%) women with hsp60 and in only five of 40 (12.5%) women lacking hsp60. Women with pelvic adhesions had an increased prevalence of immunoglobulin G antibodies to C.trachomatis compared with the other women (P = 0.01). There was no relationship between evidence of exposure to C.trachomatis and hsp60 in peritoneal fluids. These data suggest that hsp60 may be released into the peritoneal fluid as a consequence of implanted ectopic endometrium. Hsp60-mediated immune activation may be one mechanism leading to endometriosis-associated infertility

The field of preimplantation genetic diagnosis has undergone significant advances since the report of the first birth from this method in 1990. The first birth in the USA was reported in 1992, as was the first successful diagnosis and delivery of a baby free of a single gene defect disorder (cystic fibrosis and then Tay Sachs). Investigators have now reported approximately 40 births worldwide from preimplantation genetic diagnosis using the polymerase chain reaction and fluorescent in-situ hybridization methods to analyze single cells removed from early cleavage stage preimplantation embryos. The International Working Group on Preimplantation Genetics meets annually to discuss progress and pitfalls in this field. Although preimplantation genetic diagnosis offers hope to patients at risk of transmitting disease, there are many technical hazards of this experimental procedure. Technical difficulties must be overcome in order for preimplantation genetic diagnosis to become a standard clinical tool. This review will highlight some of the recent advances and problems in the field of preimplantation genetic diagnosis

A young female carrier for incontinentia pigmenti underwent preimplantation genetic diagnosis to prevent the transfer of affected embryos. FISH diagnosis was performed using X, Y, 18 and 13/21 probes. Unexpectedly, 57% of the embryos were aneuploid for these chromosomes, a rate significantly higher (P < 0.005) than expected (9.3%). The patient achieved pregnancy but spontaneously aborted a trisomy 9 fetus.

PURPOSE: The prevalence of Ureaplasma urealyticum and Mycoplasma hominis in the endocervix at the time of oocyte collection in women undergoing in vitro fertilization (IVF) was examined using the polymerase chain reaction (PCR). METHODS: All women were treated with tetracycline following sample collection. RESULTS: U. urealyticum was identified in 56 (17.2%) of 326 women while M. hominis was present in only 5 (2.1%) of 235 women. U. urealyticum was detected at a higher frequency (P = 0.01) in those women whose IVF cycle failed prior to embryo transfer. This organism was present in 8 of 19 (42.1%) women with either no fertilization or no embryo transfer, 19 of 148 (12.8%) who had no evidence of pregnancy following embryo transfer, 6 of 30 (20.0%) who had only a transient (biochemical) pregnancy, 5 of 14 (35.7%) with a spontaneous abortion, and 18 of 115 (15.6%) with a term birth. Of the eight women with U. urealyticum who had no embryos transferred, male factor was the cause of infertility in five cases, two women had tubal occlusions while in one woman the diagnosis was idiopathic. Therefore, poor sperm quality, and not a U. urealyticum infection, might explain the failure of most of these cases to proceed to the stage of embryo transfer. Analysis of all patients revealed no association between male factor infertility and U. urealyticum in the cervix. CONCLUSIONS: U. urealyticum, but not M. hominis, is present in the cervices of many culture-negative women. Its presence, however, does not influence IVF outcome subsequent to embryo transfer in women treated with tetracycline after oocyte retrieval

Among the many clinical applications of the polymerase chain reaction (PCR) is its potential use in preimplantation diagnosis of genetic disorders. Performing PCR on single blastomeres from early cleavage stage (six- to eight-cell) human embryos should, in principle, enable reliable determination of disease status for certain inherited conditions. However, reports of misdiagnoses using this technique have diminished enthusiasm for its widespread clinical use. One principal source of error is the propensity for genome-targeted PCR to exclusively amplify one allele in reactions assaying a single heterozygous diploid cell. Complete reaction failure is also common. Employing the Marfan syndrome (MFS) as a paradigm, we have developed a reliable, reverse transcription-PCR-based method of genotyping single cells that overcomes these obstacles. The technique should facilitate accurate preimplantation diagnosis of MFS and other selected genetic diseases caused by heterozygous or compound-heterozygous mutations

The prevalence of Chlamydia trachomatis in the endocervices of 307 asymptomatic culture-negative women undergoing in vitro fertilization (IVF) was evaluated. C. trachomatis was detected by polymerase chain reaction (PCR) in 20 subjects (6.5%), and there were strong correlations between a positive finding and both failure to become pregnant (P = .013) and spontaneous abortion after embryo transfer (P = .004). C. trachomatis was identified in 2 (1.8%) of 112 who had term deliveries, 3 (27.3%) of 11 who spontaneously aborted, 1 (3.3%) of 30 with biochemical pregnancies, 13 (9.6%) of 135 with no pregnancy after embryo transfer, and 1 (5.3%) of 19 whose embryos did not become fertilized. There were no relationships between PCR findings and maternal age, cause of infertility, number of oocytes retrieved or fertilized, or number of embryos transferred; 55% of PCR-positive and 40% of PCR-negative women were undergoing at least their second IVF. An undetected C. trachomatis infection may be responsible for implantation failure or spontaneous abortion after IVF and embryo transfer

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