J.A. Grifo

A fast and efficient method for simultaneous X and Y in situ hybridization of human blastomeres

PurposeTo use a 6-hr fluorescence in situ hybridization (FISH) procedure involving fluorochrome-labeled probes to determine the gender of blastomeres from arrested biopsied human embryos.ResultsSimultaneous detection of X and Y chromosomes was performed on 68 blastomeres with this technique. The FISH efficiency for gender determination was 95.5% (65/68). In addition, rehybridization with chromosome 8-specific probes was performed to determine the ploidy of blastomeres with more than two sex chromosomes.ConclusionThis technique offers an alternative to polymerase chain reaction for the preimplantation diagnosis of X-linked diseases and can also be used for ploidy assessment.

Sex determination of human embryos using the polymerase chain reaction and confirmation by fluorescence in situ hybridization

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 XYY, 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.

Interferon-γ in the diagnosis and pathogenesis of pelvic inflammatory disease

Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain: On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia , and two (6.9%) had Neisseria and Chlamydia , whereas in 15 (48.3%) patients no pathogen was isolated. Interferon-γ was present in significantly more sera ( p Neisseria , seven (87.5%) had circulating interferon-γ; three (60%) of the women with only Chlamydia , one (50%) woman with Neisseria and Chlamydia , and eight (57.1 %) with no identified pathogens were also positive for interferon-γ. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida -induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon-γ by anti-interferon-,γ antibody but not by treatment with anti-interferon-α antibody. The persistence of interferon-γ in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage.

Microsurgical fertilization procedures: The absence of stringent criteria for patient selection

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n =200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n =116 cycles) failed to fertilize in a previous cycle. Group B (n =40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n =94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.

Relation between antibodies toChlamydia trachomatis and spontaneous abortion following in vitro fertilization

BackgroundMany couples undergo in vitro fertilization due to occlusion of the fallopian tubes. Chlamydia trachomatis infections are a major cause of this tubal damage. Since this organism has also been associated with poor pregnancy outcome, we investigated whether a past exposure to C.trachomatis was associated with spontaneous abortion following in vitro fertilization and embryo transfer.MethodsSera from 145 women undergoing IVF were diluted 1:128 and tested for IgG antibodies to C.trachomatis by an immunoperoxidase assay, using infected cells fixed to slides. All subjects and their partners were negative for C.trachomatis by culture or by DNA hybridization.ResultsSerological evidence of a past chlamydial infection was observed in 33.8% of the women. The incidence of antichlamydial IgG was greater (P <0.001) in women whose infertility was due to known tubal disease (37 of 78; 47.4%) than in women whose infertility was due to other causes (12 of 67; 17.9%). Spontaneous abortions after embryo transfer occurred in 20% of the subjects. The incidence of antichlamydial IgG in aborting women (20 of 29; 69.0%) was greater (P <0.001) than the incidence in either women with successful pregnancies (9 of 38; 23.7%) or women who did not become pregnant (20 of 78; 25.6%) after IVF. No relation was observed between antichlamydial antibody status and maternal age, the number of oocytes aspirated, the number of oocytes fertilized, and the number of embryos transferred.ConclusionsA previous infection with C.trachomatis may increase susceptibility to subsequent spontaneous abortion, even in the absence of a detectable current infection.

Origin of single pronucleated human zygotes

PurposeDiploidy in embryos developing from single pronucleated zygotes can occur following parthenogenetic activation or by asynchronous inflation of pronuclei following fertilization. To distinguish between these two mechanisms, sexing was performed.ResultsThe presence of a Y chromosome in the embryo was considered proof that fertilization had occurred. Twenty-one dividing embryos originating from single pronucleated zygotes were analyzed using the polymerase chain reaction or fluorescence in situ hybridization. In total, 43% (9/21) of the embryos showed Y chromosomes.ConclusionIt can be extrapolated that more than 80% of them originated from fertilized eggs.

The parental origin of the distal pronucleus in dispermic human zygotes.

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.

Laser ablation of the mouse zona pellucida for blastomere biopsy.

PurposeA xenon-chloride non-contact laser system was evaluated for opening the mouse zona pellucida prior to embryo biopsy.MethodsA 308 nm beam was directed into the rear inlet of an inverted microscope, and exited to the target through a 100 X quartz objective. Following laser opening of the zona, blastomere removal was performed.ResultsEight-cell mouse embryos (n = 611) were isolated for this study. Ninety-one percent of laser biopsy embryos proceeded to the blastocyst stage compared to 94% for acidified Tyrodes biopsied embryos and 83% for zona-intact controls. Many of the laser exposed embryos, however, exhibited morphologic aberrations such as isolated blastomere arrest and embryo growth delay. Thirtyfour percent of the laser biopsy embryos implanted compared with 47% and 37% for acid biopsied and control embryos, respectively. A significant decrease in fetal development following laser zona opening, however, was noted.ConclusionsThough this laser system does allow for blastocyst formation and implantation following biopsy, the possible detrimental effects on embryonic and fetal development should be further evaluated.

Interferon-gamma in the diagnosis and pathogenesis of pelvic inflammatory disease

Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain. On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia, and two (6.9%) had Neisseria and Chlamydia, whereas in 15 (48.3%) patients no pathogen was isolated. Interferon-gamma was present in significantly more sera (p less than 0.025) from patients with pelvic inflammatory disease (65.5%) than from women without pelvic inflammatory disease (15.8%). Sera from 10 healthy women lacked detectable interferon-gamma. In patients with only Neisseria, seven (87.5%) had circulating interferon-gamma; three (60%) of the women with only Chlamydia, one (50%) woman with Neisseria and Chlamydia, and eight (57.1%) with no identified pathogens were also positive for interferon-gamma. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida-induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon-gamma by anti-interferon-gamma antibody but not by treatment with anti-interferon-alpha antibody. The persistence of interferon-gamma in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage.