Three-dimensional Liver Model Application for Liver Transplantation.

BACKGROUND: Children are removed from the liver transplant waitlist because of death or progressive illness. Size mismatch accounts for 30% of organ refusal. This study aimed to demonstrate that 3-dimensional (3D) technology is a feasible and accurate adjunct to organ allocation and living donor selection process. METHODS: This prospective multicenter study included pediatric liver transplant candidates and living donors from January 2020 to February 2023. Patient-specific, 3D-printed liver models were used for anatomic planning, real-time evaluation during organ procurement, and surgical navigation. The primary outcome was to determine model accuracy. The secondary outcome was to determine the impact of outcomes in living donor hepatectomy. Study groups were analyzed using propensity score matching with a retrospective cohort. RESULTS: Twenty-eight recipients were included. The median percentage error was -0.6% for 3D models and had the highest correlation to the actual liver explant (Pearson's R = 0.96, P < 0.001) compared with other volume calculation methods. Patient and graft survival were comparable. From 41 living donors, the median percentage error of the allograft was 12.4%. The donor-matched study group had lower central line utilization (21.4% versus 75%, P = 0.045), shorter length of stay (4 versus 7 d, P = 0.003), and lower mean comprehensive complication index (3 versus 21, P = 0.014). CONCLUSIONS: Three-dimensional volume is highly correlated with actual liver explant volume and may vary across different allografts for living donation. The addition of 3D-printed liver models during the transplant evaluation and organ procurement process is a feasible and safe adjunct to the perioperative decision-making process.

Utilization of Segmental Grafts Is Associated With Higher Transplant Rates in Pediatric Patients.

INTRODUCTION: In July 2017, a policy to increase the use of segmental grafts (SGs) was implemented at our institution. The aim was to compare changes in waitlist activity after implementation of this policy. METHODS: A single-center, retrospective study. Pediatric patients on the liver waiting list between January 2015 and December 2019 were screened. Patients were classified as receiving a liver transplant (LT) before (Period 1) or after (Period 2) policy changes. Primary end points were transplant rates and time to transplant. RESULTS: Sixty five first LT performed on 65 patients were included. Twenty nine LT were performed during Period 1 and 36 during Period 2. More than half (55%) of LT in Period 2 were SG, compared to 10.3% in Period 1 (P < 0.001). Forty nine and 56 pediatric candidates on the waiting list accounted for 38.78 and 24.48 person-years during Period 1 and Period 2, respectively. Transplant rates per 100 person-years on the waiting list increased from 85.09 during Period 1 to 187.87 in Period 2 (Rate ratio: 2.20; P < 0.001). Median time to receive a LT decreased from 229 d in Period 1 to 75 d during Period 2 (P = 0.013). One-year patient survival rates were 96.6% in Period 1 and 95.7% in Period 2. One-year graft survival rates were 89.7% and 88% in Period 1 and Period 2, respectively. CONCLUSIONS: A policy to increase the use of SG was associated with significantly higher transplant rates and lower waiting times. Implementation of this policy can be done successfully with no observed negative impact on patient and graft survival.

Performance of a Prospective Anticoagulation Stratification Algorithm After Liver Transplantation.

Venous thromboembolism (VTE) occurs in 0.4% to 15.5% and bleeding occurs in 20% to 35% of patients after liver transplantation (LT). Balancing the risk of bleeding from therapeutic anticoagulation and risk of thrombosis in the postoperative period is challenging. Little evidence exists regarding the best treatment strategy for these patients. We hypothesized that a subset of LT patients who develop postoperative deep vein thromboses (DVTs) could be managed without therapeutic anticoagulation. We implemented a quality improvement (QI) initiative using a standardized Doppler ultrasound-based VTE risk stratification algorithm to guide parsimonious implementation of therapeutic anticoagulation with heparin drip. Methods: In a prospective management QI initiative for DVT management, we compared 87 LT historical patients (control group; January 2016-December 2017) to 182 LT patients (study group; January 2018-March 2021). We analyzed the rates of immediate therapeutic anticoagulation after DVT diagnosis within 14 d of LT, clinically significant bleeding, return to the operating room, readmission, pulmonary embolism, and death within 30 d of LT before and after the QI initiative. Results: Ten patients (11.5%) in the control group and 23 patients (12.6%; P = 0.9) in the study group developed DVTs after LT. Immediate therapeutic anticoagulation was used in 7 of 10 and 5 of 23 patients in the control and study groups, respectively (P = 0.024). The study group had lower odds of receiving immediate therapeutic anticoagulation after VTE (21.7% versus 70%; odds ratio = 0.12; 95% confidence interval, 0.019-0.587; P = 0.013) and a lower rate of postoperative bleeding (8.7% versus 40%; odds ratio = 0.14, 95% confidence interval, 0.02-0.91; P = 0.048). All other outcomes were similar. Conclusions: Implementing a risk-stratified VTE treatment algorithm for immediate post-LT patients appears to be safe and feasible. We observed a decrease in the use of therapeutic anticoagulation and a lower rate of postoperative bleeding without adverse impacts on early outcomes.

