Failure Of Embryo Development Research Articles

P-603 Combined oral contraceptive pill (COCP) before controlled ovarian hyperstimulation (COH) in donors could negatively affect egg donation outcomes in recipients: a multicentre retrospective study

Abstract Study question Could COCP use before COH with a progestin primed ovarian stimulation protocol (PPOS) in donors also negatively influence the egg donation outcomes in recipients? Summary answer Synchronization with a combined oral contraceptive pill (COCP) before controlled ovarian hyperstimulation (COH) involving progestin for pituitary suppression shows worst outcomes in egg donation treatments. What is known already It is well known that COCP leads to a long-lasting pituitary suppression, a reversible reduction in AMH and antral follicle count with higher risk of inappropriate response to COH and GnRH-agonist trigger, potentially reducing number and quality of retrieved oocytes. This is supported by the latest international guidelines on COH that strongly discourage COCP use before GnRH antagonist protocols because of a reduced efficacy of such cycles, also, in terms of ongoing pregnancy rate (OPR). They also underlie that interactions between COCP pretreatment and PPOS protocols remain inadequately explored and more research are needed to clarify discrepancies persisting in literature. Study design, size, duration Multicentre retrospective cohort study evaluating egg donation outcomes, depending on the use or not of COCP pre-treatment in donors, in terms of OPR, Live Birth Rate (LBR), and secondly of fertilization rate (FR), blastulation rate (BR), implantation rate (IR) and miscarriage rate (MR). The treatments were realized in a Private Fertility Clinic (where donors also underwent COH with PPOS protocol) and a public hospital (transnational egg donation program) between January 2022 and September 2023. Participants/materials, setting, methods 216 egg donation cycles were studied. Excluding cases of fertilization and/or embryo development failure and cases for which embryo transfer outcomes were not available at the time of data collection, the study analysed the outcomes of 90 embryo transfers (ETs). After the first analysis on total cycles, a stratification was made to evaluate whether COCP could have a different influence depending on the oocytes type used: fresh or vitrified. Main results and the role of chance COCP-No and COCP-Yes groups show similar FR and BR, after donation of same oocytes number (6,4 ± 1,1 vs 6,5± 1). Interestingly, vitrified oocytes from COCP-Yes donors produced a statistically lower yield of viable blastocysts (22,2% versus 37,9%; p = 0,007). IR was higher in the COCP-No group, (46,4% vs. 28,6%), data confirmed also in the two subgroups (fresh: 42,9% vs. 14,3%; vitrified: 48,8% vs. 35,7%). Higher MR was observed in the COCP-Yes group, achieving statistical significance both for total oocytes (66,7% vs. 9,4%; p = 0,006) and vitrified ones (60% vs. 5%; p = 0,016). Pregnancy rate was higher in the COCP-No group (46,4% vs. 28,6%), the same trend was found regardless of the type of oocytes used (fresh: 42,9% vs. 14,3%; vitrified: 48,8% vs. 35,7%). The greatest discrepancies were observed in OPR and LBR subcategories. In fact, a statistically higher OPR was obtained in the COCP-No group (42% vs. 9,5%, p = 0,008), which leads to a statistically higher LBR (30,4% vs. 0%; p = 0,004). Notably, the LBR after using both fresh and vitrified oocytes was markedly higher in the COCP-No group (fresh: 28,6% vs. 0%, p = 0,385; vitrified: 31,7% vs. 0%, p = 0,041), presenting a significant difference when vitrified oocytes were used with potential clinical relevance. Limitations, reasons for caution The main limitation lies in the retrospective nature of the study and the small sample size, but the multicentre design gives greater reliability. Further research is needed to confirm these findings and better understand the specific circumstances in which COCP may or may not be beneficial. Wider implications of the findings COCP pretreatment in egg donors undergoing PPOS protocol may negatively impact embryo development and pregnancy outcomes in recipients, with increased MR, and reduced OPR, and LBR. Particularly, the worst outcomes involve vitrified oocytes, providing valuable insights to reconsider COCP use in transnational egg donation programs, but also fertility preservation ones. Trial registration number Not Applicable

P-258 In-vitro oocyte maturation (IVM) in the ART clinic – What can we learn from our failed cycles?

