Integrating imaging-based classification and transcriptomics for quality assessment of human oocytes according to their reproductive efficiency.

PURPOSE: Utilising non-invasive imaging parameters to assess human oocyte fertilisation, development and implantation; and their influence on transcriptomic profiles. METHODS: A ranking tool was designed using imaging data from 957 metaphase II stage oocytes retrieved from 102 patients undergoing ART. Hoffman modulation contrast microscopy was conducted with an Olympus IX53 microscope. Images were acquired prior to ICSI and processed using ImageJ for optical density and grey-level co-occurrence matrices texture analysis. Single-cell RNA sequencing of twenty-three mature oocytes classified according to their competence was performed. RESULT(S): Overall fertilisation, blastulation and implantation rates were 73.0%, 62.6% and 50.8%, respectively. Three different algorithms were produced using binary logistic regression methods based on "optimal" quartiles, resulting in an accuracy of prediction of 76.6%, 67% and 80.7% for fertilisation, blastulation and implantation. Optical density, gradient, inverse difference moment (homogeneity) and entropy (structural complexity) were the parameters with highest predictive properties. The ranking tool showed high sensitivity (68.9-90.8%) but with limited specificity (26.5-62.5%) for outcome prediction. Furthermore, five differentially expressed genes were identified when comparing "good" versus "poor" competent oocytes. CONCLUSION(S): Imaging properties can be used as a tool to assess differences in the ooplasm and predict laboratory and clinical outcomes. Transcriptomic analysis suggested that oocytes with lower competence may have compromised cell cycle either by non-reparable DNA damage or insufficient ooplasmic maturation. Further development of algorithms based on image parameters is encouraged, with an increased balanced cohort and validated prospectively in multicentric studies.

Systematic review and meta-analysis: does pre-implantation genetic testing for aneuploidy at the blastocyst stage improve live birth rate?

PURPOSE: To establish if preimplantation genetic testing for aneuploidy (PGT-A) at the blastocyst stage improves the composite outcome of live birth rate and ongoing pregnancy rate per embryo transfer compared to conventional morphological assessment. METHODS: A systematic literature search was conducted using PubMed, EMBASE and Cochrane database from 1st March 2000 until 1st March 2022. Studies comparing reproductive outcomes following in vitro fertilisation using comprehensive chromosome screening (CCS) at the blastocyst stage with traditional morphological methods were evaluated. RESULTS: Of the 1307 citations identified, six randomised control trials (RCTs) and ten cohort studies fulfilled the inclusion criteria. The pooled data identified a benefit between PGT-A and control groups in the composite outcome of live birth rate and ongoing pregnancy per embryo transfer in both the RCT (RR 1.09, 95% CI 1.02-1.16) and cohort studies (RR 1.50, 95% CI 1.28-1.76). Euploid embryos identified by CCS were more likely to be successfully implanted amongst the RCT (RR 1.20, 95% CI 1.10-1.31) and cohort (RR 1.69, 95% CI 1.29-2.21) studies. The rate of miscarriage per clinical pregnancy is also significantly lower when CCS is implemented (RCT: RR 0.73, 95% CI 0.56-0.96 and cohort: RR 0.48, 95% CI 0.32-0.72). CONCLUSIONS: CCS-based PGT-A at the blastocyst biopsy stage increases the composite outcome of live births and ongoing pregnancies per embryo transfer and reduces the rate of miscarriage compared to morphological assessment alone. In view of the limited number of studies included and the variation in methodology between studies, future reviews and analyses are required to confirm these findings.

Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes.

