Single Oocyte Research Articles

Evaluation of fertilization, usable blastocyst development and sustained implantation rates according to intracytoplasmic sperm injection operator experience.

Is intracytoplasmic sperm injection (ICSI) operator experience associated with fertilization, usable blastocyst development and sustained implantation rates (SIR) when at least two embryologists carry out ICSI for a single cohort of oocytes? A retrospective cohort study of all IVF/ICSI cycles at a single large infertility centre between 2008 and 2018. Cycles were included if a cohort of oocytes was split between two embryologists for ICSI. The embryologist's experience of ICSI was used to evaluate laboratory and clinical outcomes overall and by pairs of inseminating embryologists. Logistic regression, analysis of variance and Kruskal-Wallis testing were used where appropriate. Analysis of 14,362 ICSI procedures showed an association between least ICSI experience and lower mean fertilization rates (P < 0.0001), higher odds of failed fertilization (adjusted OR 4.3; P < 0.0001) and lower number of fertilization 'wins' per cohort (P < 0.0001). Usable blastocyst development rates (number of usable blastocysts/number of two pronuclear zygotes) were not associated with ICSI embryologist experience (P = 0.44), but the odds of obtaining no usable blastocysts were higher (adjusted OR 1.4; P < 0.0001) and the proportion of usable blastocyst 'wins' was lower (P = 0.0001) when embryologists with the least experience carried out ICSI. Increased ICSI experience was associated with higher mean SIR (P < 0.0001). Laboratory and clinical outcomes were similar among embryologists once 1000 ICSI cycles and above were carried out. Increased ICSI operator experience is associated with higher fertilization rates, SIR and a lower likelihood of failed fertilization and usable blastocyst development. Splitting a single oocyte cohort between more than one embryologist for ICSI is a quality-control measure that can be implemented.

Chromosome Missegregation in Single Human Oocytes Is Related to the Age and Gene Expression Profile.

The growing trend for women to postpone childbearing has resulted in a dramatic increase in the incidence of aneuploid pregnancies. Despite the importance to human reproductive health, the events precipitating female age-related meiotic errors are poorly understood. To gain new insight into the molecular basis of age-related chromosome missegregation in human oocytes, we combined the transcriptome profiles of twenty single oocytes (derived from females divided into two groups according to age <35 and ≥35 years) with their chromosome status obtained by array comparative genomic hybridization (aCGH). Furthermore, we compared the transcription profile of the single oocyte with the surrounding cumulus cells (CCs). RNA-seq data showed differences in gene expression between young and old oocytes. Dysregulated genes play a role in important biological processes such as gene transcription regulation, cytoskeleton organization, pathways related to RNA maturation and translation. The comparison of the transcription profile of the oocyte and the corresponding CCs highlighted the differential expression of genes belonging to the G protein-coupled receptor superfamily. Finally, we detected the loss of a X chromosome in two oocytes derived from women belonging to the ≥35 years age group. These aneuploidies may be caused by the detriment of REEP4, an endoplasmic reticulum protein, in women aged ≥35 years. Here we gained new insight into the complex regulatory circuit between the oocyte and the surrounding CCs and uncovered a new putative molecular basis of age-related chromosome missegregation in human oocytes.

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus-oocyte-complex (COC).

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non-invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell-safe with no inherent cell toxicity or light-induced phototoxicity indicated by negative DNA damage staining. Pre-measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01-0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.

Chemotherapy causes primordial follicle death in the human ovary via multiple apoptotic pathways and not by “burn out”

