Aneuploidy and recombination in the human preimplantation embryo. Copy number variation analysis and genome-wide polymorphism genotyping.

RESEARCH QUESTION: What are the incidence and patterns of meiotic trisomies and recombination separately and in relation to each other at the blastocyst stage via single nucleotide polymorphism genotyping combined with array comparative genomic hybridization. DESIGN: Single nucleotide polymorphism microarrays were carried out on a total of 1442 blastocyst stage embryos derived from 268 fertile couples undergoing preimplantation genetic diagnosis for the purposes of avoiding transmittance of known single gene disorders to their offspring; 24-chromosome aneuploidy screening via array comparative genomic hybridization was carried out in parallel. RESULTS: One hundred per cent of meiotic trisomies identified in these embryos were of maternal origin and their incidence increased significantly with advancing maternal age (P < 0.0001). A total of 55.8% of meiotic trisomies were meiosis I-type and 44.2% were meiosis II-type. Certain chromosomes were affected more by meiosis I-type errors, whereas others experienced more meiosis II-type errors. A detailed recombination analysis was carried out for 11,476 chromosomes and 17,763 recombination events were recorded. The average number of recombination sites was 24.0 ± 0.3 for male meiosis and 41.2 ± 0.6 for female meiosis (autosomes only). Sex-specific differences were observed in the locations of recombination sites. Comparative analysis conducted between 190 euploid embryos and 69 embryos presenting maternal meiotic trisomies showed similar recombination rates (P = 0.425) and non-recombinant chromatid rates (P = 0.435) between the two categories; differences, however, were observed when analysing embryos affected with specific maternal meiotic trisomies. CONCLUSIONS: This study yielded unique data concerning recombination and the origin of aneuploidies observed during the first few days of life and provides a novel insight into these important biological processes.

Short-term outcomes in patients with colon cancer treated with robotic right colectomy.

PURPOSE: To report a single surgeon series of consecutive robotic right colectomies (RRC) performed for non-metastatic right colon cancer. METHODS: A retrospective review of a prospectively maintained database of patients who underwent elective robotic right colectomy for right colon adenocarcinoma was conducted. Patients with stage 0-III disease were included in the study. Outcomes evaluated included operative time, number of lymph nodes harvested, estimated blood loss, time to return of bowel function, length of hospital stay, complications and a minimum of 6-month follow up. RESULTS: Forty-five consecutive patients were included in this study. The mean operative time was 175 min, the mean lymph nodes harvested were 22 and the mean length of hospital stay was 5 days. The mean time to normal bowel function restoration and to discontinuation of patient-controlled analgesia was 2 days. The hospital post-operative courses were complicated in two patients by ileus and fever due to pulmonary atelectasia, respectively. No conversions to laparotomy, reoperations or 90-day deaths were recorded. CONCLUSIONS: Robotic colorectal surgery has gained a lot of supporters through the years although a debate still exists concerning the outcomes. The present study is one of the largest evaluating short-term results of RRCs performed by a single surgeon. We believe we demonstrated the safety and efficacy of RRC in the treatment of right colon nonmetastatic adenocarcinoma.

Live birth derived from oocyte spindle transfer to prevent mitochondrial disease.

Mutations in mitochondrial DNA (mtDNA) are maternally inherited and can cause fatal or debilitating mitochondrial disorders. The severity of clinical symptoms is often associated with the level of mtDNA mutation load or degree of heteroplasmy. Current clinical options to prevent transmission of mtDNA mutations to offspring are limited. Experimental spindle transfer in metaphase II oocytes, also called mitochondrial replacement therapy, is a novel technology for preventing mtDNA transmission from oocytes to pre-implantation embryos. Here, we report a female carrier of Leigh syndrome (mtDNA mutation 8993T > G), with a long history of multiple undiagnosed pregnancy losses and deaths of offspring as a result of this disease, who underwent IVF after reconstitution of her oocytes by spindle transfer into the cytoplasm of enucleated donor oocytes. A male euploid blastocyst wasobtained from the reconstituted oocytes, which had only a 5.7% mtDNA mutation load. Transfer of the embryo resulted in a pregnancy with delivery of a boy with neonatal mtDNA mutation load of 2.36-9.23% in his tested tissues. The boy is currently healthy at 7 months of age, although long-term follow-up of the child's longitudinal development remains crucial.

Live births following Karyomapping of human blastocysts: experience from clinical application of the method.