Autosomal aneuploidy is the leading cause of embryonic and foetal death in humans. This arises mainly from errors in meiosis I or II of oogenesis. A largely ignored source of error stems from germinal mosaicism, which leads to premeiotic aneuploidy. Molecular cytogenetic studies employing metaphase fluorescence in situ hybridization and comparative genomic hybridisation suggest that premeiotic aneuploidy may affect 10-20% of oocytes overall. Such studies have been criticised on technical grounds. We report here an independent study carried out on unmanipulated oocytes that have been analysed using next generation sequencing (NGS). This study confirms that the incidence of premeiotic aneuploidy in an unselected series of oocytes exceeds 10%. A total of 140 oocytes donated by 42 women gave conclusive results; of these, 124 (88.5%) were euploid. Sixteen out of 140 (11.4%) provided evidence of premeiotic aneuploidy. Of the 140, 112 oocytes were immature (germinal vesicle or metaphase I), of which 10 were aneuploid (8.93%); the remaining 28 were intact metaphase II - first polar body complexes, and six of these were aneuploid (21.4%). Of the 16 aneuploid cells, half contained simple errors (one or two abnormal chromosomes) and half contained complex errors. We conclude that germinal mosaicism leading to premeiotic aneuploidy is a consistent finding affecting at least 10% of unselected oocytes from women undergoing egg collection for a variety of reasons. The importance of premeiotic aneuploidy lies in the fact that, for individual oocytes, it greatly increases the risk of an aneuploid mature oocyte irrespective of maternal age. As such, this may account for some cases of aneuploid conceptions in very young women.

Preimplantation Genetic Testing for Aneuploidy: Current Perspectives.

Despite improvements in assisted reproduction techniques (ARTs), live birth rates remain suboptimal, particularly in women with advanced maternal age (AMA). The leading cause of poor reproductive outcomes demonstrated in women with AMA, as well as women with recurrent miscarriage and repetitive implantation failure, is thought to be due to high rates of embryonic aneuploidy. Preimplantation genetic testing for aneuploidies (PGT-A) aims to select an euploid embryo for transfer and therefore improve ART outcomes. Early PGT-A studies using fluorescent in situ hybridization on mainly cleavage-stage biopsies failed to show improved delivery rates and, in certain cases, were even found to be harmful. However, the development of comprehensive chromosome screening, as well as improvements in culture media and vitrification techniques, has resulted in an emerging body of evidence in favor of PGT-A, demonstrating higher implantation, pregnancy, and live birth rates. While there are concerns regarding the potential harm of invasive biopsy and the cost implications of PGT-A, the introduction of noninvasive techniques and the development of new high-throughput methods which lower costs are tackling these issues. This review aims to assess the evidence for PGT-A, address possible concerns regarding PGT-A, and also explore the future direction of this technology.

Chromosomal Analysis of Cumulus Cells as a Future Predictor for Oocyte Aneuploidy: A Case Report.

BACKGROUND: Within the ovary, the optimal growth of the follicle, oocyte maturation and ovulation are highly conditioned by the two-way cross talk and interactions between the oocyte and the immediate somatic cells, known as cumulus cells (CCs). This biological communication between cell lines triggered the interest in the study of CCs as a biomarker of oocyte competence. CASE PRESENTATION: The findings of a 45,X mosaic pattern on CCs from a female patient with unremarkable medical history are reported in this study. The patient came to the Centre for Reproductive and Genetic Health, London on 14th August 2019 for her first visit and the follow up procedures were done for her to determine underlying genetic status. For this purpose, four sources of DNA including CCs, blood lymphocytes, buccal cells and immature oocytes were analyzed in the present report. CONCLUSION: In the present case study, the hypothesis of the female patient being mosaic 45,X was confirmed although the degree of mosaicism and whether this was affecting the germinal line could not be determined. In the event of the discovery of a cell line with an apparently abnormal genetic makeup, genetic counselling is important in order to understand the implications from somatic to germinal cells for patients exploring fertility journeys.

Chromosome Y as a marker for sex discrepancies in patients with organ transplants: a case report.

BACKGROUND: Organ transplantations cause discrepancy in results from cell-free DNA (cfDNA) testing, but scientific literature is scarce. CASE: A 33-year old gravida underwent cfDNA testing, which showed high levels of Y chromosome (ChrY) in the maternal bloodstream. The ChrY pattern was comparable to an adult male reference. As a result, cfDNA testing was only informative for autosomes. Routine 20-week ultrasound scan showed no structural alterations and the presence of female external genitalia. Post-clinical research revealed that the patient received a bone marrow transplant from a male donor several years before. Fluorescence in situ hybridization showed that 100% of nuclei analysed from the patient's lymphocytes presented a ChrY. CONCLUSION: This case demonstrates ChrY can be used as a marker to avoid sex discrepancies in certain patients with organ transplants.

