Developmental Incompetence Research Articles

Time will tell: time-lapse technology and artificial intelligence to set time cut-offs indicating embryo incompetence.

Can more reliable time cut-offs of embryo developmental incompetence be generated by combining time-lapse technology (TLT), artificial intelligence, and preimplantation genetics screening for aneuploidy (PGT-A)? Embryo developmental incompetence can be better predicted by time cut-offs at multiple developmental stages and for different ranges of maternal age. TLT is instrumental for the continual and undisturbed observation of embryo development. It has produced morphokinetic algorithms aimed at selecting embryos able to generate a viable pregnancy, however, such efforts have had limited success. Regardless, the potential of this technology for improving multiple aspects of the IVF process remains considerable. Specifically, TLT could be harnessed to discriminate developmentally incompetent embryos: i.e. those unable to develop to the blastocyst stage or affected by full-chromosome meiotic aneuploidies. If proven valuable, this application would prevent the non-productive use of such embryos, thereby improving laboratory and clinical efficiency and reducing patient stress and costs due to unnecessary embryo transfer and cryopreservation. The training dataset involved embryos of PGT-A cycles cultured in Embryoscope with a single media (836 euploid and 1179 aneuploid blastocysts and 1874 arrested embryos; 2013-2020). Selection criteria were ejaculated sperm, own (not donated) fresh oocytes, trophectoderm biopsy and comprehensive-chromosome-testing to diagnose uniform aneuploidies. Out-of-sample (30% of training), internal (299 euploid and 490 aneuploid blastocysts and 680 arrested embryos; 2021-2022) and external (97 euploid, 110 aneuploid and 603 untested blastocysts and 514 arrested embryos, 2018 to early 2022) validations were conducted. A training dataset (70%) was used to define thresholds. Several models were generated by fitting outcomes to each timing (tPNa-t8) and maternal age. ROC curves pinpointed in-sample classification values associated with 95%, 99% and 99.99% true-positive rate for predicting incompetence. These values were integrated with upper limits of maternal age ranges (<35, 35-37, 38-40, 41-42, and >42 years) in logit functions to identify time cut-offs, whose accuracy was tested on the validation datasets through confusion matrices. For developmental (in)competence, the best performing (i) tPNa cut-offs were 27.8 hpi (error-rate: 0/743), 32.6 hpi (error rate: 0/934), 26.8 hpi (error rate: 0/1178), 22.9 hpi (error-rate: 1/654, 0.1%) and 17.2 hpi (error rate: 4/423, 0.9%) in the <35, 35-37, 38-40, 41-42, and >42 years groups, respectively; (ii) tPNf cut-offs were 36.7 hpi (error rate: 0/738), 47.9 hpi (error rate: 0/921), 45.6 hpi (error rate: 1/1156, 0.1%), 44.1 hpi (error rate: 0/647) and 41.8 hpi (error rate: 0/417); (iii) t2 cut-offs were 50.9 hpi (error rate: 0/724), 49 hpi (error rate: 0/915), 47.1 hpi (error rate: 0/1146), 45.8 hpi (error rate: 0/636) and 43.9 hpi (error rate: 0/416); (iv) t4 cut-offs were 66.9 hpi (error rate: 0/683), 80.7 hpi (error rate: 0/836), 77.1 hpi (error rate: 0/1063), 74.7 hpi (error rate: 0/590) and 71.2 hpi (error rate: 0/389); and (v) t8 cut-offs were 118.1 hpi (error rate: 0/619), 110.6 hpi (error rate: 0/772), 140 hpi (error rate: 0/969), 135 hpi (error rate: 0/533) and 127.5 hpi (error rate: 0/355). tPNf and t2 showed a significant association with chromosomal (in)competence, also when adjusted for maternal age. Nevertheless, the relevant cut-offs were found to perform less well and were redundant compared with the blastocyst development cut-offs. Study limits are its retrospective design and the datasets being unbalanced towards advanced maternal age cases. The potential effects of abnormal cleavage patterns were not assessed. Larger sample sizes and external validations in other clinical settings are warranted. If confirmed by independent studies, this approach could significantly improve the efficiency of ART, by reducing the workload and patient impacts (extended culture and cleavage stage cryopreservation or transfer) associated with embryos that ultimately are developmentally incompetent and should not be considered for treatment. Pending validation, these data might be applied also in static embryo observation settings. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