The clinical application of a new, widely applicable method known as Karyomapping to carry out a total of 55 clinical cases of preimplantation genetic diagnosis (PGD) for single gene disorders is reported. Conventional polymerase chain reaction (PCR) testing was carried out in parallel to the new method for all cases. Clinical application of Karyomapping in this study resulted in three live births and nine clinical pregnancies out of 20 cases with a transfer. All in all, results presented in this study indicate that Karyomapping is a highly efficient, accurate and robust method for PGD of single gene disorders. Karyomapping can offer a more comprehensive assessment of the region of interest than conventional PCR analysis, allowing for more embryos to receive diagnosis (99.6% versus 96.8%), whereas its wide applicability reduces substantially the time that patients have to wait before starting their in vitro fertilization (IVF) cycle. Nonetheless, inclusion of elements of conventional PCR methodology, such as direct mutation detection, may be required in cases in which the gene of interest is in a region with reduced single nucleotide polymorphism (SNP) coverage (e.g. telomeric regions), when offering PGD for consanguineous couples, or in cases where no samples from additional family members are available.

Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders.

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach ('karyomapping') was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith-Lemli-Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD.

A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility.

BACKGROUND: Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζ(H398P)), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS: Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζ(H233L)), which is predicted to disrupt local protein interactions in a manner similar to PLCζ(H398P) and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζ(H233L) and PLCζ(H398P) exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS: Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms.

Re-Operative Laparoscopic Colorectal Surgery: A Systematic Review.

INTRODUCTION: Re-operative laparoscopic colorectal surgery is becoming increasingly common. It can be a challenging procedure, but its benefits can outweigh the associated risks. METHODS: A systematic review of the literature reporting re-operative laparoscopic surgery was carried out. Retrospective and prospective cohort studies and case series were included, with case reports being excluded. RESULTS: Seventeen articles dated from 2007 to 2020 were included in the systematic review. In total, 1555 patients were identified. Five hundred and seventy-four of them had a laparoscopic procedure and 981 an open re-operation. One hundred and eighty-three women had a laparoscopic operation. The median age ranged from to 44.9 years to 68.7 years. In seven studies, the indication of the index operation was malignancy, one study regarded re-laparoscopy for excision of lateral pelvic lymph nodes, and one study looked at redo surgery of ileal J pouch anal anastomosis. There were 16 mortalities in the laparoscopic arm (2.78%) and 93 (9.4%) in the open surgery arm. One hundred and thirty-seven morbidities were recorded in the open arm and 102 in the laparoscopic arm. Thirty-nine conversions to open occurred. The median length of stay ranged from 5.8 days to 19 days in laparoscopy and 9.7 to 34 days in the open surgery arm. CONCLUSIONS: Re-operative laparoscopic colorectal surgery is safe when performed by experienced hands. The management of complications, recurrence of malignancy, and lateral pelvic floor dissection can be safely performed. The complication rate is low, with conversion to open procedures being relatively uncommon.

Clinical application of sequencing-based methods for parallel preimplantation genetic testing for mitochondrial DNA disease and aneuploidy.

OBJECTIVE: To validate and apply a strategy permitting parallel preimplantation genetic testing (PGT) for mitochondrial DNA (mtDNA) disease and aneuploidy (PGT-A). DESIGN: Preclinical test validation and case reports. SETTING: Fertility centers. Diagnostics laboratory. PATIENTS: Four patients at risk of transmitting mtDNA disease caused by m.8993T>G (Patients A and B), m.10191T>G (Patient C), and m.3243A>G (Patient D). Patients A, B, and C had affected children. Patients A and D displayed somatic heteroplasmy for mtDNA mutations. INTERVENTIONS: Embryo biopsy, genetic testing, and uterine transfer of embryos predicted to be euploid and mutation-free. MAIN OUTCOME MEASURES: Test accuracy, treatment outcomes, and mutation segregation. RESULTS: Accuracy of mtDNA mutation quantification was confirmed. The test was compatible with PGT-A, and half of the embryos tested were shown to be aneuploid (16/33). Mutations were detected in approximately 40% of embryo biopsies from Patients A and D (10/24) but in none from Patients B and C (n = 29). Patients B and C had healthy children following PGT and natural conception, respectively. The m.8993T>G mutation displayed skewed segregation, whereas m.3243A>G mutation levels were relatively low and potentially impacted embryo development. CONCLUSIONS: Considering the high aneuploidy rate, strategies providing a combination of PGT for mtDNA disease and aneuploidy may be advantageous compared with approaches that consider only mtDNA. Heteroplasmic women had a higher incidence of affected embryos than those with undetectable somatic mutant mtDNA but were still able to produce mutation-free embryos. While not conclusive, the results are consistent with the existence of mutation-specific segregation mechanisms occurring during oogenesis and possibly embryogenesis.