Epidemiology and outcomes of surgical site infections among pediatric liver transplant recipients.

INTRODUCTION: Surgical site infections (SSI) are a significant cause of morbidity in liver transplant recipients, and the current data in the pediatric population are limited. The goal of this study was to identify the incidence, classification, risk factors, and outcomes of SSIs among children undergoing liver transplantation (LT). METHODS: A single-center, retrospective descriptive analysis was performed of patients age ≤18 years undergoing LT between September 2007 and April 2017. SSI identified within the first 30 days were analyzed. Primary endpoints included incidence, classification, risk factors, and outcomes associated with SSIs. RESULTS: We included 86 patients, eight patients (9.3%) developed SSIs. Among segmental grafts (SG) recipients, 7/61 (11.4%) developed SSI. Among whole grafts recipients, 1/25 (4%) developed SSI. SSIs were associated with the presence of biliary complications (35% vs. 3%, p < .01; odds ratios 24, 95% CI: 3.41-487.37, p<.01). There were no differences in long term graft or patient survival associated with SSI. Patients who developed SSI were more likely to undergo reoperation (50% vs. 16.7%, p = .045) and had an increased total number of hospital days in the first 60 days post-transplant (30.5 vs. 12.5 days, p = .001). CONCLUSIONS: SSIs after pediatric LT was less frequent than what has been previously reported in literature. SSIs were associated with the presence of biliary complications without an increase in mortality. SG had an increased rate of biliary complications without an association to SSIs but, considering its positive impact on organ shortage barriers, should not be a deterrent to the utilization of SGs.

Role of Female Research at the Asociacion Mexicana de Cirugia General Annual Meeting: A Retrospective Analysis From 2013 to 2019.

Background: Academic surgery has been a traditionally male-dominated field. Female contribution remains challenging. In Mexico, there is no published evidence regarding gender disparity in academic surgery. We aimed to analyze the female role in clinical research submitted to the Asociación Mexicana de Cirugía General (AMCG). Methods: Retrospective study evaluating abstracts submitted to AMCG annual meetings from 2013 to 2019. Categorical variables were compared using χ2 test. Univariate logistic regression was performed to calculate odds ratios (OR) followed by a log-binomial logistic regression model to obtain the adjusted relative risk (aRR) for acceptance as an oral presentation. Results: Overall, 7,439 abstracts were analyzed of which 24.2% were submitted by females. Female-submitted abstracts increased from 22.5% to 25.3% during 2013-2019 (p = 0.15). The proportion of 47 abstracts submitted by females was higher in the resident group (27.7% vs. 18.8%; p < 0.001). The percentage of females' abstracts selected for oral presentation was less than the percentage of males' 49 abstracts selected for presentation (9% vs. 11.5%; p = 0.002). Females' abstracts submitted have a 50 23.5% decreased chance of being selected for oral presentation (OR = 0.765, CI 95%, 0.639-0.917, 51 p = 0.003). However, after adjusting for research type and trainee status, the gender of the oral 52 presenting author showed no association (aRR = 0.95, CI 95%, 0.8-1.1, p = 0.56). Conclusion: In Mexico, the female role in academic surgery is still limited. These results should 55 encourage professors and program directors to identify and address factors contributing to gender 56 disparities.

Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients.

BACKGROUND: MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. METHODS: Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR. RESULTS: Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level. CONCLUSIONS: Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

Longitudinal variance of Donor-Derived Cell-Free DNA (dd-cfDNA) in Stable Kidney Transplant (KTx) patients are influenced by donor/recipient variables.