P-149 Time is over: time-lapse technology to set time cut-offs defining embryo developmental incompetence

Abstract Study question Can time-lapse technology (TLT) discriminate developmentally incompetent embryos through time cut-offs of fertilization and cleavage stages? Summary answer TLT identifies developmentally incompetent embryos with high precision, through cut-offs of tPNa, tPNf, t2, t4 and t8. No clinically-applicable cut-offs were found for blastocyst euploidy. What is known already TLT is instrumental for continued and undisturbed observation of embryo development. This has produced morphokinetic algorithms aimed at selecting embryos able to generate a viable pregnancy. Such efforts have had limited success. Regardless, the potential of this technology for improving multiple aspects of the IVF process remains considerable. Specifically, TLT could be harnessed to discriminate developmentally incompetent embryos: i.e., those unable to develop to the blastocyst stage or affected by full-chromosome meiotic aneuploidies. If developed, this application would prevent the non-productive use of such embryos, improving laboratory and clinical efficiency and reducing costs derived from unproductive embryo transfer and cryopreservation. Study design, size, duration The training dataset involved embryos of PGT-A cycles cultured in Embryoscope with single- media (836 euploid blastocysts, 1179 aneuploid and 1874 arrested embryos; 2013-2020). Selection criteria were ejaculated sperm, fresh own eggs, trophectoderm biopsy, and comprehensive-chromosome-testing to diagnose non-mosaic aneuploidies. Out-of-sample (30% of training), internal (299 euploid blastocysts, 490 aneuploid and 680 arrested embryos; 2021- 2022) and external (97 euploid, 110 aneuploid, 603 untested blastocysts and 514 arrested embryos, 2018 to early-2022) validations were conducted. Participants/materials, setting, methods A training dataset (70%) was used to define thresholds. Several models were generated by fitting outcomes to each timing (tPNa-t8) and maternal age. ROC-curves pinpointed in-sample classification values associated with 95%, 99% and 99.99% true-positive-rate for predicting incompetence. These values were integrated with upper limits of maternal age ranges (&amp;lt;35, 35-37, 38-40,41-42 and &amp;gt; 42 years) in logit functions to identify time cut-offs, whose accuracy was tested on the validation datasets through confusion matrices. Main results and the role of chance For developmental (in)competence, the best performing (i) tPNa cut-offs were 27.8hpi (error-rate: 0/743), 32.6hpi (error-rate: 0/934), 26.8hpi (error-rate: 0/1178), 22.9hpi (error-rate: 1/654,0.1%), and 17.2hpi (error-rate: 4/423,0.9%) in the &amp;lt;35, 35-37, 38-40, 41-42 and &amp;gt; 42 years groups; (ii) tPNf cut-offs were 36.7hpi (error-rate: 0/738), 47.9hpi (error-rate: 0/921), 45.6hpi (error-rate: 1/1156,0.1%), 44.1hpi (error-rate: 0/647), and 41.8hpi (error-rate: 0/417) in the &amp;lt;35, 35-37, 38-40, 41-42 and &amp;gt; 42 years groups; (iii) t2 cut-offs were 50.9hpi (error-rate: 0/724), 49hpi (error-rate: 0/915), 47.1hpi (error-rate:0/1146), 45.8hpi (error-rate: 0/636), and 43.9hpi (error-rate: 0/416) in the &amp;lt;35, 35-37, 38-40, 41-42 and &amp;gt; 42 years groups; (iv) t4 cut-offs were 66.9hpi (error-rate: 0/683), 80.7hpi (error-rate: 0/838), 77.1hpi (error-rate:0/1063), 74.7hpi (error-rate: 0/590), and 71.2hpi (error-rate: 0/389) in the &amp;lt;35, 35-37, 38-40, 41-42 and &amp;gt; 42 years groups; (v) t8 cut-offs were 118.1hpi (error-rate: 0/619), 110.6hpi (error-rate: 0/772), 140hpi (error-rate: 0/969), 135hpi (error-rate: 0/533), and 127.5hpi (error-rate: 0/355) in the &amp;lt;35, 35-37, 38-40, 41-42 and &amp;gt; 42 years groups. tPNf and t2 showed a significant association with chromosomal (in)competence, also when adjusted for maternal age. Nevertheless, relevant cut-offs were not clinically applicable. Limitations, reasons for caution Study limits are its retrospective design and datasets unbalanced towards advanced maternal age cases. The potential effects of abnormal cleavage patterns were not assessed. Larger sample size and external validations in other clinical settings are warranted. Wider implications of the findings If confirmed by independent studies, this approach could significantly impact on the efficiency of ART reducing the workload (extended culture, cryopreservation, and transfer) associated with embryos that ultimately are developmentally incompetent and should not be considered for treatment. Pending validation, these data might be applied also in static settings. Trial registration number N.A.