Abstract Study question Why do some IVM cycles remain unsuccessful? Which factors contribute to failure in producing a usable embryo in IVM cycles? Summary answer Failure to obtain usable embryos after IVM was associated with oocyte dysfunction, inappropriate patient selection and unanticipated low oocyte yield. What is known already In-vitro maturation of oocytes retrieved from antral follicles has been offered as a mild-ART-approach to subfertile women with increased functional ovarian reserve, ovarian resistance to gonadotropins and in the context of fertility preservation. When using monophasic IVM culture systems, oocyte maturation, embryological development and pregnancy outcomes are generally lower compared to conventional ART (cART). Women who yield high numbers of cumulus oocyte complexes from a single egg retrieval procedure, such as those with polycystic ovary syndrome (PCOS), have been considered the best candidates for IVM, to compensate for the lower maturation rate and lower embryo yield from IVM cycles. Study design, size, duration This was a single-tertiary center, retrospective cohort study between May 2018 and February 2022 including 277 patients: 53 patients with failed IVM and 224 patients with a successful IVM cycle (inter-patient analysis). All ART cycles from patients with one failed IVM were investigated (intra-patient analysis). Failed IVM cycles were grouped: (i) ≤20% oocyte maturation; (ii) fertilization failure; (iii) embryo development failure. Patients with FSH resistance, fertility preservation or egg donation were excluded. Participants/materials, setting, methods Only patients with AMH >2.27 ng/ml were included. Oocytes retrieved in minimally stimulated non-hCG triggered IVM cycles were matured in a monophasic IVM system. Embryos were vitrified at cleavage stage and transferred in a hormonally substituted (HRT) cycle. Baseline patient characteristics, endocrinology and embryological data were analyzed. Intra-patient analysis was done using all ART cycles (failed IVM, IVM and cART) performed in one patient. Main results and the role of chance Inter-patient analysis revealed similar age, BMI and free testosterone, whereas AMH was lower in patients with a failed cycle (8.8 ± 3.5 vs 10.9 ± 6.4 ng/mL, p = 0.03). As expected, maturation, fertilization and useable embryos differed significantly in failed vs successful cycles: respectively 31% vs 48%; 40% vs 65%; 0% vs 50%; p < 0.0001. Out of 53 patients with a failed IVM cycle, 6 had oocyte maturation problems, 11 had failed fertilization and 36 had impaired embryological development. Intra-patient analysis revealed that in the failed oocyte maturation group, 2/6 patients had maturation problems that persisted in cART cycles, indicating oocyte dysfunction. Similarly, recurrent fertilization failure was noted in 3/11 patients, due to insufficient sperm quality (n = 2). In 8/36 patients, insufficient embryo quality was observed both after IVM and cART. Hence 13/53 (24.5%) failed IVM cycles were attributed to intrinsic patient characteristics. A subgroup of patients with a failed IVM cycle had normal maturation (3/6), fertilization (7/11) or good embryos (18/36) in other IVM or cART cycles. Factors associated with cycle failure included technical issues during egg retrieval resulting in unanticipated low oocyte yield (n = 14/40), and suboptimal inclusion criteria (no PCOS, n = 3/40). Limitations, reasons for caution This was a retrospective study and holds the possibility of unmeasured confounding factors. Wider implications of the findings Risk factors for IVM failure should be identified (e.g. non-PCOS, poor operator experience) and any underlying intrinsic patient conditions should be managed within a multi-disciplinary team. Optimal patient selection is key. Analysis of failed cycles helps clinicians to estimate the prognosis of future ART cycles in women with elevated AMH. Trial registration number not applicable

Lipid peroxidation in avian semen.

The main cause of sperm chromatin damage is oxidative stress related to embryo development failure and adult infertility in mammals and also avian. Oxidative stress results in lipid peroxidation (LPO) causing cell damage. Lipid peroxidation is the oxidation of polyunsaturated fatty acids (PUFAs) in biological systems and causes changes in the physical structure and characteristics of the cell membrane. Due to the high amounts of PUFAs in the avian sperm membrane, its sperm seem susceptible to pe-roxidative damage and is a substantial factor in the fertilization capacity of sperm. The most commonly used methods for measuring LPO or its by-products, such as malondialdehyde (MDA) and 4-hydroksy-2-nonenal (4-HNE), in bird semen are based on the colorimetric method TBARS (thiobarbituric acid reactive substances) and on the use of a fluorescence probe (CC 11-BODIPY 581/591) as a marker to evaluate membrane lipid peroxidation. This review aims first to introduce LPO in avian semen and its effects on avian sperm and second to summarize the commonly applied methods of evaluating LPO and its damage in fresh and stored avian semen.