It has been proposed that gonadotoxic chemotherapy results in the “burn out” of primordial follicle reserve by activating PI3K/PTEN/Akt signaling pathway. Others have challenged this concept and put forward DNA damage and apoptosis as the main mechanism of follicle loss. We conducted this study to answer this controversy and conclusively determine the mechanism of chemotherapy-induced damage to ovarian reserve in women. Ovarian cortical pieces from organ donors aged ≤32 years were xenografted subcutaneously to SCID mice (n=12 mice/ tissue from 4 donors each). After 10 days, the mice were given an injection of cyclophosphamide (75mg/kg) or the vehicle. The tissues were recovered 12 hours later. The recovered xenografts were assessed for apoptosis by anti-caspase-3 (AC3) and DNA double strand breaks by γ-H2AX immunostaining as well as follicle growth initiation rate (FGIR) by primary/primordial follicle ratios. Single primordial follicle oocytes were laser captured for RNA sequencing and single cell quantitative real time PCR (qRT-PCR) to determine the signaling pathways activated in response to chemotherapy exposure. Immunofluorescence microscopy and image analysis were used to assess phosphorylation of PI3K/PTEN/Akt pathway and Bad-Bcl2 co-localization. There was no difference in the primordial follicle growth initiation rate (FGIR) between the vehicle and the cyclophosphamide-treated xenografts (p=0.35). However, cyclophosphamide caused a significant increase in the percentage of apoptotic (65.02±3.55% vs. 28.27±5.77%; p=0.001) and DNA-damaged (82± 2.7% vs. 23.82±8.66%;p=0.0001) primordial follicles . Ingenuity Pathway Analysis (IPA) of the RNA sequencing data from laser captured primordial follicles showed significant decrease in the expression of PECAM (p=0.00009), IKBKE (P=0.0001), and ANGPT1 (p=0.003) after cyclophosphamide treatment. These are positive regulators of the Akt pathway and hence results indicate that cyclophosphamide suppresses rather than activating Akt. In addition, IPA predicted that suppression of these pathways favor apoptosis in single primordial follicles. By qRT-PCR analyses, there was no change in the expression of Akt (p=0.9). Further, cyclophosphamide treatment did change the expression of the phospho-Akt (P=0.14), phospho-Foxo3a (p=0.17), or phospho-rpS6 (0.48) compared to the vehicle. By qRT-PCR, chemotherapy increased the expression of the anti-apoptotic Bcl2 (p=0.0003) accompanied by enhanced colocalization of the pro-apoptotic BAD-Bcl2 complex (p=0.006) in the primordial follicles, confirming that cyclophosphamide induces follicle death via apoptosis. This single cell transcriptomic and immunohistochemical analysis of human primordial and primary follicles prove that gonadotoxic chemotherapy agents do not cause follicle activation; they rather create a pro-apoptotic state resulting in massive loss of ovarian reserve. Future research on pharmacological fertility preservation should target preventing DNA damage and apoptosis rather than follicle activation.

The number of oocytes associated with maximum cumulative live birth rates per aspiration depends on female age: a population study of 221 221 treatment cycles.

The number of oocytes associated with maximum cumulative live birth rates per aspiration depends on female age: a population study of 221 221 treatment cycles.

36. TWO HEALTHY BABIES BORN AFTER IMPLANTATION OF MULTIPLE CHROMOSOMALLY-RESCUED OOCYTES

Introduction Meiotic rescue is a well-known phenomenon but little is known about live births after oocyte rescue, because of the shortage of data on PB biopsy. PGT-A has been performed for many years to increase the chance of pregnancy, especially in older women. Embryo aneuploidy is a major cause of pregnancy failure and is largely of maternal meiotic origin (>95%), with the risk increasing exponentially from approximately 35 years of age. Molecular analysis of polar bodies provides indirect assessment of an oocyte's chromosomal status by determining whether the chromosomes correctly segregated during meiosis I and II. In about one third of cases, missegregation of one or two chromosomes in meiosis I can be rescued in meiosis II, leading to a euploid oocyte. We present two cases of healthy babies born after single and double chromosome-rescued oocytes Material and methods A 42- (case 1) and a 44-year old woman (case 2) underwent ART treatment with PGT-A PB, after histories of 3 and 2 prior ART failures respectively. Both couples underwent ICSI with first and second PB biopsy and comprehensive chromosome analysis by Array-CGH. The mechanisms leading to missegregation in PB1 and PB2 (nondisjunction or premature separation of sister chromatids (PSSC)) were analysed using chromosome-specific STRs. Results and conclusions Case 1: a single euploid fertilized oocyte was transferred, after double rescue of chromosomes 3 and 18 (PB1 -3, -18; PB2: +3, +18) at day 2, resulting in a singleton pregnancy. Case 2: a single euploid fertilized oocyte was transferred after rescue of chromosome 16 (PB1: -16; PB2:+16) at day 3. In case 1, the aneuploidy of chromosomes 3 and 18 in the first PB was caused by PSSC followed by the extrusion of 2 homologous chromosomes in the second PB. In case 2, chromosome 16 underwent PSSC and extrusion of a single chromatid in PB1, followed by nondisjunction in meiosis II and extrusion of two sister chromatids in PB2. In both cases, the rescued oocyte was the only euploid oocyte out of 12 retrieved and they were competent for generating a pregnancy. The two women gave birth at term to healthy babies (a girl of 3280 g and a boy of 3500 g). To our knowledge, this is the first report of healthy births after a multiple chromosomally-rescued oocyte. PGT-A of PBs is a valid method with the limitation of detecting only maternal aneuploidies and so is appropriate for screening of older women. Analysis of the 2 polar bodies by aCGH is valuable not only for aneuploidy screening in ART for older women, but also to understand the mechanisms leading to missegregation and rescue in oocytes.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Study of the mechanical properties of fresh and cryopreserved individual human oocytes.