Live birth rate is associated with oocyte yield and number of biopsied and suitable blastocysts to transfer in preimplantation genetic testing (PGT) cycles for monogenic disorders and chromosomal structural rearrangements.

OBJECTIVES: To investigate whether live birth (LB) is associated with oocyte yield and number of biopsied and suitable blastocyst to transfer following preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR). STUDY DESIGN: All couples underwent controlled ovarian stimulation, blastocyst biopsy, vitrification and transfer of suitable embryo(s) in a frozen embryo transfer (FET) cycle. RESULTS: Of 175 couples who underwent PGT treatment, 249 oocytes retrievals were carried out and 230 FET were subsequently undertaken. 122/230 (53%, 95% CI 47-59) FET resulted in a LB and 16/230 (7%, 95% CI 4-11) have resulted in ongoing pregnancies. 21/230 (9%, 95% CI 6-14) FET resulted in miscarriage and 69/230 (30%, 95% CI 24-36) concluded with failed implantation. Two (1%, 95% CI 0-3) transfers underwent termination for congenital malformation, with no evidence of misdiagnosis by prenatal testing. The relationship between number of oocytes retrieved and number of blastocysts biopsied and suitable embryos to transfer were significant (p = 0.00; Incidence rate ratio (IRR) 1.05; 95% 1.04-1.06; p = 0.00; IRR 1.04; 95%, 1.03-1.06), respectively. The number of oocytes collected (p = 0.007; OR 1.06; 95% CI 1.01-1.10), the number of blastocysts biopsied (p = 0.001; OR 1.14; 95% 95% CI 1.06-1.23) and the number of suitable embryos to transfer (p = 0.00; OR 1.38; 95% CI 1.17-1.64) were all significantly associated with the odds of achieving a LB. There is a 14% and 38% increased chance of a LB per additional blastocyst biopsied and suitable embryo to transfer, respectively. CONCLUSIONS: PGT-M and PGT-SR outcomes are significantly associated with egg yield, number of blastocysts to biopsy and suitable embryos to transfer.

Optimal bone fracture repair requires 24R,25-dihydroxyvitamin D3 and its effector molecule FAM57B2.

The biological activity of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] remains controversial, but it has been suggested that it contributes to fracture healing. Cyp24a1-/- mice, synthesizing no 24R,25(OH)2D3, show suboptimal endochondral ossification during fracture repair, with smaller callus and reduced stiffness. These defects were corrected by 24R,25(OH)2D3 treatment, but not by 1,25-dihydroxyvitamin D3. Microarrays with Cyp24a1-/- callus mRNA identified FAM57B2 as a mediator of the 24R,25(OH)2D3 effect. FAM57B2 produced lactosylceramide (LacCer) upon specific binding of 24R,25(OH)2D3. Fam57b inactivation in chondrocytes (Col2-Cre Fam57bfl/fl) phenocopied the callus formation defect of Cyp24a1-/- mice. LacCer or 24R,25(OH)2D3 injections restored callus volume, stiffness, and mineralized cartilage area in Cyp24a1-null mice, but only LacCer rescued Col2-Cre Fam57bfl/fl mice. Gene expression in callus tissue suggested that the 24R,25(OH)2D3/FAM57B2 cascade affects cartilage maturation. We describe a previously unrecognized pathway influencing endochondral ossification during bone repair through LacCer production upon binding of 24R,25(OH)2D3 to FAM57B2. Our results identify potential new approaches to ameliorate fracture healing.

Karyomapping: a single centre's experience from application of methodology to ongoing pregnancy and live-birth rates.