Overexpression of the Ginkgo biloba dihydroflavonol 4-reductase gene GbDFR6 results in the self-incompatibility-like phenotypes in transgenic tobacco

ABSTRACT Although flavonoids play multiple roles in plant growth and development, the involvement in plant self-incompatibility (SI) have not been reported. In this research, the fertility of transgenic tobacco plants overexpressing the Ginkgo biloba dihydroflavonol 4-reductase gene, GbDFR6, were investigated. To explore the possible physiological defects leading to the failure of embryo development in transgenic tobacco plants, functions of pistils and pollen grains were examined. Transgenic pistils pollinated with pollen grains from another tobacco plants (either transgenic or wild-type), developed full of well-developed seeds. In contrast, in self-pollinated transgenic tobacco plants, pollen-tube growth was arrested in the upper part of the style, and small abnormal seeds developed without fertilization. Although the mechanism remains unclear, our research may provide a valuable method to create SI tobacco plants for breeding.

Insights on seed abortion (endosperm and embryo development failure) from the transcriptome analysis of the wild type plant species Paeonia lutea

Paeonia lutea is a wild peony (an endangered flowering plant species) found in China. Seed abortion (endosperm and embryo development failure) is linked to several endangered plant species. Therefore, it is of interest to complete a comparative analysis of transcriptome between the normal active seeds (Population A) and the endangered abortion seeds (Population H). Data from GO assignments of differentially expressed genes (DEGs) shows that "metabolic process", "binding", "cellular process", "catalytic activity", "cell" and "cell part" are commonly prevalent in these popuations. DEGs between the populations are found to be connected with metabolic pathways, biosynthesis of secondary metabolites, purine metabolism and ribosome. We used quantitative RT-PCR to validate 16 DEGs associated with these populations. It is found that histone genes and proline-rich extensin genes are predominant in the common groups. Histone genes (H2A, H2B, H3, H4 and linker histone H1) show 3 to 4 folds log2FC higher expession in population A than in population H in stage I unlike in stage II and III. Increased activity of proline-rich extensin genes in population A than in population H corresponding to seed abortion in the later population is implied. These preliminary data from the transcriptome analysis of the wild type plant species Paeonia lutea provide valuable insights on seed abortion.

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

Background:Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects.Objectives:Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects.Methods:Female mice were exposed to GEN on postnatal days (PND)1–5 and uterine tissues collected on PND5, PND22–26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators.Results:GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice.Discussion:Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336

Certain morphological characteristics of the spermatozoon used for intracytoplasmic sperm injection (ICSI) are associated with a higher likelihood of developmental arrest and embryo degeneration

Around 25% of in-vitro cultured human embryos arrest their development and degenerate during the first 5 days. The functional and morphological characteristics of the spermatozoa, selected for the intracytoplasmic sperm injection (ICSI) could account for the subsequent embryo development failure. The objective of the present study was to assess the relationship between the morphological characteristics of the spermatozoon used for ICSI and the increased risk of developmental arrest and embryo degeneration. Prospective observational study. A total of 83 spermatozoa from 68 men used in ICSI procedures were studied. Inclusion criteria were: 1) patient age < 39 years, 2) patient body mass index (BMI) < 25 kg/m2, 3) excellent quality oocytes. To find out which sperm morphological features could be responsible for the embryo degeneration the morphology of each individual spermatozoon used for ICSI was recorded. The morphology of all individual spermatozoa was evaluated using high magnification (×6100) immediately before the injection. Seven sperm morphological parameters and the presence of sixteen head, neck and tail abnormalities were evaluated. All 83 embryos were cultured in-vitro in Global one-step medium. The frequencies of occurrence of sperm morphological defects were compared between the resulted viable and degenerated embryos. Data was analyzed using the IBM SPSS software, v.21.0. Among 83 ICSI performed, 63 embryos (75.9%) had successful development and 20 embryos (24.1%) arrest their development before day 5. The incidence of pyriform head, thick midpiece and cytoplasmic residues among the spermatozoa that resulted in embryos with developmental arrest were significantly higher in comparison to spermatozoa that led to successfully developed embryos (20% vs. 13%, 40% vs. 21% and 20% vs. 6%, respectively, p<0.05, Chi-square test). In addition, the spermatozoa resulted in embryos with successful development had significantly shorter heads and tails in comparison with those that resulted in embryo development arrest (4.46 vs. 4.63 μm and 41.42 vs. 43.65 μm, p<0.05, Student t-test, respectively). When receiver operating curve (ROC) analysis was performed to find the optimum cut-off value to predict embryo development sperm head length had the best AUC (AUC = 0.66, CI 95%: 0.51–0.76). The optimum cut-off value for head length was 4.25 μm (sensitivity 89.5%, specificity 60.3%). The recorded spermatozoa tail length had lower AUC (0.63, CI 95%: 0.49–0.77) and the optimal cut-off value of 42.5 μm (sensitivity 63.2%, specificity 42.5%). Specific sperm morphological characteristics, such as head length and tail length and the presence of certain sperm abnormalities (pyriform head, thick midpiece and residual cytoplasmic droplets) are associated with an increased risk of developmental arrest and embryo degeneration. Selection of human spermatozoa with head length < 4.25 μm, tail length < 42.5 μm and without pyriform head, thick midpiece and cytoplasmic residues significantly increase the chance for successful embryo development.

Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis.

Does the molecular and metabolic profile of human mural granulosa cells (GCs) correlate with oocyte fate? A close relation between the metabolic profile of mural GCs and the fate of the corresponding oocyte was revealed by the analysis of selected biomarkers defined by GC Fourier transform infrared microspectroscopy (FTIRM) analysis. In ART, oocyte selection is mainly based on the subjective observation of its morphological features; despite recent efforts, the success rate of this practice is still unsatisfactory. FTIRM is a well-established vibrational technique recently applied to evaluate oocytes quality in several experimental models, including human. GCs retrieved from single-follicle aspirates were obtained with informed consent from 55 women undergoing controlled ovarian stimulation for IVF treatment. GCs were analysed by FTIRM to retrospectively correlate their spectral features with the fate of the companion oocytes. The study has been conducted between March 2016 and September 2017. Patients were selected according to the following inclusion criteria: age <40 years; non-smokers; no ovarian infertility diagnosis (only tubal, idiopathic and male infertility); regular ovulatory menstrual cycles (25-30 days) with FSH < 10 IU/I on Day 3 of the menstrual cycle; sperm sample with a total motility count after treatment ≥300.000; number of retrieved oocytes ≥8. Based on the clinical outcome of the corresponding oocyte, GCs were retrospectively classified into the following experimental groups: clinical pregnancy (CP), fertilization failure (FF), embryo development failure (EDF) and implantation failure (IF). All samples were analysed by the FTIRM technique. The spectral biomarker signature of different oocyte fates was derived by several feature selection procedures ('Leave-one-out' method on factorial discriminant analysis (FDA), variable characterization method and logistic regression method with the multinomial Logit model). ANOVA, permutational multivariate ANOVA, FDA and canonical analysis of principal co-ordinates statistical tools were also applied to validate the identified spectral biomarkers. In total, 284 GCs samples were retrieved and retrospectively classified as FF: (N = 92), EDF (N = 113), IF (N = 56) and CP (N = 23). From the spectral profiles of GCs belonging to CP, FF, EDF and IF experimental groups, 17 spectral biomarkers, were identified by several feature selection procedures (P < 0.0001). These biomarkers were then validated by applying multivariate tools, to evaluate their ability to segregate GCs samples into the four experimental groups. FDA showed a clear separation along the F1-axis (62.75% of discrimination) between GCs from oocytes able (CP, IF groups) or not (FF, EDF groups) to develop into embryos; the F2-axis (24.14% of discrimination) segregated the embryos that gave pregnancy (CP) from those that failed implantation (IF). The confusion matrix (total percentage of correctness = 80.25%) obtained from this analysis pinpointed that GCs from oocytes unable to develop into embryos (FF, EDF) were better characterized than those from oocytes able to give viable embryos (CP, IF). ANOVA (P < 0.05) analysis pinpointed that: each experimental group showed specific macromolecular traits, ascribable to different biological and metabolic characteristics of GCs; these metabolic features were likely associated with different oocytes fates, but not to patient characteristics, since from the same patient we obtained GCs with different metabolic profiles. The study is based on a small sample size but provides proof of concept that the GCs' metabolic profile is associated with the companion oocyte fate. The generated model should be further tested on a larger cohort of patients, classified in a similar manner, to assess the potential predictive value of this approach. Ultimately, validity of the proposed approach should be tested in a RCT. For the first time, the FTIRM analysis of human GCs has demonstrated an approach to better understand the molecular crosstalk between follicular cells and oocytes and has identified potential spectral biomarkers for improving human IVF success rate. The study was funded by GFI 2014 grant. The authors declare that there is no conflict of interest.