Mitochondrial DNA Deficiency and Supplementation in Sus scrofa Oocytes Influence Transcriptome Profiles in Oocytes and Blastocysts

Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell division. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.

Single-Cell Transcriptomics of Human Oocytes: Environment-Driven Metabolic Competition and Compensatory Mechanisms During Oocyte Maturation.

Aims: The mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro maturation (IVM) oocytes, remain to be elucidated. Here we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression.Results: Three key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Furthermore, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP+ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy.Innovation: The present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence.Conclusion: In vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.

Oocyte Cryopreservation: microSecure Vitrification (µS-VTF) No Worse Than the Rest, But is it an Experimental Procedure?

Objective When vitrified human oocytes yield viable blastocysts, live birth success is comparable to fresh oocytes. However, despite ASRM classifying oocyte cryopreservation as “non-experimental” in 2012, little progress has been made to understand developmental inconsistencies associated with some egg batches. Our aim was to contrast developmental incompetence of oocytes vitrified by µS-VTF in a DMSO-free solution compared to other conventional open VTF/EG-DMSO systems. Design Based on our limited clinical experience, we performed a retrospective analysis of our 2014 – 2018 oocyte warming-ET cycles. VTF oocytes (n = 416) from 28 patients were grouped according to VTF system: open device (n = 87; outside Lab source) or closed µS VTF devices (n = 329; Lab control), vitrified in EG/DMSO or EG only (Innovative Cryo Enterprises, NJ) cryoprotective solutions, respectively. Differences in fertilization, cleavage, blastocyst and pregnancy rates were statistically compared between groups. In particular, we aimed to assess differences in developmental issues. Materials and Methods All human oocytes were vitrified and warmed using standardized, published protocols. All oocytes were ICSI’ed 2-3h post-warming and cultured in Life Global medium + protein supplementation under tri-gas, humidified incubation (37°C) conditions up to Day 7. Blastocysts underwent ET, biopsy/PGS and/or µS-VTF/VFET. Results Developmental delay and reduced mean blastocyst production is common in vitrified-warmed oocytes compared to fresh oocytes (50-65%), independent of VTF method. Yet, when blastocysts are produced normal pregnancy outcomes occurred. Development incompetence between batches however, independent device-solution treatments, continues to be a serious problem (see Table results). Conclusion Delayed and compromised blastocyst development with cryopreserved oocytes continues to be a batch-to-batch problem ignored in most publications. Little experimental progress in the IVF industry is or will be made unless we initiate an ongoing scientific dialog. Ethically speaking, patients should be properly informed and consented prior to elective freeze preservation. Disclosures MCS developed µS-VTF without any commercial interests. Funding None.

Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study

Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest? Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth. Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca(2+) chelator could be restored by means of ionophores in an animal model. This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent. Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (≤15%). Severe male factor patients and patients with <30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, in vitro culture was performed as in the control cycles, up to blastocyst stage when achievable. Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (P < 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (P < 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (P < 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy. The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos. This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest. No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare.

Ovary transcriptome profiling via artificial intelligence reveals a transcriptomic fingerprint predicting egg quality in striped bass, Morone saxatilis.

