Cumulative live birth rates after one ART cycle including all subsequent frozen-thaw cycles in 1050 women: secondary outcome of an RCT comparing GnRH-antagonist and GnRH-agonist protocols.

STUDY QUESTION: Are cumulative live birth rates (CLBRs) similar in GnRH-antagonist and GnRH-agonist protocols for the first ART cycle including all subsequent frozen-thaw cycles from the same oocyte retrieval? SUMMARY ANSWER: The chances of at least one live birth following utilization of all fresh and frozen embryos after the first ART cycle are similar in GnRH-antagonist and GnRH-agonist protocols. WHAT IS KNOWN ALREADY: Reproductive outcomes of ART treatment are traditionally reported as pregnancies per cycle or per embryo transfer. However, the primary concern is the overall chance of a live birth. After the first ART cycle with fresh embryo transfer, we found live birth rates (LBRs) of 22.8% and 23.8% (P = 0.70) for the GnRH-antagonist and GnRH-agonist protocols, respectively. But with CLBRs including both fresh and frozen embryos from the first oocyte retrieval, chances of at least one live birth increases. There are no previous randomized controlled trials (RCTs) comparing CLBRs in GnRH-antagonist versus GnRH-agonist protocols. Previous studies on CLBR are either retrospective cohort studies including multiple fresh cycles or RCTs comparing single embryo transfer (SET) with double embryo transfer (DET). STUDY DESIGN, SIZE, DURATION: CLBR was a secondary outcome in a Phase IV, dual-center, open-label, RCT including 1050 women allocated to a short GnRH-antagonist or a long GnRH-agonist protocol in a 1:1 ratio over a 5-year period using a web-based concealed randomization code. The minimum follow-up time from the first IVF cycle was 2 years. The aim was to compare CLBR between the two groups following utilization of all fresh and frozen embryos from the first ART cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women referred for their first ART cycle at two public fertility clinics, <40 years of age were approached. A total of 1050 subjects were allocated to treatment and 1023 women started standardized ART protocols with recombinant human follitropin-ß (rFSH) stimulation. Day-2 SET was planned and additional embryos were frozen and used in subsequent frozen-thawed cycles. All pregnancies generated from oocyte retrieval during the first IVF cycle including fresh and frozen-thaw cycles were registered. Ongoing pregnancy was determined by ultrasonography at gestational week 7-9 and live birth was irrespective of the duration of gestation. CLBR was defined as at least one live birth per allocated woman after fresh and frozen cycles. Subjects were censored out after the first live birth. Cox proportional hazard model was used to evaluate the relative prognostic significance of female age, BMI, the number of retrieved oocytes and the diagnosis of infertility in relation to the CLBR. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were similar and equal proportions of patients continued with frozen-thaw (frozen embryo transfer, FET) cycles after their fresh ART cycle in the GnRH-antagonist and GnRH-agonist arms. When combining all fresh and frozen-thaw embryo transfers from first oocyte retrieval with a minimum of 2-year follow-up, the CLBR was 34.1% (182/534) in the GnRH-antagonist group versus 31.2% (161/516) in the GnRH-agonist group (odds ratio (OR):1.14; 95% CI: 0.88-1.48, P = 0.32). Mean time to the first live birth was 11.0 months in the GnRH-antagonist group compared to 11.5 months in the GnRH-agonist group (P < 0.01). The total number of deliveries from all FET cycles where embryos were thawed were higher in the antagonist group 64/330 (19.4%) compared to the agonist group 43/355 (12.1%) ((OR): 1.74; 95% CI: 1.14-2.66, P = 0.01). The evaluation of prognostic factors showed that more retrieved oocytes were associated with a significantly higher CLBR in both treatment groups. For the subgroup of obese women (BMI >30 kg/m2), the CLBR was significantly higher in the GnRH-antagonist group (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: The duration of the trial is a possible limitation with introduction of new methods as 'Freeze all' and 'GnRH-agonist triggering', but as these treatments were used in only few women, a systematic bias is not likely. Blastocyst culture of surplus embryos for freezing was introduced to both groups simultaneously, thereby minimizing the risk of bias. Furthermore, with a minimum of 2-year follow-up, a minority (<1%) still had cryopreserved embryos and no live birth at the end of the trial. The post hoc prognostic covariate analyses with multiple strata should be interpreted with caution. Finally, the physicians were not blinded to GnRH treatment group after randomization. WIDER IMPLICATIONS OF THE FINDINGS: With the improvement of embryo culture, freezing and thawing methods as well as a strategy of elective SET, CLBR until first live birth provides an all-inclusive success rate for ART. When comparing GnRH-antagonist and GnRH-agonist protocols, we find similar CLBRs, despite more oocytes being retrieved in the GnRH-agonist protocol. STUDY FUNDING/COMPETING INTERESTS: An unrestricted research grant is funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: EudraCT #: 2008-005452-24. ClinicalTrial.gov: NCT00756028. TRIAL REGISTRATION DATE: 18 September 2008. DATE OF FIRST PATIENT'S ENROLLMENT: 14 January 2009.