In assisted reproduction technologies, the cryopreservation of oocytes is a common procedure used to circumvent female infertility. However, some morphological and functional alterations of oocytes have been observed depending on the protocol applied. In this work, the mechanical response of individual human oocytes before and after a freeze-thawing procedure was characterised. Oocytes, immediately after retrieval, were morphologically evaluated by bright-field optical microscopy and their elasticity measured by indentation measurements using atomic force microscopy. Oocytes were then frozen according to the open-vitrification protocol and stored in liquid nitrogen. Afterwards, the same oocytes were thawed and the indentation measurements repeated. Using this approach, we can follow the elasticity of a set of single oocytes from retrieval up to the freeze-thawing procedure. The analysis of the resulting data shows that the retrieved healthy oocytes, which preserve their healthy morphological features after cryopreservation, maintain unchanged also in stiffness values. In contrast, oocytes having dysmorphic characteristics, before and/or after freeze-thawing, show significant variations in their mechanical response. In addition, the dysmorphic oocytes are generally observed to be softer than the healthy oocytes. Our results indicate that stiffness of healthy oocytes is not considerably affected by the open-vitrification-thawing procedure, and that distinct elasticity ranges can be identified for healthy and dysmorphic oocytes. These findings indicate that the mechanical characterization of oocytes represents an opportunity to detect cellular defects, and assess the quality and bio-viability of processes such as cryopreservation.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Trackingcomputer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and washbuffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

DNA methylation analysis and editing in single mammalian oocytes

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy )-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy -related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.

Reproduction of invasive Loricariichthys platymetopon Isbrücker &amp; Nijssen, 1979 (Actinopterygii, Loricariidae) on the upper Paraná River floodplain

Loricariichthys platymetopon ocorria originalmente nos rios Uruguai, Paraguai e baixo Paraná. Com a formação do reservatório de Itaipu em 1982, invadiu e colonizou o alto rio Paraná. Usando os dados obtidos trimestralmente em nove pontos de amostragem na planície de inundação do alto rio Paraná, foram caracterizadas as fases reprodutivas e células germinativas das fêmeas de L. platymetopon. Além disso, foram verificados: a variação no diâmetro dos oócitos, a fecundidade dos lotes, a fecundidade relativa dos lotes por comprimento padrão e peso total e os locais de reprodução na planície de inundação do alto rio Paraná. Foram registrados os estágios: oogônia única, cistos de oogônias e oócitos, oócitos de crescimento primário inicial, intermediário e final, oócitos de crescimento completo e oócitos maduros. As fases reprodutivas foram validadas pelos atributos das células germinativas e as fêmeas foram registradas nas seguintes fases: desenvolvimento, apto à desova, regressão e regeneração. O diâmetro dos oócitos variou de 100 a 4.100 µm. A fecundidade do lote variou de 372 a 1.392 oócitos.g-1 e a estimativa da fecundidade relativa do lote por comprimento padrão foi de 16 a 49 oócitos.cm-1 e, em peso foi de 3,6 a 6,4 oócitos.g-1. Os padrões reprodutivos de L. platymetopon na planície de inundação do alto rio Paraná mostraram os locais onde a atividade reprodutiva é mais intensa (lagoas do Guaraná, das Garças e Fechada e rio Baía) e locais onde é menos intensa (rios Ivinheima e Paraná). Assim, a resistência biótica do rio Ivinheima, devido à integridade desses ambientes nessa área, que faz parte da Área de Proteção Ambiental das Ilhas e Várzeas do Rio Paraná, tem impedido o sucesso reprodutivo desta invasora.