Inherited gene transcripts deposited in oocytes direct early embryonic development in all vertebrates, but transcript profiles indicative of embryo developmental competence have not previously been identified. We employed artificial intelligence to model profiles of maternal ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in the striped bass (Morone saxatilis), a farmed species with serious egg quality problems. In models developed using artificial neural networks (ANNs) and supervised machine learning, collective changes in the expression of a limited suite of genes (233) representing <2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in gene expression (<0.2-fold), with most individual transcripts making a small contribution (<1%) to the overall prediction of egg quality. These findings indicate that the predictive power of the transcriptome as regards egg quality resides not in levels of individual genes, but rather in the collective, coordinated expression of a suite of transcripts constituting a transcriptomic “fingerprint”. Correlation analyses of the corresponding candidate genes indicated that dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence. The affected gene networks are centrally involved in regulation of early development in all vertebrates, including humans. By assessing collective levels of the relevant ovarian transcripts via ANNs we were able, for the first time in any vertebrate, to accurately predict the subsequent embryo developmental potential of eggs from individual females. Our results show that the transcriptomic fingerprint evidencing developmental dysfunction is highly predictive of, and therefore likely to regulate, egg quality, a biologically complex trait crucial to reproductive fitness.

Blastocyst implantation failure relates to impaired translational machinery gene expression

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes

Cumulus cells (CCs) maintain strict functional relationships with the enclosed antral oocyte and are thought to reflect its developmental competence. Several studies have described a correlation between CC gene expression and oocyte quality. Herein, we tested whether CC-specific FSH and LH receptors (FSHR and LHR, respectively) are differentially expressed in CCs enclosing developmentally competent or incompetent oocytes. To this end, mouse fully grown cumulus-oocyte complexes were isolated and their CCs and oocytes analysed separately. Based on their chromatin organisation, oocytes were classified as those with a surrounded nucleolus (SN) or a non-surrounded nucleolus (NSN), the former being developmentally competent, whereas the latter arrest at the 2-cell stage. The CCs were then analysed to compare the pattern of expression of the Fshr and Lhr genes and their proteins. Quantitative reverse transcription-polymerase chain reaction analysis revealed that only Lhr is significantly differentially expressed. Immunofluorescence analysis revealed that both FSHR and LHR proteins are significantly upregulated in CCs surrounding oocytes arrested at the 2-cell stage, reflecting their developmental incompetence.

Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes

BackgroundThe cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts.ResultsGlobal gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR.ConclusionsThe developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte.

&lt;i&gt;In Vitro&lt;/i&gt; Culture of Feline Embryos Increases Stress-induced Heat Shock Protein 70 and Apoptotic Related Genes

Developmental competence and quality of in vitro produced embryos has been demonstrated to be lower than in vivo derived embryos. This study aimed specifically to determine the effects of in vitro culture of feline embryos using various culture densities on developmental competence and expression of stress- and apoptotic-related genes in terms of heat shock protein 70 (HSP70) and apoptotic-related (BAX and BCL-2) gene expressions. In experiment 1, we characterized the inducible form of a feline-specific HSP70 mRNA sequence, as it has not been previously reported. The primers for feline HSP70 mRNA were synthesized and tested on heat-treated cat fibroblasts. In experiment 2, feline embryos were cultured at different culture densities (embryo:culture volume; 1:1.25, 1:5 and 1:20). The developmental competence was determined along with HSP70, BAX and BCL-2 transcript abundances using quantitative RT-PCR. In vivo derived embryos were used as a control group. A partial cat HSP70 mRNA sequence (190 bp) was characterized and exhibited high nucleotide identity (93 to 96%) with other species. Cleaved embryos cultured at high density (1:1.25) developed to blastocysts at a lower rate than those generated from lower densities. Irrespective of the culture densities used, in vitro cultured blastocysts showed increased levels of HSP70 and BAX transcripts compared with in vivo counterparts. Blastocysts derived from the highest culture density (1:1.25) showed higher levels of upregulation of HSP70 and BAX transcripts than those cultured at lower culture densities (1:5 and 1:20). In conclusion, increased levels of pro-apoptotic (BAX) and stress-response (HSP70) transcripts correlated with developmental incompetence of embryos cultured at high embryonic density, indicating that stress accumulated during in vitro embryo culture affected the fate for embryo development and quality.