Risk of severe ovarian hyperstimulation syndrome in GnRH antagonist versus GnRH agonist protocol: RCT including 1050 first IVF/ICSI cycles.

STUDY QUESTION: Is the risk of severe ovarian hyperstimulation syndrome (OHSS) similar in a short GnRH antagonist and long GnRH agonist protocol in first cycle IVF/ICSI patients less than 40 years of age?. SUMMARY ANSWER: There is an increased risk of severe OHSS in the long GnRH agonist group compared with the short GnRH antagonist protocol. WHAT IS KNOWN ALREADY?: In the most recent Cochrane review, the GnRH antagonist protocol was associated with a similar live birth rate (LBR), a similar on-going pregnancy rate (OPR), and a lower incidence of OHSS (odds ratio (OR) = 0.43 95% confidence interval (CI): 0.33-0.57) compared with the traditional GnRH agonist protocol. Previous trials comparing the two protocols mainly included selected patient populations, a limited number of patients and the applied OHSS criteria differed, making direct comparisons difficult. In two recent large meta-analyses, no significant differences in LBR (OR = 0.86; 95% CI: 0.72-1.02) or in the incidence of severe OHSS were reported, while others found a lower LBR (OR = 0.82; 95% CI: 0.68-0.97) and a reduced risk of severe OHSS using the GnRH antagonist protocol (OR = 0.60; 95% CI: 0.40-0.88). STUDY DESIGN, SIZE, DURATION: Phase IV, dual-centre, open-label, RCT including 1050 women allocated to either short GnRH antagonist or long GnRH agonist protocol in a 1:1 ratio and enrolled over a 5-year period using a web-based concealed randomization code. This is a superiority study designed to detect a difference in severe OHSS, the primary outcome, between the two groups with a power of 80% and stratified for age, assisted reproductive technology (ART) clinic and planned fertilization procedure (IVF/ICSI). The secondary aims were to compare rates of mild and moderate OHSS, positive plasma (p)-hCG, on-going pregnancy and live birth between the two arms. None of the women had undergone previous ART treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: All infertile women referred for their first IVF/ICSI at two public fertility clinics, less than 40 years of age and with no uterine malformations were asked to participate. A total of 1099 subjects were randomized, including women with poor ovarian reserve, polycystic ovary syndrome and irregular cycles. A total of 49 women withdrew their consent, thus 1050 subjects were allocated to the GnRH antagonist (n = 534) and agonist protocol (n = 516), respectively. In total 1023 women started recombinant human follitropin-ß (rFSH) stimulation, 528 in the GnRH antagonist group and 495 in the GnRH agonist group. All subjects were given a fixed rFSH dose of 150 IU or 225 IU according to age ≤36 years or >36 years, with the option to adjust dose at stimulation day 6. Clinical OHSS parameters were collected at oocyte retrieval, and Days 3 and 14 post-transfer. On-going pregnancy was determined by transvaginal ultrasonography at gestational weeks 7-9. In the intention-to-treat (ITT) analysis for reproductive outcomes, 1050 subjects were included. For the ITT analyses on OHSS 1023 subjects who started gonadotrophin stimulation were included. MAIN RESULTS AND THE ROLE OF CHANCE: The incidence of severe OHSS [5.1% (27/528) versus 8.9% (44/495) (difference in proportion percentage point (Δpp) = -3.8pp; 95% CI: -7.1 to -0.4; P = 0.02)] and moderate OHSS [10.2% (54/528) versus 15.6% (77/495) (Δpp = -5.3pp; 95% CI: -9.6 to -1.0; P = 0.01) ] was significantly lower in the GnRH antagonist group compared with the agonist group, respectively. In the GnRH antagonist and agonist group, respectively, 4.7% (25/528) versus 8.5% (42/495) women were seen by a physician due to OHSS (P = 0.01), and 1.7% (9/528) versus 3.6% (18/495) were admitted to hospital due to OHSS (P = 0.06). No women had ascites-puncture in the GnRH antagonist group versus 2.0% (10/495) in the GnRH agonist group (P < 0.01). LBRs were 22.8% (122/534) versus 23.8% (123/516) (Δpp = -1.0pp; 95% CI: -6.3 to 4.3; P = 0.70) and OPRs were 24.9% (133/528) versus 26.2% (135/516) (Δpp = -1.3pp; 95% CI: -6.7 to 4.2; P = 0.64) per randomized subject in the GnRH antagonist versus agonist group, with a mean number of 1.1 versus 1.2 embryos transferred in the two groups. Pregnancy rates (PR) per randomized subject, per started gonadotrophin stimulation and per embryo transfer were all similar in the two groups. LIMITATIONS, REASONS FOR CAUTION: A possible limitation is the duration of the trial, with new methods, such as 'freeze all' and 'GnRH agonist triggering', being developed during the trial, the new methods were sought avoided, however a total number of 32 women had 'freeze all' and 'GnRH agonist triggering' was performed in three cases. Ultrasonic measurements were performed by different physicians and inter-observer bias may be present. Measures of anti-Mullerian hormone and antral follicle count, to estimate ovarian reserve and thus predict risk of OHSS, were not performed. Finally, the physicians were not blinded to GnRH treatment group after randomization. WIDER IMPLICATIONS OF THE FINDINGS: The short GnRH antagonist protocol should be the protocol of choice for patients undergoing their first ART cycle in females <40 years of age including both low and high responders when an age-dependent initially fixed gonadotrophin dose is used, as an increased risk of severe OHSS and the associated complications is seen in the long GnRH agonist group and as PRs and LBRs are similar in the two groups. Patients at risk of OHSS particularly benefit from the short GnRH antagonist treatment as GnRH agonist triggering can be used. STUDY FUNDING/COMPETING INTERESTS: An unrestricted research grant is funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: EudraCT #: 2008-005452-24. ClinicalTrial.gov: NCT00756028. Trial registration date: 18 September 2008. Date of first patient's enrolment: 14 January 2009.