MP91-05 DO ALCOHOL, SMOKING AND MALE AGE AFFECT SEMEN PARAMETERS AND IVF/ICSI OUTCOMES?

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation1 Apr 2016MP91-05 DO ALCOHOL, SMOKING AND MALE AGE AFFECT SEMEN PARAMETERS AND IVF/ICSI OUTCOMES? Fahad AlMashat, Saad Abumelha, Chris Poullis, Tet Yap, Nagla Rushwan, Hossam Abdalla, Meen Yau Thum, and Suks Minhas Fahad AlMashatFahad AlMashat More articles by this author , Saad AbumelhaSaad Abumelha More articles by this author , Chris PoullisChris Poullis More articles by this author , Tet YapTet Yap More articles by this author , Nagla RushwanNagla Rushwan More articles by this author , Hossam AbdallaHossam Abdalla More articles by this author , Meen Yau ThumMeen Yau Thum More articles by this author , and Suks MinhasSuks Minhas More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2592AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To determine the effects of alcohol, smoking and male age on semen parameters and the outcomes from IVF/ICSI. METHODS Records of couples attending a single IVF Unit were retrospectively reviewed (2013 to 2014). The age, number of cigarettes smoked/day and number of units of alcohol consumed/week were recorded along with semen parameters performed on the day of IVF/ICSI. Couples with the female age over 37 years were excluded. Smoking was sub-categorized into (1-9, 10-20 and > 20) cigarettes/day, and alcohol consumption (1-4, 5-10, 11-20 and >20) units/week. A multivariate analysis of the effects of age, alcohol consumption and smoking on semen parameters and outcome from IVF/ICSI was performed. RESULTS 1257 couples attended for fertility assessment. Mean age was 36.4 (Range 23-76). Mean Female age was 34 (Range 20-37). 13% (n=162) of males were smokers (mean 9 cigs/day). 77% (n=964) of males had a history of alcohol consumption (mean 9.8 units/week). 35.8% (n=450) of couples underwent IVF treatment, 57% (n=717) underwent ICSI and 7.2% (n=90) had failure of embryo development. Increasing male age was associated with a reduction in semen volume, sperm count, motility and morphology (p< 0.05). Fertilization rate, pregnancy and live birth rate were unaffected (p=0.603, p=0.895 and p=0.088 respectively). Smoking in all groups showed a reduction in sperm count only. There were no differences in ICSI outcome between smokers and non-smokers. With IVF, smoking reduced pregnancy rates (p=0.049) but not fertilization or life birth rates (p=0.081 and p=0.219 respectively). Alcohol affected sperm motility and morphology (p < 0.0001) but not volume or count (p=0.544 and p=0.232 respectively). In IVF, pregnancy rate was reduced (p<0.048) but not fertilisation and live birth rates (p=0.847and p=0.063 respectively). In ICSI, fertilization rates were reduced (p<0.013) but not pregnancy and live births (p<0.132 and p<0.099 respectively). No correlation between the number of cigarettes or alcohol units and the outcome of IVF/ICSI was established. CONCLUSIONS Age, smoking and alcohol impair semen parameters. The number of cigarettes and amount of alcohol consumed did not correlate with IVF/ICSI outcome. Live birth rates from IVF/ICSI are not affected by these factors suggesting that these technologies improve fertility outcomes by optimising sperm selection. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1154-e1155 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Fahad AlMashat More articles by this author Saad Abumelha More articles by this author Chris Poullis More articles by this author Tet Yap More articles by this author Nagla Rushwan More articles by this author Hossam Abdalla More articles by this author Meen Yau Thum More articles by this author Suks Minhas More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Increased cleavage rate of human nuclear transfer embryos after 5-aza-2′-deoxycytidine treatment