Spontaneously resolving macular cyst in an infant

The purpose of this study is to describe transient macular cysts in an infant and correlate their occurrence with normal development events. A newborn Caucasian girl presented with a protruding corneal mass in her left eye at birth. She underwent a complete ophthalmic examination. A keratinized staphylomatous malformation involving the entire cornea and precluding further visualization of the anterior and posterior segment was observed in the left eye. Spectral domain optical coherence tomography (SD-OCT) of the right eye performed when the child was approximately 6-week-old had revealed an unexpected finding of macular cysts involving the inner nuclear and outer retinal layers. Corneal transplant in the left eye was performed a month later. Ocular examination under anesthesia just prior to surgery revealed normal intraocular pressure, anterior segment and retina in the right eye. SD-OCT was normal in both eyes and showed complete resolution of the cysts in the right eye. The patient had not been on any medications at that time. Although clinical retinal examination might be unremarkable, SD-OCT may reveal cystic spaces in the macula. In the absence of conditions known to be associated with macular edema, transient macular cysts may arise due to a developmental incompetence of the blood-retinal barrier or may represent transient spaces created during normal migration of retinal cells. Further study is warranted to delineate the entity of transient macular cysts in infancy.

54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis. This work was supported by VEGA 1/0077/11.

Developmental Incompetence to Stand Trial in Juvenile Courts*

Juveniles' competency to participate in delinquency proceedings has received increased attention in recent years. Developmental incompetence, whereby juveniles' incompetency is based upon their immaturity, as opposed to a mental disorder or developmental disability, is an evolving and important aspect of this area of law. The following paper reviews theories used to support the notion of developmental incompetence, as well as the extant empirical research on juveniles' competency-related abilities. Using a LexisNexis search, statutory and case laws pertaining to juvenile competency were identified across the 50 states and the District of Columbia. Only six states clearly allow developmental incompetence, whereas 17 have laws that do not include developmental immaturity as an acceptable basis of incompetence in juvenile courts. Developmental incompetence is likely to affect a relatively small proportion of juvenile cases, but has important implications for juvenile forensic practice. Recommendations are offered for forensic practitioners conducting this type of evaluation.

Age-associated alteration of telomere biology in mouse oocytes

Telomere shortening in cultured somatic cells triggers replicative senescence, characterized by cell cycle arrest and apoptosis. To our best knowledge, the effect of maternal aging on telomere length in oocytes has not been yet demonstrated. Therefore we focused on age-related alteration in telomere biology of mouse oocytes. Telomere length in oocytes from young mice and aged mice was studied. The expression pattern of telomere reverse transcriptase (Tert) and telomerase activity were also measured. Three subsets of ≤100 oocytes were collected from super-ovulated C57BL/6J mice aged 6-8 weeks for young and 48-52 weeks for aged for Q-PCR and Trap assay. Telomerase expression was analyzed by Q-PCR. For immunochemistry, samples were visualized by confocal microscopy (LSM510, Zeiss). Telomere length was measured by Q-PCR analysis. Telomerase activity was measured by a TRAP assay using a the Telomerase PCR ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany). A transcript level of Tert was significantly reduced in aged oocytes (6.0 ± 1.4), compared with that in young oocytes (11.5 ± 1.6). Immunofluorescent staining clearly demonstrated that a pattern and intencity of TERT protein expression in oocytes altered during maternal aging. A telomerase activity in aged oocytes (0.30 ± 0.11) was significantly lower than that in young oocytes (0.70 ± 0.11). Relative telomere length, expressed as a T/S ratio by Q-PCR, was remarkably shorter in aged oocytes (0.84 ± 0.10) than that in young oocytes (1.74 ± 0.20). These results suggest that the low expression and low activity of telomerase may contribute to telomere shorteninig, which leads to developmental incompetence of aged oocytes.

Basal ovarian stromal blood flow, antral follicles count and anti-müllerian hormone level are associated with oocyte quality in stimulated cycles

OBJECTIVE: To evaluate whether cycle day 3 basal ovarian blood flow, antral follicle count and anti-Müllerian hormone (AMH) level are useful tools to predict diminished oocyte quality and developmental incompetence in infertile women.

Gene expression profiling of human oocytes following in vivo or in vitro maturation

Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo (P < 0.05, range 4.98 x 10(-2) -2.22 x 10(-4)) and 162 of these are expressed at 10-fold or greater levels (P < 0.05, range 4.98 x 10(-2)-1.38 x 10(-3)). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.