The Rove Beetle Creophilus maxillosus as a Model System to Study Asymmetric Division, Oocyte Specification, and the Germ-Somatic Cell Signaling.

Creophilus maxillosus (Staphylinidae, Coleoptera, Polyphaga) has a meroistic-telotrophic ovary composed of tropharium, which contains trophocytes (nurse cells) and vitellarium, which contains growing oocytes. The trophocytes are connected to the oocytes by cytoplasmic nutritive cords, which deliver nutrients to the oocytes. The formation/differentiation of the oocytes and trophocytes takes place in the pupal ovary within linear chains of sibling cells. Each chain is composed of a single oocyte connected to a linear chain of sister trophocytes. The nuclei of the oocytes contain an extrachromosomal DNA body (extra DNA body) consisting of amplified ribosomal DNA (rDNA). During oogenesis, the prospective oocyte, located at the base (posterior) of each chain, is the only cell within the chain that amplifies rDNA and retains permanent contact with the somatic pre-follicular cells. The oogonial divisions leading to the formation of the oocyte/trophocytes chain are asymmetric, and during consecutive divisions, the rDNA body always segregates basally (posteriorly) to the prospective oocyte abutted on the somatic cells. However, the segregation of rDNA is imperfect, and within each oocyte/trophocytes chain, there is a gradient of rDNA: the prospective oocyte has the highest amount of rDNA and the trophocyte that is most distant (most anterior) from the oocyte has no or the lowest amount of rDNA. In addition, the divisions within each chain are parasynchronous, with the pro-oocyte being the most mitotically advanced cell in the chain. These observations indicate the presence of a signaling gradient emanating from the somatic cells and/or oocyte; this gradient diminishes in strength with the increasing distance from its source, i.e., the oocyte/somatic cells. Because of this phenomenon, C. maxillosus is the perfect model in which to study the germ-somatic cell interactions and signaling. This chapter describes the methods for the collection and laboratory culture of C. maxillosus and the analysis of divisions and signaling in the C. maxillosus ovary.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

62 Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0mM, pyruvate 0.3/0.1mM, lactate 10.0/6.0mM, NEEA 1×/1× MEM, EAA 0.25×/0.5× MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean±s.e.m.) were analysed using ANOVA (P&amp;lt;0.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1±3.3%) and 6.25-25% (46.6±3.2%) compared with 100-100% (34.2±3.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6±2.5) and 6.25-25% (26.0±2.6) was reduced compared with 100-100% (41.4±4.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision.

To test or not to test? A framework for counselling patients on preimplantation genetic testing for aneuploidy (PGT-A).

