Euploidy rates in donor egg cycles significantly differ between fertility centers.

Study question: Do external factors affect euploidy in egg donor cycles? Summary answer: The study demonstrates that during human assisted reproduction, embryonic chromosome abnormalities may be partly iatrogenic. What is known already: Chromosome abnormalities have been linked in the past to culture conditions such as temperature and Ph variations, as well as hormonal stimulation. Those reports were performed with older screening techniques (FISH), or ART methods no longer in use, and the subjects studied were not a homogeneous group. Study design, size, duration: A total of 1645 donor oocyte cycles and 13 282 blastocyst biopsies from 42 fertility clinics were included in this retrospective cohort study. Samples from donor cycles with PGS attempted between September 2011 and July 2015 were included. Participants/materials, setting, methods: PGS cycles from multiple fertility clinics referred to Reprogenetics (Livingston, NJ) that involved only oocyte donation were included in this study. Testing was performed by array comparative genomic hybridization (aCGH). Ploidy data were analyzed using Generalized Linear Mixed Models with logistic regression using a logit link function considering a number of variables that represent fixed and random effects. Main results and the role of chance: Euploidy rate was associated with the referring center and independent of almost all the parameters examined except donor age and testing technology. Average euploidy rate per center ranged from 39.5 to 82.5%. The mean expected rate of euploidy was 68.4%, but there are variations in this rate associated with the center effect. Limitations, reasons for caution: Data set does not include details of the donor selection process, donor race or ethnic origin, ovarian reserve or ovarian responsiveness. Due to the retrospective nature of the study, associations are apparent, however, causality cannot be established. Discrepancies in regard to completeness and homogeneity of data exist due to data collection from over 40 different clinics. Wider implications of the findings: This is the first study to show a strong association between center-specific ART treatment practices and the incidence of chromosome abnormality in human embryos, although the meiotic or mitotic origin of these abnormalities could not be determined using these technologies. Given the widespread applications of ART in both subfertile and fertile populations, our findings should be of interest to the medical community in general as well as the ART community in particular. Study funding/competing interest(s): No external funds were used for this study. S. Munne is a founding principle of Reprogenetics/current employee of Cooper Genomics. M Alikani's spouse is a founding principle of Reprogenetics/current consultant for Cooper Genomics. The remaining authors have no conflicts to declare.

First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridization and microarray analysis.

BACKGROUND: Balanced chromosomal rearrangements represent one of the most frequent indications for preimplantation genetic diagnosis (PGD). Although fluorescence in situ hybridization (FISH) has been successfully employed for diagnosis in such cases, this approach usually restricts assessment of the chromosomes involved in the rearrangement. Furthermore, with FISH-based strategies, it is sometimes necessary to create patient-specific protocols, increasing the waiting time and costs. In the current study, we explored the use of two comprehensive chromosome screening methods, conventional metaphase comparative genomic hybridization (CGH) and microarray-CGH (aCGH), as alternatives for PGD of chromosome rearrangements. METHODS: The study included 16 patients who underwent 20 cycles of PGD for a variety of chromosome rearrangements (reciprocal or Robertsonian translocations or inversions). Testing was performed at various embryonic stages using CGH (9 cases) or aCGH (11 cases). RESULTS: Results were obtained for 121 out of 132 samples (91.7%). Of the diagnosed samples, 48.8% were found to carry abnormalities associated with the rearrangement, either alone or in combination with other chromosomal abnormalities. A further 28.9% of samples were normal/balanced for the rearranged chromosomes, but affected by aneuploidy for other chromosomes. Only 22.3% of samples were chromosomally normal. Of the 15 patients who completed their treatment cycles, 5 became pregnant after one or two cycles resulting in four healthy births. The delivery rate per cycle was 21% (27% per embryo transfer). CONCLUSIONS: This is the first study to describe the clinical application of comprehensive chromosome screening applied to polar bodies, blastomeres or trophectoderm cells from patients carrying inversions and translocations. Using these techniques, most patients requesting PGD for a chromosome rearrangement can be treated using a single protocol. Additionally, the detection of abnormalities affecting chromosomes unrelated to the rearrangement may assist in the selection of viable embryos for transfer.

Improved detection of aneuploid blastocysts using a new 12-chromosome FISH test.