Mental distress and personality in women undergoing GnRH agonist versus GnRH antagonist protocols for assisted reproductive technology.

STUDY QUESTION: Do mental distress and mood fluctuations in women undergoing GnRH agonist and GnRH antagonist protocols for assisted reproductive technology (ART) differ depending on protocol and the personality trait, neuroticism? SUMMARY ANSWER: ART treatment did not induce elevated levels of mental distress in either GnRH antagonist or agonist protocols but neuroticism was positively associated with increased mental distress, independent of protocols. WHAT IS KNOWN ALREADY: ART treatment may increase mental distress by mechanisms linked to sex hormone fluctuations. General psychological characteristics, such as personality traits indexing negative emotionality, e.g. neuroticism, are likely to affect mental distress during ART treatment. STUDY DESIGN, SIZE, DURATION: A total of 83 women undergoing their first ART cycle were consecutively randomized 1:1 to GnRH antagonist (n = 42) or GnRH agonist (n = 41) protocol. The study population was a subgroup of a larger ongoing Danish clinical randomized trial and was established as an add-on in the period 2010-2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women in the GnRH antagonist protocol received daily injections with recombinant follicle-stimulating hormone, Puregon(®) and subcutaneous injections with GnRH antagonist, Orgalutran(®). Women in the GnRH agonist protocol received nasal administration of the GnRH agonist, Synarela(®) and subcutaneous injections with FSH, Puregon(®). The study design did not allow for a blinding procedure. All women self-reported the Profile of Mood States, the Perceived Stress Scale, the Symptom Checklist-92-Revised, and the Major Depression Inventory questionnaires, at baseline, at ART cycle day 35, on the day of oocyte pick-up, and on the day of hCG testing. Also, a series of Profile of Mood States were reported daily during pharmacological treatment to monitor mood fluctuations. The personality trait Neuroticism was assessed at baseline by the self-reported NEO-PI-R questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: ART did not induce within- or between-protocol changes in any of the applied measures of mental distress. However, the GnRH antagonist protocol was associated with more pronounced median mood fluctuations during the stimulation phase (antagonist, 11.0 SD, [IQR = 21.1-6.1]; agonist, 8.9 SD, [IQR = 11.3-5.7], P = 0.025). This association became non-significant after applying a Bonferroni-Holm correction. Neuroticism was highly positively associated with increased levels of mental distress throughout treatment independent of protocols (all P-values <0.006), and cross-sectional analysis revealed that women with high or low Neuroticism scores at baseline showed a significant trend towards lower chances of a positive pregnancy test (P-value =0.028). LIMITATIONS, REASONS FOR CAUTION: Information on prognostic factors such as preceding length of infertility, number of retrieved oocytes and number of prior insemination treatments was not accounted for in the analyses. The stratification of protocols by age in the subgroups of women included in this study was suboptimal. Women with prior or current use of antidepressant medication were excluded from our study. WIDER IMPLICATIONS: Our results imply that mental distress emerging during ART treatment is not causally linked to hypogonadism per se or to the choice of protocol. Rather, our data highlight the potential importance of (i) rapid increases in ovarian steroids and (ii) addressing personality traits indexing negative emotionality, i.e. Neuroticism, in women undergoing ART treatment, to optimize both emotional adjustment and, possibly, the chances of obtaining pregnancy. STUDY FUNDING/COMPETING INTERESTS: The Danish Research Council for Independent Research and MSD, Denmark kindly supported the study. The authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: EudraCT - 2008-005452-24.