BACKGROUND: The longitudinal time-course of dd-cfDNA after kidney transplant (KTx) is not well-described. The cut off values of dd-cfDNA in KTx derive from biopsy-coupled single measurements. Meaningful interpretation necessitates understanding of: (1) time variance of dd-cfDNA levels post-KTx, (2) factors determining biologic variability, and (3) relationship to donor and recipient characteristics. We hypothesized that an understanding of the aforementioned factors would better inform clinical decision-making using dd-cfDNA. METHODS: One hundred and twenty five KTx patients with dd-cfDNA obtained longitudinally were included. Univariate analyses were directed at inter-patient variability and intra-patient inter-occasion variability of dd-cfDNA. Multivariate linear regression was used in analyses accounting for repeat measures. RESULTS: At 1-month post KTx median dd-cfDNA: (1) were higher in repeat KTx (.57%, P < .001), and dual KTx (1.10%, P = ns) versus a first KTx (.31%); (2) showed a significant difference in donor after cardiac death (DCD [.45%]) versus living related (LRD [.27%]) donors (P = .036). Longitudinal (1-3 months) dd-cfDNA measurements showed a significant downtrend for all donor types. Panel-reactive antibodies (PRA) were positively correlated with dd-cfDNA. CONCLUSIONS: Repeat Tx, dual Tx, DCD, and PRA are associated with a higher dd-cfDNA. Incorporation of donor/recipient variables and time down post transplant is material for rational interpretation of dd-cfDNA.

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/µL with a range of 768-7510 copies/µL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

Liver resection for metastatic thyroid carcinoma. Case report and literature review.

Liver resection for metastatic cancer has become the standard of care for specific groups of patients, including noncolorectal non-neuroendocrine liver metastases (NCNNELM). Liver metastasis from differentiated thyroid carcinoma is considered rare, with an approximated frequency of 0.5%. We present a case of metastatic papillary thyroid carcinoma (PTC) to the liver and literature review. Herein, we report a 72-year-old male that underwent formal left hepatectomy for 4.4 cm metastatic PTC generating left bile duct obstruction. Two months after, presented with multiple small lesions within the hepatic parenchyma and diffuse ductal dilatation of the right biliary system. Therefore, treated with a percutaneous biliary drain placement without complications. In a patient diagnosed with initial Stage II PTC, undergoing total thyroidectomy 10 years before presenting to the clinic. Bearing over a decade of treatments for local and distal recurrences. We believe approaching strategies for this specific disease should be developed to establish standard management.

A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

Retrospective cohort of pancreatic and Vater ampullary adenocarcinoma from a reference center in Mexico.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) and ampulla of Vater adenocarcinomas (AVAC) are periampullary tumors. These tumors have overlapping symptoms and a common treatment, but present differences in their survival and biology. No recent studies in Mexico have been published that describe the clinicopathological characteristics of these tumors. Therefore, the aim of this study was to describe the clinicopathological characteristics of PDAC and AVAC in patients at a reference center in Mexico. METHODS: A retrospective cohort of patients with PDAC or AVAC was analyzed at our institution (July 2007 to June 2016). Inferential analysis of the clinical data was performed with Student's t-test or a χ2 test with odds ratios (OR) and confidence intervals (CI), depending on the variables. Overall survival was compared using Kaplan-Meier curves with log-rank p values. RESULTS: Forty patients with PDAC and 76 with AVAC were analyzed, including 77 females and 39 males with a mean age of 60.6 years and a mean evolution time of 5.7 months. PDAC patients had more abdominal pain, a larger tumor size and more advanced stages than AVAC patients. In contrast, AVAC patients had more jaundice, a higher percentage of complete resections and higher overall survival. Up to 70% of patients were overweight. PDAC cohort included a higher proportion of smokers. CONCLUSIONS: Our cohort was slightly younger, had a larger percentage of females, and a greater percentage of obese patients than those in many international reports. A high proportion of PDAC patients are diagnosed in advanced stages and have a low likelihood of resectability.

The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy.

Female meiosis is comprised by two cell divisions, meiosis I (MI) and II (MII) and two different stages at which the development of the oocyte is temporarily halted. In the case of MI, this pause can potentially last for four to five decades. This added layer of complexity distinguishes female gametogenesis from its male counterpart. The single most important genetic factor impacting human reproductive success is aneuploidy. Aneuploid embryos may undergo permanent arrest during preimplantation development, fail to implant or spontaneously abort. Most aneuploidies originate during female meiosis and become increasingly common with advancing maternal age. To shed further light on the nature of aneuploidy in human oocytes, we utilized comparative genomic hybridization (CGH) to provide a detailed cytogenetic analysis of 308 first and second polar bodies (PBs). These were biopsied from fertilized oocytes, generated by 70 reproductively older women (average maternal age of 40.8 years). The total oocyte abnormality rate was 70%, and MII anomalies predominated over MI (50% aneuploidy rate versus 40.3%). Both whole chromosome non-disjunction and unbalanced chromatid predivision were seen, but the latter was the dominant MI aneuploidy-causing mechanism. Chromosome losses occurred more frequently than chromosome gains, especially during MI. Chromosomes of all sizes were found to participate in aneuploidy events, although errors involving smaller chromosomes were more common. These data reveal the spectrum of aneuploidies arising after each meiotic division, indicating that oocyte-derived abnormalities present at conception differ from those observed in established pregnancies. It is also clear that advancing maternal age had a significant adverse effect on female meiosis, and that this effect is most pronounced in MII. Indeed, our data suggest that MII may be more susceptible to age-related errors than MI.