Fluorescence in-situ hybridization (FISH) has been the principal method used for the identification and preferential transfer of chromosomally normal embryos, in the context of both preimplantation genetic diagnosis (PGD) and screening (PGS). Generally, the probe combinations used during PGS have focused on chromosomes frequently identified as abnormal in prenatal samples or material derived from first-trimester spontaneous abortions. Recent data, however, obtained with the use of comparative genomic hybridization (CGH), have suggested that commonly used PGS strategies may fail to detect a large number of aneuploidies affecting preimplantation embryos. Some chromosomes, which have been relatively neglected in PGS protocols thus far, display a disproportionate contribution to embryo aneuploidy and should be prioritized for screening. Using CGH data, it is possible to design new probe combinations that examine between 10 and 12 chromosomes and are capable of accurately diagnosing 89-91% of anomalies seen in embryos. At present, 24-chromosome tests, such as CGH, array CGH or single nucleotide polymorphism arrays, remain relatively costly and, in some cases, are yet to be fully validated. For these reasons, a cost-effective method, capable of accurately detecting almost all aneuploid embryos, represents an attractive alternative to comprehensive chromosome screening approaches.

Increased efficiency of preimplantation genetic diagnosis for aneuploidy by testing 12 chromosomes.

One of the most important factors in increasing the screening potential of preimplantation genetic diagnosis (PGD) for aneuploidy is to increase the number of chromosomes analysed. Inclusion of chromosomes 8, 14 and 20 to the standard set of chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22 allows the analysis of 12 chromosomes in three rounds of fluorescent in-situ hybridization (FISH) without decreasing the efficiency of the technique. Pregnancy rate was significantly increased when only embryos that had been diagnosed as normal for the 12 chromosomes analysed were transferred compared with transfer of embryos with any abnormality for chromosomes 8, 14 or 20 (P < 0.05). This study proves that the high efficiency and practical feasibility of FISH analysis of 12 chromosomes in PGD for aneuploidy is a superior approach than the standard nine-chromosome analysis in order to screen for abnormalities.

Preimplantation genetic diagnosis for gender selection in the USA.

Preimplantation genetic diagnosis (PGD) for gender selection for non-medical reasons has been considered an unethical procedure by several authors and agencies in the Western society on the basis that it could disrupt the sex ratio, that it discriminates against women and that it leads to disposal of normal embryos of the non-desired gender. In this study, the analysis of a large series of PGD procedures for gender selection from a wide geographical area in the USA shows that, in general, there is no deviation in preference towards any specific gender except for a preference of males in some ethnic populations of Chinese, Indian and Middle Eastern origin that represent a small percentage of the US population. In cases where only normal embryos of the non-desired gender are available, 45.5% of the couples elect to cancel the transfer, while 54.5% of them are open to have embryos transferred of the non-desired gender, this fact being strongly linked to cultural and ethnic background of the parents. In addition this study adds some evidence to the proposition that, in couples with previous children of a given gender, there is no biological predisposition towards producing embryos of that same gender. Based on these facts, it seems that objections to gender selection formulated by ethics committees and scientific societies are not well founded.

[Retroperitoneal laparoscopic treatment of symptomatic caliceal diverticular calculi]. / Traitement par laparoscopie rétropéritonéale d'un amas lithiasique intradiverticulaire caliciel symptomatique.

Treatment of urinary calculi in caliceal diverticular is indicated when they are symptomatic. Minimally invasive techniques, in particularly laparoscopic approach, occupy an increasingly important place in the urological therapeutic armamentarium and have changed from an open surgical approach to endoscopic treatment for the management of symptomatic caliceal diverticular calculi. Herein, we report the case of a woman with symptomatic calculi in an upper caliceal diverticular managed by retroperitoneal laparoscopic approach.

[Renocutaneous fistula: report of a chronic inguinal fistula]. / Fistules rénocutanées: à propos d'un cas de fistule chronique inguinale.

The authors report a new case of isolated spontaneous nephrocutaneous fistula revealed by a persisting draining sinus in the left groin. Their present causative factors are identified. The computed tomography and magnetic resonance imaging actually may resolve the diagnostic dilemma and are helpful in the surgical treatment.

[Is it possible to define a threshold for free PSA index that is useful in the daily practice of urology?]. / Est-il possible de définir un seuil de l'index de PSA libre utile en pratique urologique quotidienne?