This study aimed to determine whether karyomapping can be applied to couples requiring preimplantation genetic diagnosis (PGD) for single gene disorder (SGD) and/or chromosomal rearrangement. 75/82 (91.5%) and 6/82 (7.3%) couples were referred for autosomal SGD and X-linked disease, respectively. One couple (1.2%) was referred for SGD and chromosomal rearrangement. Of 608 embryos, 146 (24%, 95% CI 21-28) day-3 and 462 (76%, 95% CI 72-79) blastocyst biopsies were performed. A total of 81 embryo transfers were performed; 16/81 (20%) were following day-3 embryo biopsy, 65/81 (80%) were following blastocyst biopsy and cryopreserved embryo transfer. Of 81 embryo transfers with known pregnancy outcome, 51 (63%, 95% CI 52-73) were on-going pregnancies, 6/81 (7%, 95% CI 3-15) resulted in first trimester miscarriages and 24/81 (30%, 95% CI 21-40) were failed implantations. Of the 51 on-going pregnancies, 15 (29%, 95% CI 19-43) couples had a singleton live birth at the time of write up. There have been no reports of abnormal prenatal, genetic testing or diagnosis of phenotype at birth. Karyomapping is reliable, efficient and accurate for couples requiring PGD for SGD and/or chromosomal rearrangement. Additionally, it provides aneuploidy screening, minimising risks of miscarriage and implantation failure.

Meiotic outcome in two carriers of Y autosome reciprocal translocations: selective elimination of certain segregants.

BACKGROUND: Reciprocal Y autosome translocations are rare but frequently associated with male infertility. We report on the meiotic outcome in embryos fathered by two males with the karyotypes 46,X,t(Y;4)(q12;p15.32) and 46,X,t(Y;16)(q12;q13). The two couples underwent preimplantation genetic diagnosis (PGD) enabling determination of the segregation types that were compatible with fertilization and preimplantation embryo development. Both PGD and follow up analysis were carried out via fluorescence in situ hybridization (FISH) or array comparative genomic hybridization (aCGH) allowing the meiotic segregation types to be determined in a total of 27 embryos. RESULTS: Interestingly, it was seen that the number of female embryos resulting from alternate segregation with the chromosome combination of X and the autosome from the carrier gamete differed from the corresponding balanced males with derivative Y and the derivative autosome by a ratio of 7:1 in each case (P = 0.003) while from the adjacent-1 mode of segregation, the unbalanced male embryos with the combination of der Y and the autosome were seen in all embryos from couple A and in couple B with the exception of one embryo only that had the other chromosome combination of X and derivative autosome (P = 0.011). In both cases the deficit groups have in common the der autosome chromosome that includes the segment Yq12 to qter. CONCLUSION: The most likely explanation may be that this chromosome is associated with the X chromosome at PAR2 (pseudoautosomal region 2) in the sex-body leading to inactivation of genes on the autosomal segment that are required for the meiotic process and that this has led to degeneration of this class of spermatocytes during meiosis.

The impact of mosaicism in preimplantation genetic diagnosis (PGD): approaches to PGD for dominant disorders in couples without family history.

OBJECTIVES: Mosaicism in certain dominant disorders may result in a 'non-Mendelian' transmission for the causative mutation. Preimplantation genetic diagnosis (PGD) is available for patients with inherited disorders to achieve an unaffected pregnancy. We present our experience for two female patients with different dominantly inherited autosomal disorders; neurofibromatosis type 1 (NF1) and tuberous sclerosis complex type 2 (TSC2). METHODS: PGD protocol development was carried out using single cells from the patients. PGD was carried out on polar bodies and different embryonic cells. RESULTS: Protocol development for NF1 using lymphocytes from the patient suggested mosaicism for the mutation. This was supported further by quantitative fluorescent-PCR performed on genomic DNA. During PGD, polar bodies and blastomeres lacked the mutation that probably was absent or present at very low levels in the patient's germline. Single lymphocyte analysis during protocol development for TSC2 did not indicate mosaicism; however, analysis of single buccal cells and multiple embryo biopsies across two consecutive IVF/PGD cycles confirmed gonosomal mosaicism. CONCLUSIONS: The trend in PGD is for blastocyst biopsy followed by whole genome amplification, eliminating single cell analysis. In the case of certain dominantly inherited disorders, pre-PGD single cell analysis is beneficial to identify potential mosaicism that ensures robust protocols. © 2016 John Wiley & Sons, Ltd.