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation.

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

4q35 deletion and 10p15 duplication associated with immunodeficiency.

We report a familial cryptic reciprocal translocation between 4q35 and 10p15 leading to deletion of the terminal long arm of chromosome 4 and duplication of the terminal short arm of chromosome 10 in two family members who both have immunological disturbances and a similar facial appearance. The precise location and extent of the deletion and duplication was determined by fluorescence in situ hybridization (FISH). Furthermore, we investigated the deletion breakpoint of a previously reported patient with 4q34.3-qter deletion [Van Buggenhout et al. (2004); Am J Med Genet Part A 131A:186-189].

The most common chromosome aberration detected by high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma.

We analyzed genetic changes in condylomas (four cases), vulvar intraepithelial neoplasia I-III (VIN I-III, eleven cases), and primary vulvar squamous cell carcinomas (VSCC, ten cases) by high-resolution comparative genomic hybridization (HR-CGH) and flowcytometry. All samples were also human papilloma virus (HPV)-genotyped. Gain of chromosome 1, the aberration most often seen in VIN III (67%), was not seen in HPV-positive or -negative VSCCs (0%). Both VIN III and VSCC frequently showed gain of 3q (56 and 70%, respectively). The VIN III samples often demonstrated gain of 20q (56%) and 20p (44%), and the VSCC samples gain of 8q (60%), loss of 3p (50%), and 8p (40%). None of the four most frequent changes in the VSCC samples occurred exclusively in the HPV-positive or -negative samples. As expected, we did not find any cytogenetic changes in condylomas and nearly any changes in VIN I-II.

Cryptic familial t(11;18)(q25;q23) incidentally detected by interphase FISH.

During a prospective prenatal study of numerical abnormalities of chromosomes 13, 18, 21, X and Y using locus-specific probes, we incidentally found a case with only one signal for chromosome 18 per cell in a chorionic villus sampling (CVS) associated with an otherwise apparently normal G-banded karyotype. This led us to discover a cryptic t(11;18) segregating in a four-generation family. The CVS was performed because of mental retardation in the brother to the father of the fetus. A subtelomeric chromosome 18 probe revealed one signal on 18qter and one on 11qter of the father. Thus the father had a balanced reciprocal t(11;18) in spite of the apparently normal G-banded karyotype. Using the same probes, we found an unbalanced translocation 46,XX,-18,+der (18)t(11;18)-(q25;q23)pat in the fetus. Further investigation of the family showed the translocation in balanced and unbalanced form in four generations in mentally normal and retarded individuals, respectively. The study emphasizes the need for a follow-up with molecular cytogenetic techniques in dysmorphic and retarded children.

Rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization: a one-year clinical experience with high-risk and urgent fetal and postnatal samples.