OBJECTIVES: Retrospective evaluation of the use of the free PSA index before prostatic biopsies. MATERIAL AND METHODS: The authors retrospectively studied the values for total PSA, free PSA, and free PSA index (ratio of free PSA over total PSA expressed as a %) in men with a total PSA between 2 and 10 ng/ml, from a population of 391 men prior to prostatic biopsies. They also isolated a subgroup of patients in whom the free PSA index could have been used as a first-line marker to decide whether or not to perform prostatic biopsies. RESULTS: The mean values for total PSA, free PSA, and free PSA index were compared as a function of the diagnosis, age, and ultrasound prostatic volume. The yields of the various cut-off values for the free PSA index for PSA between 2 and 4 ng/ml, 4 and 10 ng/ml, and 2 and 10 ng/ml with a normal digital rectal examination are reported. Between 2 to 10 ng/ml, at a cut-off value of 30%, 94.1% of cancers would have been detected (sensitivity) and 22% of biopsies would have been avoided, 10 of which would have been useless, i.e. a 30.3% economy of useless biopsies not performed (specificity). At the cut-off value of 15%, less than half of cancers would have been detected (47.1%) and 90.9% of useless biopsies would have been avoided. Biases creating difficulties of interpretation were the assay kits, the reference population, age, storage of sera, and prostatic volume. CONCLUSION: The free PSA index would be a useful first-line parameter in only 12.7% of candidates for prostatic biopsies. The cut-off value of 30%, validated for our assay method, would be able to detect the majority of cancers in men aged 50 to 65 years, while avoiding biopsies in the third of men with no detectable cancer.

[Clinical assessment of free serum prostate specific antigen (PSA)]. / Evaluation clinique de la mesure de l'antigène spécifique de la prostate (PSA) sous sa forme libre sérique.

The prostate specific antigen (PSA) level represents all of the immunoreactive serum PSA, either free or bound to alpha-1-anti-chymotrypsin. Isolated assay of free PSA has demonstrated a higher free PSA/total PSA ratio in cases of benign prostatic hyperplasia (BPH) than in cases of cancer, suggesting the possible use of this ratio in the detection of prostatic cancer when the PSA level is between 4 and 10 ng/mL. We retrospectively assayed free PSA in 64 cases of localized prostate cancer, 90 cases of BPH before transurethral resection and 59 healthy controls. By comparing the mean values of the 3 populations and the ROC curves, we confirmed the superiority of the free PSA/total PSA ratio over total PSA in the detection of prostatic cancer, but these results, established in a retrospectively constituted population, need to be confirmed by prospective epidemiological studies. Nevertheless, in routine urological practice, we propose that free PSA assay be performed in all men with a PSA level between 4 and 10 ng/mL and a normal prostate on digital rectal examination.

[Salvage cryotherapy for recurrent prostate cancer after radiotherapy failure: principles, indications, patient selection, oncological results and morbidity]. / Cryothérapie de rattrapage après radiothérapie prostatique : principes, indications, sélection des patients, résultats oncologiques et morbidité

Potentially curative salvage options for biochemical failure after primary prostatic radiotherapy include salvage radical prostatectomy, brachytherapy, high-intensity focused ultrasound and cryotherapy. Salvage cryoablation for recurrent prostate cancer after irradiation failure is currently a well-established therapeutic option, since technical improvements have permitted better oncologic outcomes and lower complications rates over the years. This article reviews surgical technique, oncologic and functional outcomes, as well as morbidity and complications of salvage cryotherapy for local recurrence after external beam radiotherapy or brachytherapy for prostate cancer.

Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage-stage embryos.

Previous studies assessing the relationship between embryo development, maternal age and chromosome abnormalities were either small or analysed mostly embryos not suitable for replacement. The present study includes >6000 embryos, including many suitable for replacement. Embryos with the best morphology and development were 44% euploid in patients younger than 35, decreasing to 21% in patients 41 and older. The worst morphology group had only 30% normal embryos from patients younger than 35, and 12% in embryos from patients 41 and older. Thus morphological analysis was able to improve the population of normal embryos only from 30 to 44% in the best of cases. Regarding specific abnormalities, 20% of embryos were aneuploid, 32% aneuploid plus other abnormalities, and the rest had post-meiotic abnormalities. Of those, only aneuploidy increased with maternal age. There were no big differences in the frequency of chromosome abnormalities depending on patient indication, within a similar age group. In summary, previous trends detected in suboptimal embryos were also confirmed in the best embryos for replacement. Although dysmorphism and advanced maternal age are both related to chromosome abnormalities, these parameters can yield at most <50% euploid embryos, and other techniques such as preimplantation diagnosis are required to ensure that only euploid embryos are replaced.

Sequential G-banding FISH on human sperm chromosomes.

A method that allows the performance of double-colour chromosome painting (FISH) on previously G-banded human sperm metaphases has been developed. Sperm chromosomes were obtained by using the fusion technique between zona-free hamster oocytes and human spermatozoa. Single- and double-colour chromosome painting was performed using DNA libraries specific for chromosomes X, Y and 21 on either unstained or G-banded preparations. The hybridization efficiency was very high (98%). The sequential staining technique is very useful for analyses of structural (stable) and numerical chromosome aberrations in human sperm and thus can increase the efficiency of the human sperm-hamster oocytes fusion system to assess the risk to human germ cells as a result of endogenous and exogenous factors.