As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. The present study found that the use of cytochalasin B was not necessary for, and even harmful to, the enucleation of human zygotes. This study also decreased the DNA methylation levels in reconstructed embryos using a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), in an attempt to correct the abnormalities in DNA methylation that might play an important role in the failure of embryo development. After 5-aza-dC treatment and nuclear transfer (NT-Aza group), 32.7% of reconstructed embryos developed to the 8-cell stage, which is a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that of the NT group, as determined by 5-methylcytosine immunodetection. Based on the present results, this study recommends performing the enucleation procedure without cytochalasin B treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies.As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. In the present study, the effects of cytochalasin B (CB) treatment on the survival rate of the manipulated zygotes and the effects of 5-aza-2′-deoxycytidine (5-aza-dC) treatment on the cleavage rate of the reconstructed embryos were observed. Human polyspermic zygotes were enucleated in metaphase with or without CB treatment and injected with human cumulus cells. The reconstructed embryos were cultured in medium supplemented with or without 5-aza-dC for developmental potential comparison. The DNA methylation levels of the embryos were determined using 5-methylcytosine immunodetection. CB treatment lowered the survival rate of manipulated zygotes. After the 5-aza-dC treatment (NT-Aza group), 32.7% of the reconstructed embryos developed to the 8-cell stage, a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that in the NT group. Based on the present results, we recommend performing the enucleation procedure without CB treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies.

담수순화 감성돔 Acanthopagrus schlegelii 동결보존 정자의 수정능력

Fertilization rates were over 90% when sperm from black porgy Acanthopagrus schlegelii acclimated and raised in freshwater and black porgy raised in seawater were fertilized with eggs from female black porgy raised in seawater, although cryopreserved sperm were significantly lower in fertilization rates than non-cryopreserved sperm. From the observations of embryos development at different salinities (0, 10, 20 and 32 psu) within 3 hours upon fertilization (16 cell stage), all were dead at 0 psu. However, the development process and speed at 10 and 20 psu were the same as at 32 psu. But, many developing embryos were egg envelope injury at low-salinities of 10 and 20 psu unlike at 32 psu. This is attributable to osmotic shock. Hatching rate of embryos at 32 psu was 60% or so, whereas low as 5% or less were successfully hatched at 10 and 20 psu, implying that osmotic shock could be responsible for the failure of embryo development.

Dynamic distribution of NuMA and microtubules in human fetal fibroblasts, developing oocytes and somatic cell nuclear transferred embryos

The nuclear mitotic apparatus (NuMA) plays a central role in the assembly and maintenance of spindle poles. Somatic cell nuclear transfer (SCNT) studies on non-human primates have shown that meiotic spindle removal during enucleation causes depletion of NuMA and the minus-end-directed motor protein (HSET) from the ooplasm, and this in turn leads to failure of embryo development. To determine whether NuMA from somatic cells could compensate for NuMA loss during enucleation, the distribution of NuMA and microtubule organization were investigated in human fibroblasts, developing oocytes and SCNT embryos. Human fetal fibroblasts, oocytes at various maturation stages and human embryos reconstructed by different SCNT methods were analyzed for NuMA and α-tubulin using immunofluorescent confocal microscopy. NuMA was detected in interphase nuclei of fibroblasts and oocytes. During mitosis and meiosis, NuMA relocated to the domain surrounding the two spindle poles. During the enucleation process, NuMA was removed along with the meiotic spindle. At 2 h after injection into a donor cell, transitory bipolar spindles were organized and NuMA was detected in the reformed poles. NuMA could be detected spreading uniformly across the nucleoplasm of one pseudo-pronucleus in SCNT embryos but was excluded from the nucleolus. Regardless of the method used for SCNT (enucleation-injection or injection-pronuclei enucleation), NuMA aggregated and relocated to the reformed spindle poles at metaphase of the first mitotic event. At interphase, NuMA relocated throughout the nucleus in developmentally arrested SCNT embryos. Our results show that donor cell nuclei contain NuMA, which might contribute to the maintenance of spindle morphology in SCNT embryos. Normal spindle and NuMA expression were found in human SCNT embryos at different developmental stages.

The role of embryopathology in the study of first trimester spontaneous miscarriage

The study of first trimester spontaneous miscarriage is the object of increasing attention not only for clinicians but also for patients and their family. Embryopathology concerns the study of pathologic conditions and malformations like early development failure, development alterations and true malformative lesions. It is well known that the most severe genetic abnormalities emerge as an interruption in embryogenesis and that the most complex developmental abnormalities caused by external factors - like infections, toxic and environmental agents - have their onset in the early stages of embryo formation and are often also the cause of pregnancy loss. In order to improve the monitoring of at risk couples we have expanded the study of basic research with those of research applied to human pathology. The role of embryopathology is fundamental to understand the timing at which the embryo development failure and its subsequent death actually occur and the main defect in embryo formation underlying development abnormalities. From 1994 to 2010 we examined 256 early spontaneous abortions in which an intact gestational sac was present. The introduction of acrylic resin embedding technique in 1995 added many advantages such as the possibility of obtaining semi-thin and uniformly thick sections with better histological and cytostructural details, assuring the almost total absence of coartation, and the preservation of antigenic properties. This technique therefore permits improvement in morphological obervation in order to reinterpret the classification of spontaneous aborted embryo. This enables a better definition of the real stage and time when development was interrupted. That is the basis to understand the role of external factor as a cause of pregnancy loss.