OBJECTIVES: To evaluate the clinical utility of rapid prenatal and postnatal detection of common chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) analysis with DNA probes. DESIGN: Four hundred and seventy-seven high-risk and/or urgent amniotic fluid, chorionic villus and fetal and postnatal blood samples were prospectively examined by FISH with probes specific for chromosomes 13, 18, 21, X, and Y and results were reported within 48 hours. All FISH results were followed by conventional chromosome analysis, if possible. SETTING: Cytogenetic service laboratory at the tertiary referral center, Rigshospitalet in Copenhagen. MAIN OUTCOME MEASURES: The fraction of clinically significant chromosome aneuploidies that was detected by FISH analysis, and the fraction of terminations that was based on FISH and ultrasound results rather than on conventional cytogenetic results. RESULTS: The FISH assay detected 76% of the clinically significant chromosome abnormalities as determined by subsequent cytogenetic analysis. Seventy-two percent of the terminations of the chromosomally abnormal pregnancies were based on FISH and ultrasound results rather than on conventional cytogenetic results. CONCLUSION: FISH analysis is a clinically useful adjunctive tool to conventional pre- and postnatal cytogenetic analysis. The assay rapidly detects the majority of clinically significant chromosome abnormalities, thus facilitating difficult pre- and postnatal clinical decisions.

Comparative genomic hybridization reveals a recurrent pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and in advanced-stage cervical carcinoma.

We analyzed 17 cases of dysplasia/carcinoma in situ (CIS) of the cervix and 29 advanced-stage cervical squamous cell carcinomas by comparative genomic hybridization (CGH). A comparable recurrent pattern of aberrations was detected in both preinvasive and invasive cases, although the total number of aberrations was much higher in the latter category. The most consistent chromosomal gain was mapped to chromosome arm 3q in 35% of preinvasive cases and in 72% of invasive cases. Chromosome aberrations were detected in 13/17 preinvasive cases with a total of 61 involved chromosome arms. In the invasive cases, frequent gains also occurred on 1q (45%), 8q (41%), 15q (41%), 5p (34%), and Xq (34%), and frequent losses were mapped to chromosome arms 3p (52%), 11q (48%), 13q (38%), 6q (38%), and 4p (34%). A recurrent pattern of aberrations has not previously been described in preinvasive lesions of the cervix. Our finding is surprising considering that only few preinvasive lesions are expected to progress to invasive cancer.

Prenatal detection of chromosome aneuploidies by fluorescence in situ hybridization: experience with 2000 uncultured amniotic fluid samples in a prospective preclinical trial.

Successful rapid prenatal detection of selected numerical chromosome abnormalities by using fluorescence in situ hybridization (FISH) on uncultured amniotic fluid samples has been described by Klinger et al. (1992) and Ward et al. (1993, 1997). Using essentially the same FISH protocol and identical probes specific for chromosomes 21, 18, 13, X, and Y, we prospectively compared the results of FISH and conventional cytogenetics on 2000 amniotic fluid cell samples. The 1-day FISH assay yielded discrete differences in the signal profiles between cytogenetically disomic, i.e., normal, and trisomic samples. Due to intermittent absent Y-signals, the assay differentiated less well between samples with cytogenetically normal and abnormal sex chromosome complements. The assay efficiency, and thus the clinical utility, was affected by (1) unsuccessful hybridizations (7 per cent of all hybridizations), (2) hybridizations with less than 50 scorable nuclei (19 per cent of all hybridizations), and (3) visibly contaminated samples with possible maternal cell contamination (14 per cent of all samples). As a result, we were not able to reproduce the results of Klinger et al. (1992) and Ward et al. (1993, 1997).

Prenatal detection of chromosome aneuploidies in uncultured chorionic villus samples by FISH.

We developed a 1-d FISH assay for detection of numerical chromosome abnormalities in uncultured chorionic villus samples (CVS). Probes specific for chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. Aneuploidy detection using this assay was directly compared with the results obtained by conventional cytogenetic analysis in a consecutive, clinical study of 2,709 CVS and placental samples. The FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. On the basis of these results, we generated FISH-assay cutoff values that discriminated between karyotypically normal and aneuploid samples. Samples with mosaicism and a single sample with possible heritable small chromosome X probe target were exceptions and showed poor agreement between FISH results and conventional cytogenetics. We conclude that the FISH assay may act as a more accurate and less labor-demanding alternative to "direct" CVS analysis.

Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.

[Rapid detection of numerical aberrations of chromosomes in the first trimester of pregnancy by using fluorescence in situ hybridization (FISH)].