Vitamin D metabolism, cartilage and bone fracture repair.

The 1,25-(OH)(2)D metabolite mediates the endocrine actions of vitamin D by regulating in the small intestine the expression of target genes that play a critical role in intestinal calcium absorption. The major role of the vitamin D hormone on bone is indirect and mediated through its endocrine function on mineral homeostasis. However, genetic manipulation of the expression of Cyp27b1 or the VDR in chondrocytes strongly support a direct role for locally synthesized 1,25(OH)(2)D, acting through the VDR, in vascular invasion and osteoclastogenesis during endochondral bone development. Cells from the growth plate respond to the 24,25-(OH)(2)D and 1,25-(OH)(2)D metabolites in a cell maturation-dependent manner and the effects of 1,25-(OH)(2)D are thought to be mediated through binding to the membrane-associated receptor PDIA3 (protein disulfide isomerase associated 3). The physiological relevance of membrane-mediated 1,25-(OH)(2)D signaling is emerging and is discussed. Finally, preliminary results suggest that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing and support a role for 24,25-(OH)(2)D in mammalian fracture repair.

Chondrocyte-specific modulation of Cyp27b1 expression supports a role for local synthesis of 1,25-dihydroxyvitamin D3 in growth plate development.

The Cyp27b1 enzyme (25-hydroxyvitamin D-1alpha-hydroxylase) that converts 25-hydroxyvitamin D into the active metabolite, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is expressed in kidney but also in other cell types such as chondrocytes. This suggests that local production of 1,25(OH)(2)D(3) could play an important role in the differentiation of these cells. To test this hypothesis, we engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. Inactivation of both alleles of the Cyp27b1 gene led to decreased RANKL expression and reduced osteoclastogenesis, increased width of the hypertrophic zone of the growth plate at embryonic d 15.5, increased bone volume in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. The expression of the angiogenic marker VEGF was decreased, accompanied by decreased platelet/endothelial cell adhesion molecule-1 staining in the neonatal growth plate, suggesting a delay in vascularization. In parallel, we engineered strains of mice overexpressing a Cyp27b1 transgene in chondrocytes by coupling the Cyp27b1 cDNA to the collagen alpha(1)(II) promoter. The transgenic mice showed a mirror image phenotype when compared with the tissue-specific inactivation, i.e. a reduction in the width of the hypertrophic zone of the embryonic growth plate, decreased bone volume in neonatal long bones, and inverse expression patterns of chondrocytic differentiation markers. These results support an intracrine role of 1,25(OH)(2)D(3) in endochondral ossification and chondrocyte development in vivo.

No association between the -1031 polymorphism in the TNF-alpha promoter region and type 1 diabetes.

Tumor necrosis factor alpha (TNF-alpha) is an important immunomodulator and is believed to be involved in the development or progression of type 1 diabetes. In the following study, we evaluated TNF-alpha promoter polymorphisms at positions -863 and -1031 and their association with type 1 diabetes in a group of 210 diabetic patients from Lebanon. Our results show that in our population, the C allele is predominant at position -863, whereas the A allele is very rare (2%). At position -1031, however, the C and T allele distribution was similar in both the patient (17.8% vs 82.2%, respectively) and the control (21.4% vs 79.6%) groups. No association of TNF-alpha genotype at position 1031 with type 1 diabetes was found as demonstrated by the family-based association test and the transmission disequilibrium test. However, when patient genotypes were compared, the recessive CC genotype was only found in type 1 diabetic males but not in type 1 diabetic females. This observation, however, requires further investigation in a larger sample before conclusive association to gender is suggested. In conclusion, our results demonstrate that no association between TNF-alpha polymorphism and type 1 diabetes seems to exist in our population.