292 EVALUATION OF THREE PROTEIN SOURCES FOR THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

Bovine embryo development in vitro can be affected by many factors, including protein source, which can cause embryo development failure. The use of in vitro culture media supplemented with serum-free compounds could allow a better understanding of embryo requirements during the preimplantation stages by eliminating a highly variable and undefined compound such as serum. The objective of this study was to evaluate the effect of 3 different protein supplements used during IVM, IVF, and IVC on embryo production. Ovaries were collected from slaughtered cows and then aspirated to obtain oocytes for in vitro embryo production procedures. A total of 2056 oocytes were used, from which 685 were processed with maturation medium supplemented with 10% serum replacement (SR) (Gibco Knockout Serum Replacement, Invitrogen, Carlsbad, CA, USA), a defined serum-free formulation (TCM-199 + SR), fertilization medium with SR (TALP + SR), and culture medium with SR (SOF + SR). These were compared with 675 and 696 oocytes processed with the same IVM, IVF, and IVC media, but supplemented with 10% FCS or 10% heat-inactivated estrous cow serum (ECS), respectively. Data obtained from the variables studied were processed by analysis of variance and means were compared by Tukey’s test. The percentages of embryos produced with FCS (52.4%) and ECS (52.7%) were significantly higher compared with the percentage obtained with SR (41.5%) (P &lt; 0.05). The percentages of morulae were similar in the groups supplemented with FCS (36.5%) and SR (36.7%), but significantly higher than the percentage in the ECS group (26.9%) (P &lt; 0.05). For blastocysts, the percentages of embryos developed with FCS (35.2%) and ECS (35.6%) were significantly higher than that obtained with SR (29.2%) (P &lt; 0.05). When evaluating expanded blastocysts, the percentage obtained in the FCS (45.9%) group was significantly higher than that in the ECS group (33.2%), and this was significantly higher than that obtained in SR (21%), with all these differences being significant (P &lt; 0.05). It is concluded that it is possible to produce bovine embryos in vitro using FCS, ECS, or SR as supplements in IVM, IVF, and IVC media. Significant differences were found in different embryo stages, with the highest proportion of embryos developing with the addition of FCS, whereas supplementation with SR only improved the production of morulae. We thank Consejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico) for the graduate student’s scholarship.

229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment. Supported by MEC (AGL2006-03495).

Role of mammalian sperm nuclear structure in fertilization and embryo development.

Although intact spermatozoon is successfully collected from infertile patients, repeated implantation failure or pregnancy loss are often experienced. Sperm nuclear defects have been thought to be one of the most important reasons for repeated assisted reproductive technology failure. In comparison with other mammalians, characteristic heterogeneity has been found in each mature human sperm nuclei, therefore it is necessary to investigate the significance between fertilization failure, developmental disability and structural abnormality of human sperm nuclei. Furthermore, if close relationships between the heterogeneity of human ejaculated sperm nuclei and DNA fragmentation are defined by analyzing sperm nucleoproteins, it would be clearly shown that impaired sperm chromatin leads to failure of embryo development in vitro or in vivo, so called late paternal effect on embryo development. It will be necessary in the near future to study the strategy for more novel methodology than those previously reported in terms of sperm selection. The present report reviews the roles of mammalian sperm nuclear structure, especially in humans, in fertilization and embryo development after the insemination procedure. (Reprod Med Biol 2006; 5: 161-168).