What is the treatment path and cumulative live birth (CLB) rate from a single oocyte retrieval of patients who intend to pursue PGT-A at the start of an IVF cycle compared to matched controls? The choice of PGT-A at the start of the first IVF cycle decreases the CLB per oocyte retrieval for patients <38 years of age, however patients ≥38 years of age benefit significantly per embryo transfer (ET) when live birth (LB) is evaluated. PGT-A has been shown to reduce the practice of transferring multiple embryos and to confer a higher live birth rate per transfer. This is a retrospective cohort study from December 2014 to September 2016, involving 600 patients: those intending PGT-A for their first IVF cycle (N = 300) and their matched controls. Post-hoc power calculations (alpha of 0.05, power of 0.80) indicated that our study was powered adequately to demonstrate significant differences in CLB per retrieval and LB per transfer. The study was performed at a large academically affiliated infertility practice where approximately 80% of patients have insurance coverage for fertility care. Patients were identified through electronic medical records, and those who intended to pursue PGT-A at the start of stimulation were assessed. Patients were matched by age, time of oocyte retrieval and oocyte yield to the same number of controls. CLB outcomes per single retrieval, including the fresh and frozen transfers arising from the initial stimulation cycle, were calculated. PGT-A was not beneficial when CLB rate was assessed per retrieval, however its benefits were significant when LB rate was assessed per transfer. First cycle, <38 year-old patients who intended to have PGT-A had a significantly (P < 0.001) lower CLB rate per oocyte retrieval compared to controls (49.4% vs. 69.1%). Conversely, patients ≥ 38 years in the PGT-A group had similar CLB rates compared to controls per oocyte retrieval, while LB rates per transfer were doubled compared to controls (62.1% vs. 31.7%; P < 0.001). Of the first-cycle PGT-A and control patients, 25.3% and 2.3% failed to achieve a transfer, respectively. This is not a true intention-to-treat study, due to its retrospective nature. Additionally, the number of patients with two or more previous miscarriages was significantly greater in the PGT-A group as compared to controls, however a sub-analysis showed that this failed to impact outcomes. The findings indicate that PGT-A may be detrimental for those <38 years old undergoing their first IVF cycle. PGT-A has the greatest clinical impact when a transfer is achieved in the ≥38 years old population. This study evaluates the typical treatment path following a patient's choice to pursue PGT-A at the cycle start, and can be used as a guide for counselling patients in relation to age and cycle number. None. N/A.

Variable IGF1R mRNA Expression in Individual GV and In Vitro Matured M2 Human Oocytes

Objective Characterization of human insulin-like growth factor 1 receptor (IGF1R) mRNA expression in individual human oocytes to better understand the role of biologically significant mRNAs in establishing oocyte quality following ovarian hyperstimulation for in vitro fertilization (IVF) treatment. Design Germinal vesicle oocytes (GV) were identified after removal of all cumulus cells prior to ICSI and collected from consenting patients with IRB-approval. Materials and Methods Single GVs were placed directly into lysis buffer (5μl). Oocytes in vitro matured (IVM) to the M2 stage were placed into individual tubes containing lysis buffer after 24 to 48 hours (h) in culture. Total RNA isolated from cumulus granulosa cells (GC) using the TRIzol reagent (Invitrogen) as stated in the manufacturer’s protocol served as a positive control. Reverse Transcription (RT) was performed using anchored oligo-dT primers (IDT, Coralville, IA) and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) on the entire 5μl oocyte lysate at 37°C for 1.5h or 1mg total GC RNA 42°C for 1h. Quantitative polymerase chain reaction (qPCR) was performed combining standards or 1μl cDNA with PCR buffer containing SYBR Green I (Sigma Chemical Co, St. Louis, Missouri), Taq polymerase (Genscript, Piscataway, NJ), and primers for IGF1R. Melting curves were routinely determined to ascertain generation of only the expected product. Results qPCR performed on GC cDNA demonstrated detection of the expected product under the reaction conditions in use. Gel electrophoresis of the PCR product confirmed the size of the DNA fragment generated. qPCR analysis detected a signal for IGF1R mRNA in 9 of the 15 GVs analyzed from 9 patients. Moreover, for 3 patients with multiple GVs, differences in IGF1R rnRNA expression were seen between individual GV oocytes, as well as between oocytes from different patients. Similarly, 8 of 12 IVM M2 oocytes from 11 patients gave a signal for the presence of IGF1R mRNA. Conclusions These results report the use of a direct-lysis method for the extraction and analysis of mRNA from single human oocytes. These data suggest that GV oocytes failing to respond to the hCG trigger during ovarian stimulation for IVF vary in their biological integrity. Furthermore, we observed that GV oocytes advanced to the M2 stage in vitro in the absence of any demonstrable IGF1R mRNA expression. Molecular assessment of single human oocytes can be used to better understand the low per-oocyte success rate in human IVF. Disclosures Nothing to disclose. Funding None.

