Faculty Bibliography

Although round spermatids have been studied extensively using staining techniques and electron microscopy, little information is available about their appearance in living conditions. We describe a method of collecting and identifying round spermatids from ejaculates and testicular biopsies. The validity of the selection procedure was confirmed by fluorescence in-situ hybridization. Based on cell size, morphological characteristics of nucleus and cytoplasm, and on the nucleus/cytoplasm ratio, we harvested a population of cells that was 84% haploid. This procedure can be applied to select spermatids for clinical or research purposes

PGD has been successfully used for several years. Over 40 babies have been born worldwide by use of these techniques. Unfortunately, a number of misdiagnoses have been made, a distressing consequence of a new frontier. Significant advances have been made to improve the efficiency and accuracy of PCR and FISH. The widespread use of this technology awaits further documentation of safety and accuracy. Other issues must also be addressed. First, the cost-effectiveness of the techniques relative to the traditional alternatives must be evaluated. A number of ethical issues regarding embryo screening must be addressed including what diseases are serious enough to warrant the procedure. Another concern is the use of this technology for nongenetic disorders such as gender selection. Finally, the experimental nature of these procedures must continually be discussed with patients, and long-term follow-up studies must be undertaken. Development of more accurate and less expensive assays coupled with improved IVF success rates may make PGD a more widely used clinical tool. The future awaits these developments

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.

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

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

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)