Sperm chromosome analysis in the father of a child with a de-novo reciprocal translocation t(11;15)(q12;q22) by G-banding and fluorescence in-situ hybridization.

Analysis of sperm chromosomes by G-banding and two-colour fluorescence in-situ hybridization (FISH) was carried out in the father of a child with a de-novo reciprocal translocation t(11;15)(q12;q22). Sperm chromosome complements were obtained after in-vitro fusion of zona-free hamster oocytes and donor spermatozoa. A total of 112 sperm complements was first analysed by G-banding. The frequency of structural chromosome aberrations (9.8%) and the conservative frequency of aneuploidy (0.0%) were not significantly different from those obtained in our control donors. The proportions of X-bearing (53.2%) and Y-bearing (46.8%) spermatozoa were not significantly different from the expected 1:1 ratio. A total of 313 sperm complements was analysed by two-colour FISH. The frequency of structural abnormalities for chromosomes 11 and 15 was 3.2 and 0.3% respectively. The frequency of rearrangements for chromosome 11 was statistically significant when compared with control donors (0.4%) (P < 0.0001). No spermatozoa with the t(11;15)(q12;q22) translocation were observed, showing no evidence for a germ-cell mosaicism. These results suggest that the de-novo involvement of chromosome 11 in a structural rearrangement is not random, and that in this patient an increased risk of de-novo structural chromosome abnormalities in further offspring does exist.

Cytogenetic analysis of spermatozoa in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23).

Analysis of sperm chromosomes was carried out in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23) by G-banding and chromosome painting. Sperm metaphases were obtained using the zona-free hamster oocyte-human sperm fusion technique. A total of 138 complements were sequentially analysed by G-banding and fluorescence in-situ hybridization (FISH). The frequency of spermatozoa with structural chromosome abnormalities (5.1%) and the estimated conservative aneuploidy (1.4%) were within the range obtained in our control donors (6.9 and 4%). The sex ratio (45.3% X versus 54.7% Y) was not significantly different from the theoretical 1:1. A total of 309 sperm complements was analysed by FISH, 138 sequentially analysed by G-banding-FISH and another 171 analysed by FISH only. The frequencies of structural chromosome abnormalities for chromosomes 7 and 9 (0.6 and 0% respectively) were not significantly different from those obtained in our control donors (0.6 and 0.8%). No spermatozoa with the t(7;9) (q22;p23) were observed, showing no evidence for a germ-cell mosaicism. A statistically significant, positive association between sperm breakpoints and fragile sites (P = 0.0225) was observed. However, the coincidence between fragile sites and sperm breaks (80%) was not significantly different from that obtained in our control donors (79.2%). These results suggest that in this case the risk of structural chromosome abnormalities in further offspring is not increased, although an association between fragile sites and sperm chromosome breaks in the father does exist.

Chromosome segregation in a man heterozygous for a pericentric inversion, inv(9)(p11q13), analyzed by using sperm karyotyping and two-color fluorescence in situ hybridization on sperm nuclei.

Analysis of sperm karyotypes and two-color fluorescent in situ hybridization (FISH) on sperm nuclei were carried out in a man heterozygous for the pericentric inversion inv(9)(p11q13). Sperm chromosome complements were obtained after in vitro fusion of zona-free hamster oocytes and donor sperm. A total of 314 sperm complements was analyzed: 153 (48.7%) carried the inverted chromosome 9 and 161 (51.3%) carried the normal one. None of the sperm complements contained a recombinant chromosome 9, suggesting that no chiasmata were formed in the heterochromatic region. The frequency of structural chromosome aberrations unrelated to the inversion (8.3%) and the frequency of conservative aneuploidy (3.2%) were within the limits observed in our control donors. The proportions of X-bearing (47.3%) and Y-bearing sperm (52.7%) were not significantly different from the expected 1:1 ratio. The percentage of disomy for chromosome 21 was analyzed by two-color FISH in 10336 sperm nuclei. The disomy rate for chromosome 21 (0.30%) was not significantly different from that found in our controls. These results suggest that the risk for this man of producing chromosomally abnormal offspring or spontaneous abortions was not increased, and do not support the existence of an interchromosomal effect for chromosome 21.

Comparison of the Bard Trak test with voided urine cytology in the diagnosis and follow-up of bladder tumors.