Preimplantation genetic testing for aneuploidy is cost-effective, shortens treatment time, and reduces the risk of failed embryo transfer and clinical miscarriage

To determine if preimplantation genetic testing for aneuploidy (PGT-A) is cost-effective for patients undergoing invitro fertilization (IVF). Decision analytic model comparing costs and clinical outcomes of two strategies: IVF with and without PGT-A. Genetics laboratory. Women ≤ 42years of age undergoing IVF. Decision analytic model applied to the above patient population utilizing a combination of actual clinical data and assumptions from the literature regarding the outcomes of IVF with and without PGT-A. The primary outcome was cumulative IVF-related costs to achieve a live birth or exhaust the embryo cohort from a single oocyte retrieval. The secondary outcomes were time from retrieval to the embryo transfer resulting in live birth or completion of treatment, cumulative live birth rate, failed embryo transfers, and clinical losses. 8,998 patients from 74 IVF centers were included. For patients with greater than one embryo, the cost differential favored the use of PGT-A, ranging from $931-2411 and depending upon number of embryos screened. As expected, the cumulative live birth rate was equivalent for both groups once all embryos were exhausted. However, PGT-A reduced time in treatment by up to four months. In addition, patients undergoing PGT-A experienced fewer failed embryo transfers and clinical miscarriages. For patients with > 1 embryo, IVF with PGT-A reduces healthcare costs, shortens treatment time, and reduces the risk of failed embryo transfer and clinical miscarriage when compared to IVF alone.

A multi-sensor system for measuring bovine embryo metabolism

This paper presents the development of a multi-sensor platform capable of simultaneous measurement of dissolved oxygen (DO) concentration, glucose and lactate concentrations in a micro-chamber for real-time evaluation of metabolic flux in bovine embryos. A micro-chamber containing all three sensors (DO, glucose, and lactate) was made to evaluate metabolic flux of single oocytes or embryos at different stages of development in ≤ 120 µL of respiration buffer. The ability of the sensor to detect a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis was demonstrated in embryos by an ablation of oxygen consumption and an increase in lactate production following addition of oligomycin, an inhibitor of mitochondrial adenosine triphosphate (ATP) synthesis. An increased reliance upon glycolysis relative to OXPHOS was demonstrated in embryos as they developed from morula to hatched blastocysts by a progressive increase in the lactate/oxygen flux ratio, consistent with isolated metabolic assessments reported previously. These studies highlight the utility of a metabolic multi-sensor for integrative real-time monitoring of aerobic and anaerobic energy metabolism in bovine embryos, with potential applications in the study of metabolic processes in oocyte and early embryonic development.

Live baby rate per oocyte with no stimulation

What is the intrinsic fecundity rate of the human oocyte in relation to age? Ovarian hyperstimulation has been found to yield a live baby rate per oocyte of only about 4 to 6%. Thus on average more than 20 to 25 oocytes would be required to produce a single live baby. But this is in stimulated cycles. The live baby rate per egg in natural cycle single embryo transfer IVF would estimate better the intrinsic fertility of the human oocyte. 13,949 oocyte retrievals were performed in a natural cycle program with single embryo transfer in an attempt to determine what is the fertility potential of a single human oocyte that has not been submitted to ovarian hyperstimulation. Infertile women undergoing natural cycle with a single embryo transfer were studied retrospectively. The primary data of live baby per oocyte were approximated with a logistic curve r+1(a+exp[b(t-c)]) where r is live baby rate per oocyte and t is age in years. The coefficients were evaluated using gradient method as implemented in statistical package R (version 3.2.5). This allowed the construction of a robust logistic curve fit. The primary outcome of interest was live baby rate per oocyte. For women ≤42 years, the overall live baby rate per oocyte was 18%, which translated into only 5.5 oocytes needed to produce one baby. For women 42 years of age, every oocyte would have a 4% chance of becoming a baby, which means for a 42 year old woman it would require 22.7 rather than 5.5 eggs to produce a baby. The drop in intrinsic fertility per oocyte is summarized remarkably robustly in a logistic curve. There is at first a steady (almost horizontal) maintenance of fertility per oocyte (26%), followed after age 34 with a sharp linear decline until age 42 (4%), with a 10% loss of fertility every year. This decline slows down after age 43, with only 3% of original fertility remaining at age 45. The logistic model explains virtually all observed variation in live baby per oocyte rate: adjusted R-square is 99%. For all transfers the basic integrity of the logistic curve was remarkably robust. Live baby rate per single unstimulated oocyte is 26% until age 35, and then declines 10% per year until age 42, when it is only 4%. This is approximately a five times greater live baby rate per egg than has been reported with stimulated cycles.