Simultaneous assessment of aneuploidy, polymorphisms, and mitochondrial DNA content in human polar bodies and embryos with the use of a novel microarray platform.

OBJECTIVE: To develop a microarray platform that allows simultaneous assessment of aneuploidy and quantification of mitochondrial DNA (mtDNA) in human polar bodies and embryos. DESIGN: Optimization and validation applied to cell lines and clinical samples (polar bodies, blastomeres, and trophectoderm biopsies). SETTING: University research laboratory and a preimplantation genetic diagnosis (PGD) reference laboratory. PATIENT(S): Samples from 65 couples who underwent PGD for aneuploidy and/or a single-gene disorder. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 1) Comparison of aneuploidy screening results obtained with the use of the new microarray with those derived from two well established cytogenetic techniques. 2) mtDNA quantification. 3) Analysis of single-nucleotide polymorphisms. RESULT(S): The fully optimized microarray was estimated to have an accuracy of ≥97% for the detection of individual aneuploidies and to detect 99% of chromosomally abnormal embryos. The microarray was shown to accurately determine relative quantities of mtDNA. Information provided from polymorphic loci was sufficient to allow confirmation that an embryo was derived from specific parents. CONCLUSION(S): It is hoped that methods such as those reported here, which provide information on several aspects of oocyte/embryo genetics, could lead to improved strategies for identifying viable embryos, thereby increasing the likelihood of successful implantation. Additionally, the provision of genotyping information has the potential to reveal DNA contaminants and confirm parental origin of embryos.

Successful Live Birth following Preimplantation Genetic Diagnosis for Phenylketonuria in Day 3 Embryos by Specific Mutation Analysis and Elective Single Embryo Transfer.

Phenylketonuria (PKU) is an autosomal recessive inherited metabolic disorder caused by a complete or near-complete deficiency of the liver enzyme phenylalanine hydroxylase (PAH), which converts the amino acid phenylalanine to tyrosine, leading to the increase of blood and tissue concentration of phenylalanine to toxic levels. PKU is not life threatening but is treated through lifelong dietary management. If untreated, it can lead to severe learning disability, brain function abnormalities, behavioural and neurological problems. The non-life threatening nature of PKU has until now caused some debate on whether to licence its detection by preimplantation genetic diagnosis (PGD). We report the first successful live birth in the UK following single cell embryo biopsy and PGD for the detection of two different mutations in the (PAH) gene. This case highlights both an important scientific development as well as the ethical challenge in offering couples who carry PKU this new reproductive option when starting their family.

Characterization of two heterozygous mutations of the oocyte activation factor phospholipase C zeta (PLCζ) from an infertile man by use of minisequencing of individual sperm and expression in somatic cells.

OBJECTIVE: To examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLCζ(H398P), histidine > proline) and PLCζ(H233L) (histidine > leucine) mutations were previously identified. DESIGN: Laboratory-based study. SETTING: University laboratory. PATIENT(S): An infertile 38-year-old man with significantly impaired oocyte activation ability. INTERVENTION(S): Minisequencing of individual sperm for PLCζ(H398P) and PLCζ(H233L), and investigation of localization patterns arising from the expression of fluorescently tagged PLCζ isoforms in HEK293T cells. MAIN OUTCOME MEASURE(S): The presence/absence of PLCζ(H398P) and PLCζ(H233L) determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCζ isoforms quantified in HEK293T cells. RESULT(S): Sperm possessed either PLCζ(H233L) or PLCζ(H398P), but never both at the same time. Fluorescent PLCζ(H233L) and PLCζ(H233L+H398P) (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLCζ(WT) > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCζ(H233L+H398P) exhibited a statistically significantly reduced level of fluorescence compared with PLCζ(H398P) at 48 hours but not 56 hours. CONCLUSION(S): Both H398P and H233L mutations are present on different alleles and do not alter PLCζ localization in HEK293T cells. Loss-of-activity mutations in PLCζ may contribute not only toward male infertility but also male subfertility in cases where PLCζ is mutated on a single allele.