OBJECTIVES: To compare the results of the BTA Trak test with voided urine cytology (VUC) in the diagnosis and follow-up of bladder tumors. PATIENTS AND METHODS: Urine samples were obtained from 53 patients with bladder tumor (77 samples) and 53 patients treated for bladder tumor with no evidence of disease on the basis of cystoscopic evaluation (88 samples). Urine samples were collected prior to cystoscopy. The BTA assay was performed by the BTA Trak test according to the manufacturer's recommendations. A value >14 U/ml was considered abnormal. RESULTS: There was a statistically significant increase in median BTA value with increasing stage of tumor: 11.9, 57.9 and 391.0 U/ml respectively for stages pTa, pT1 and pT2/3 (p<0.0001, Kruskal-Wallis test). There was also a correlation between increasing grade and median BTA values measured at 6.9, 13.1 and 235.0 U/ml in grades 1, 2 and 3 tumors respectively (p<0.0001, Kruskall-Wallis test). The overall sensitivity of the BTA Trak test was 58.4% compared to 46.7% for VUC, a difference of 11.7%, which was statistically significant (McNemar test, p<0.005). The sensitivity of both tests combined was 63.6%. The specificity of the VUC (94.3%) was significantly higher than that of the BTA Traktrade mark (75.0%) (p<0.005, McNemar test). The accuracy of the Bard Trak test (67.3%) was similar to that of VUC (66.9%). CONCLUSION: The BTA Trak test is more sensitive than urinary cytology in the detection of bladder tumors but the improvement involved is insufficient to consider decreasing the frequency of endoscopic examinations in the follow-up of superficial bladder tumor.

An analysis of human sperm chromosome aneuploidy.

A sperm chromosome analysis of 24 men with normal or balanced karyotypes was carried out to study the frequency of sperm chromosome aneuploidy. A total of 3,446 human sperm complements (36-315 per donor) was analyzed after in vitro penetration of hamster eggs. Two sets of donors were studied at two different centers in the United States (center 1) and Spain (center 2). The frequencies of hyperhaploidy and hypohaploidy in control donors were similar between center 1 (1.9% vs. 7.7%) and center 2 (1.8% vs. 10.3%). In carrier donors there were no significant differences between the two centers in the frequency of hyperhaploidy (0.8% vs. 1.9%), but that of hypohaploidy was significantly higher in center 2 (11.0%) than in center 1 (4.6%). A significant excess of hypohaploid complements, as compared to hyperhaploid complements, was found in both centers in both control and carrier donors. The sex ratio was similar in both centers and did not differ significantly from a 1:1 sex ratio. The larger chromosomes in the complement (1, 2, 3, 4, 5, 7, and 10) presented a significantly lower frequency of hypohaploidy, while some of the smaller chromosomes (13, 19, and 21) showed a higher frequency of hypohaploidy than expected. Chromosome 21 and the sex chromosomes showed an increase in the percentage of hyperhaploidy, as compared to other chromosomes, that was close to statistical significance (P = 0.08). Our results reflect a preferential loss of small chromosomes during slide preparation and suggest that chromosome 21 and the sex chromosomes could be more frequently involved in aneuploidy.

Predictability of preimplantation genetic diagnosis of aneuploidy and translocations on prospective attempts.

The aim of this study was to determine if the outcomes of aneuploidy and translocation testing by preimplantation genetic diagnosis (PGD) at the 8-cell stage have a predictive value for new genetic diagnosis cycles. In total, 83 cycles (39 patients) undergoing PGD of translocations and 378 cycles (176 patients) of aneuploidy were included. Predictability, defined as having similar rate (+/-20%) of euploid embryos in the first and successive cycles, was found in 66% of patients undergoing aneuploidy testing. Predictability was found significantly more often in patients undergoing PGD of translocations (90%, P = 0.006). In addition, patients with 0, <30 or > or =30% euploid embryos in the first cycle were compared and groups 0 and <30% had significantly fewer euploid embryos in the second cycle (22-26%) than those of the group with > or =30% (37%) (P < 0.05). Patients who did not become pregnant after the first attempt were stimulated more aggressively than those becoming pregnant, producing significantly more embryos in the second than in the first cycle (P < 0.001). Therefore, correlation between euploidy rate and pregnancy rate could not be assessed objectively between cycles. In conclusion, the PGD results of a first cycle can predict the results of the second cycle, but this is likely to be of more value when the condition investigated is translocation rather than aneuploidy. The chance of pregnancy is usually related to the number of euploid embryos.

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis.

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.