Effects of exercise on in vitro fertilization (IVF) outcomes

To determine if regular exercise prior to IVF effects IVF outcomes. Observational study. Couples undergoing treatment for infertility from 1994–2003 at three IVF clinics in the greater Boston area were invited to complete a self-administered questionnaire and consent to review of their medical records. 65% of those approached agreed to participate. The couples included in this analysis were all undergoing their first cycle of IVF. The IVF outcomes of interest included (1) failed ovarian stimulation, (2) failure of embryo development after insemination, (3) cancellation of the cycle with frozen embryos, (4) failure of implantation (failure to develop a clinical or chemical pregnancy after embryo transfer), (5) failure to have a live birth after chemical pregnancy, and (6) successful live births. This final group, the successful live births, were used as the controls for each of the failure case groups. We used unconditional logistic regression to estimate relative risks (odds ratios (OR)) and 95 percent confidence intervals (CI) for failure at each stage associated with exercise. Of the 2572 women enrolled in this study, 2567 women answered questions about exercise. Of these women, 979 did not regularly exercise. Of the remaining 1588 women who did report regular exercise, 1563 reported hours of exercise per week, and 1512 reported the number of years they have been exercising. Among women who reported regular exercise, the mean hours of exercise reported per week was 4.3 hours (range 1 to 40 hours). The mean number of years exercised was 11.4 years (range 1 to 30 years). After adjusting for age, body mass index, and year of IVF procedure we observed no difference in IVF outcomes comparing women who regularly exercised to those who did not, nor did we observe a relation with number of hours exercised per week. However, we observed that compared with non-exercisers, women who had been exercising regularly for 1–5 years were at greater risk for failure of cycle stimulation (OR = 1.9; CI = 1.2- 3.2), failure of implantation (OR = 1.5; CI 1.1–2.0) and failure of development of a live birth after a chemical pregnancy (OR = 1.9; CI = 1.3–2.8). In addition, risk of cycle stimulation failure was also significantly elevated among women who had been exercising regularly for more than 10 years (OR = 2.0; CI = 1.2–3.5). Exercising in general and hours of exercise per week did not effect IVF outcomes. However, we observed a complex relationship between number of years of regular exercise and IVF outcomes.

The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development.

The integrity of sperm DNA is important for the success of natural or assisted fertilization, as well as normal development of the embryo, fetus and child. ICSI, by bypassing sperm selection mechanisms, increases the risk of transmitting damaged DNA and the significance of this requires investigation. DNA damage in sperm from an unselected group of 60 men undergoing IVF treatment was measured by single cell gel electrophoresis (Comet assay) and correlated with semen and treatment cycle parameters. Wide spectra of sperm DNA damage were found both within and between men but no specific subgroups were identified. Semen and treatment cycle parameters were not different in men grouped according to high or low sperm DNA damage. However, regression analysis showed that DNA damage was positively associated with age (29-44 years), abnormal sperm and motility and negatively associated with sperm concentration. In ICSI cycles DNA damage was positively associated with impairment of post-fertilization embryo cleavage. This study contributes to the evidence of DNA damage within sperm. High loads of DNA damage measured by the Comet assay were predictive of failure of embryo development after ICSI. As it is likely that sperm with DNA damage contributed to successful fertilization and in-vitro development, potential adverse effects remain to be clarified.

Cytological analyses of in vitro somatic embryogenesis in Brimeura amethystina salisb. ( Liliaceae)

Somatic embryogenesis was obtained through in vitro culture of roots, leaves, mesocotyls and bulb scales of Brimeura amethystina Salisb. Embryogenesis was achieved both on the primary explant and after callus formation. The highest frequencies of both callus and embryo formation were obtained when using media containing 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA). Complete plantlets were obtained by inducing adventitious rooting on hormone free medium. A cytological and cytophotometric study of embryos and plantlets was undertaken to explain the observed frequent failure of embryo development after the globular stage: a great variability of nuclear DNA content was found among non-developing embryos in respect of normal ones.

An ultrastructural study of embryo and endosperm development during in vitro culture of maize ovaries (Zea mays)

A culture method is described which allows the continuous supply of fresh liquid medium and which prevents the accumulation of toxic metabolites. Development of maize embryos and endosperm after various periods of in vitro ovary culture was studied by light and electron microscopy. Using this method the ultrastructural features of embryo development in vitro were similar to those of in vivo embryos. In contrast, the formation of endosperm was irregular with the absence of cellularization of the inner endosperm being frequent. In some cases, only the endosperm developed without any indication of embryo formation. In a calcium-depleted medium, embryo development was normal but again, endosperm formation was aberrant. No cells were formed in the central part of the endosperm and near the placental region degeneration took place, resulting in vacuoles with dark inclusions, clumps of rough endoplasmic reticulum membranes, and cellular breakdown. The events occurring after in vitro culture strongly resemble those taking place after intergeneric crosses or crosses between diploid and tetraploid strains. It is concluded that defective endosperm development is probably the main factor for the failure of embryo development.