Fluorescence in situ hybridization (FISH) has been applied for rapid prenatal diagnosis of common numerical aberrations of chromosomes. We used FISH with chromosome 13, 18 and 21 specific probes on 528 uncultured mesenchymal chorionic villi cell samples to detect the chromosomal abnormalities, and we also performed the conventional chromosome analysis of cultured cells from parallel samples. The results showed, in samples disomic with respect to the probed chromosomes, and average of 1 percent (range 0-18 percent) had three hybridization signals. By contrast, in the samples trisomic or triploidic for the probed chromosomes, an average of 70 percent (52-84 percent) (chromosome 13), 73 percent (68-84 percent) (chromosome 18), and 76 percent (54-90 percent) (chromosome 21, including one case of mosaic trisomy 21) of the nuclei displayed three signals. The whole test took about 24 hours. We concluded that FISH can provide a rapid and accurate method for the first trimester prenatal idetification of selected numerical aberrations of chromosomes.

Image analysis in comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a new technique by which genomic imbalances can be detected by combining in situ suppression hybridization of whole genomic DNA and image analysis. We have developed software for rapid, quantitative CGH image analysis by a modification and extension of the standard software used for routine karyotyping of G-banded metaphase spreads in the Magiscan chromosome analysis system. The DAPI-counterstained metaphase spread is karyotyped interactively. Corrections for image shifts between the DAPI, FITC, and TRITC images are done manually by moving the three images relative to each other. The fluorescence background is subtracted. A mean filter is applied to smooth the FITC and TRITC images before the fluorescence ratio between the individual FITC- and TRITC-stained chromosomes is computed pixel by pixel inside the area of the chromosomes determined by the DAPI boundaries. Fluorescence intensity ratio profiles are generated, and peaks and valleys indicating possible gains and losses of test DNA are marked if they exceed ratios below 0.75 and above 1.25. By combining the analysis of several metaphase spreads, consistent findings of gains and losses in all or almost all spreads indicate chromosomal imbalance. Chromosomal imbalances are detected either by visual inspection of fluorescence ratio (FR) profiles or by a statistical approach that compares FR measurements of the individual case with measurements of normal chromosomes. The complete analysis of one metaphase can be carried out in approximately 10 minutes.

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (FISH).

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates. Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.

Prenatal diagnosis by fluorescence in situ hybridization on chorionic villus cells: nonsignificance of maternal cell contamination.

We assessed the effect of maternal cell contamination on the sensitivity of prenatal diagnosis by fluorescence in situ hybridization (FISH) on overnight attached mesenchymal chorionic villus cells. Double targets FISHs with X and Y chromosome-specific probes were performed on parallel samples from the same pregnancies: (1) samples of assumed fetal tissue retained during dissection, and (2) samples of suspected maternal tissue discarded during dissection. In the karyotypically male samples the tissue retained during dissection contained 0-3% nuclei with two X-specific signals each. In the tissue discarded from the karyotypically male samples 0-50% of the nuclei had two X-specific signals each. We conclude that given thorough dissection of chorionic villi, the sensitivity of prenatal diagnosis of aneuploidies by FISH on overnight attached samples of this tissue is not critically affected by maternal cell contamination.

Fluorescence in situ hybridization with a chromosome 21-specific cosmid contig: 1-day detection of trisomy 21 in uncultured mesenchymal chorionic villus cells.

We present a modified, fast trisomy 21 detection assay using fluorescence in situ hybridization (FISH) on uncultured mesenchymal chorionic villus cells. The whole test takes about 24 h. We used a cosmid contig as a probe and modified an in situ sample preparation method first described by Klinger et al. (1992). The assays saves time and cost of culture in comparison with a previously described trisomy 21 detection FISH assay (Bryndorf et al., 1993). A small blind clinical study comparing the modified and the previously described FISH assays using mesenchymal chorionic villus cells showed comparable results and concordance with conventional cytogenetic analysis. The frequency of nuclei with three hybridization signals from samples disomic for chromosome 21 ranged from 0 to 8 per cent with both assays, while trisomic samples had 60-80 and 54-90 per cent of the mesenchymal nuclei with three signals in the modified and previously described assays, respectively. Normal (disomic) and trisomic mesenchymal chorionic villus samples can be distinguished clearly and rapidly without culture in the modified assay.