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos.

OBJECTIVE: To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS). DESIGN: Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). SETTING: Reference laboratory. PATIENT(S): Patients donating embryos or undergoing PGS. INTERVENTION(S): Embryo biopsy, array-CGH, FISH reanalysis. MAIN OUTCOME MEASURE(S): Diagnosis, no result rate and error rate. RESULT(S): Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy. CONCLUSION(S): Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

Preimplantation genetic diagnosis of single-gene disorders: experience with more than 200 cycles conducted by a reference laboratory in the United States.

OBJECTIVE: To evaluate trends and outcomes from preimplantation genetic diagnosis (PGD) cycles. DESIGN: Retrospective data review. SETTING: A reference laboratory specializing in the provision of PGD services. PATIENT(S): One hundred sixty-two patients at risk of transmitting a serious monogenic disorder to their children. INTERVENTION(S): In vitro fertilization and PGD. MAIN OUTCOME MEASURE(S): Results of PGD cycles. RESULT(S): Two hundred twenty-four PGD cycles were referred by 59 different IVF centers. Forty-six different disorders were diagnosed, including several not previously diagnosed at the preimplantation stage. Cystic fibrosis was the most common reason for referral (73 cases). A diagnosis was obtained for 84.4% of tested embryos, with results available 6 to 36 hours from sample receipt. Only 10.7% of cycles had no transfer. The pregnancy rate per cycle with ET was 43.4%. CONCLUSION(S): Unlike previous reports of multiple PGD cycles, all of the cases in this study involved shipping of biopsied cells to a specialist reference laboratory for diagnosis. This approach, sometimes referred to as "transport PGD," accounts for the vast majority of PGD cycles in the United States. Preimplantation genetic diagnosis was shown to be an effective alternative to prenatal diagnosis for patients with an ethical or a religious objection to pregnancy termination and for infertile patients carrying a genetic disorder. Demand for this service at our center doubled in each of the last 4 years. Pregnancy rates per ET were encouraging, almost half of all patients undergoing their first PGD cycle achieving a birth or ongoing pregnancy.

A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing.

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or small-range mutation in the F8 gene, without the need for further custom-made modifications.

Multiplex fluorescent analysis of four short tandem repeats for rapid haemophilia A molecular diagnosis.

Indirect molecular diagnosis of hemophiliaA (HA) is carried out by analyzing intragenic polymorphic markers described along the coagulation factorVIII (FVIII) gene. Several studies have demonstrated that the two commonly used intronic short tandem repeats (STR13 and STR22) located in the FVIII gene are highly informative for this task. Two extragenic markers closely linked to FVIII (DXS1073 and DXS1108) have also been described as valuable tools for gene tracking. The objective of the present work was to develop a rapid, single-tube automated method to simultaneously analyze these four STRs. Consistent amplification was achieved by quadruplex fluorescent PCR and the products were analyzed by capillary electrophoresis. Validation of the method included DNA analysis of 88 individuals from a control population, 45 HA patients and 32 individuals from 10 HA-affected families. Statistical study showed that the STR13, STR22 and DXS1108 loci were in significant linkage disequilibrium, whereas DXS1073 was not. Nevertheless, the combination of the four markers offered a high heterozygosity rate (>90%) that improved tracing of FVIII gene inheritance. Optimal results with application to single cells in a HA preimplantation genetic diagnosis (PGD) protocol demonstrated the sensitivity of the technique. In conclusion, the automated fluorescent method described is an extremely rapid, simple and highly informative one that is easy to standardize and allows direct comparison of results among different groups working with genetic counseling, prenatal diagnosis and PGD in HA-affected families.

Multiple mutation analysis of the cystic fibrosis gene in single cells.

PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes.

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.