Null Mutation in Transforming Growth Factor β1 Disrupts Ovarian Function and Causes Oocyte Incompetence and Early Embryo Arrest

TGFbeta1 is implicated in regulation of ovarian function and the events of early pregnancy. We have investigated the effect of null mutation in the Tgfbeta1 gene on reproductive function in female mice. The reproductive capacity of TGFbeta1 null mutant females was severely impaired, leading to almost complete infertility. Onset of sexual maturity was delayed, after which ovarian function was disrupted, with extended ovarian cycles, irregular ovulation, and a 40% reduction in oocytes ovulated. Serum FSH and estrogen content were normal, but TGFbeta1 null mutant mice failed to display the characteristic proestrus surge in circulating LH. Ovarian hyperstimulation with exogenous gonadotropins elicited normal ovulation rates in TGFbeta1 null mutant mice. After mating with wild-type stud males, serum progesterone content was reduced by 75% associated with altered ovarian expression of mRNAs encoding steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase-1 and P450 17 alpha-hydroxylase/C17-20-lyase. Embryos recovered from TGFbeta1 null mutant females were developmentally arrested in the morula stage and rarely progressed to blastocysts. Attempts to rescue embryos by exogenous progesterone administration and in vitro culture were unsuccessful, and in vitro fertilization and culture experiments demonstrated that impaired development is unlikely to result from lack of maternal tract TGFbeta1. We conclude that embryo arrest is due to developmental incompetence in oocytes developed in a TGFbeta1-deficient follicular environment. This study demonstrates that TGFbeta1 is a critical determinant of normal ovarian function, operating through regulation of LH activity and generation of oocytes competent for embryonic development and successful initiation of pregnancy.

Hybridization in the sea: gametic and developmental constraints on fertilization in sympatric species of Pseudechinus (Echinodermata: Echinoidea)

The constraints of gametic compatibility and developmental competence on hybrid production were examined in three sympatric species of the echinoid Pseudechinus in southern New Zealand. Differences were noted in embryonic temperature sensitivity between the species. Pseudechinus novaezealandiae embryos appeared to be better adapted to cooler temperatures than those of Pseudechinus huttoni, while Pseudechinus albocinctus embryos tolerated a wider range of temperatures than either of its sympatric congeners. Such differences were mirrored by the kinetics of gamete interaction between all possible reciprocal crosses of the three species. There appeared to be some physiological incompatibility between the eggs of P. novaezealandiae and the spermatozoa of the other two species. P. novaezealandiae spermatozoa were, however, capable of fertilizing both P. huttoni and, to a lesser extent, P. albocinctus ova. Hybrid echinoplutei resulting from the P. huttoni (female)× P. novaezealandiae (male) cross did not, however, become competent to metamorphose. The probabilities of settlement of echinoplutei resulting from the other reciprocal crosses involving the gametes of P. novaezealandiae were also low. It appears that a combination of physiological (gametic incompatibility) and genetic (developmental incompetence) factors contribute to maintaining the integrity of the P. novaezealandiae lineage. These factors were not useful in understanding how P. albocinctus and P. huttoni remain distinct species. This species-pair was characterized by reciprocal gamete compatibility as well as developmental competence to settlement. Thus, gamete incompatibility, while important for some groups, cannot provide the basis for speciation in the New Zealand members of the genus Pseudechinus.

Oocyte maturation and ovum quality in pigs

Oocyte quality in pigs is defined as the potential of that oocyte to develop into a viable offspring. There is increasing evidence that the programming of the oocyte must be completed before leaving the ovarian follicle, including both nuclear and cytoplasmic maturation. Pig oocytes matured in vitro under basic conditions are deficient in some, as yet unidentified, cytoplasmic factors and thus developmentally incompetent. This developmental incompetence can be overcome to some extent by follicular supplementation (with follicular fluid or granulosa cells) of the culture medium, emphasizing the importance of somatic signals during oocyte maturation. Furthermore, evidence is accumulating that the status of the follicle has a critical impact on the competence of the oocyte in vitro and in vivo and this has been demonstrated by co-culture with follicles at different maturational stages or from breeds with enhanced embryo survival. It now also appears that manipulation of maternal nutrition before mating or oocyte collection can enhance embryo survival in vivo and oocyte developmental competence in vitro, presumably by altering follicular secretions and hence the environment in which the oocyte is nurtured. Identification of both the key follicular factors influencing pig oocyte quality and reliable markers of oocyte quality will undoubtedly yield major improvements in embryo survival in vivo and embryo production in vitro.