Polycystic ovary syndrome phenotype does not have impact on oocyte morphology

Purpose The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype. Methods We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)). Results A total of 1496 metaphase II oocytes (n = 602 for phenotype A combining PCOM + HA + OA, n = 462 oocytes for phenotype C: PCOM + HA, and n = 432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), respectively: adjusted p = 0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype. Conclusion The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.

The Evolution of Alternative Buoyancy Mechanisms in Freshwater Fish Eggs

Alternative reproductive tactics (ARTs) are behavioural, morphological, and physiological traits associated with alternative reproductive phenotypes within a population or species. ARTs are widespread in nature, and are a particular feature of teleost fishes. However, few studies have examined egg buoyancy mechanisms in the context of the evolution of ARTs in freshwater fishes. In marine fishes, egg buoyancy is achieved chiefly through hydration. While the buoyancy of freshwater fish eggs has been suggested to be determined primarily through the presence of oil droplets, the majority (60%) of freshwater pelagic eggs do not possess an oil droplet. We applied a physical model of buoyancy to understand the contributions of oil droplets and hydration to the buoyancy of pelagic freshwater fish eggs. We further used phylogenetic regression to estimate the effect of the relative size of the perivitelline space, habitat and parental care on the occurrence of oil droplets, while controlling for non-independence among species due to phylogenetic relatedness. Our analysis demonstrates that the probability of oil droplets in freshwater pelagic eggs exhibits a significant negative relationship with the size of perivitelline space, which may reflect a trade-off relating to energy allocation in contrasting habitats. We also demonstrate a positive association between the probability of oil droplets and the provision of parental care and occupancy of lentic habitats. These findings indicates the evolution of contrasting buoyancy mechanisms as novel ARTs in freshwater fishes. A theoretical model in combination with empirical analysis indicate the evolution of novel ARTs in freshwater fishes as adaptive responses to flow conditions.

Vitamin D receptor gene polymorphisms and the effectiveness of assisted reproductive technology

Introduction. Vitamin D administration is recommended for women with vitamin D insufficiency, who are planning to undergo assisted reproductive treatment (ART). The action of the active form of vitamin D is mediated through its vitamin D receptor (VDR). The presence of VDR in the cells and tissues of the female reproductive system suggests that vitamin D plays an important role in human reproduction. However, the effect of VDR gene polymorphisms on human reproduction has not been adequately studied.Aim. To assess the effect of VDR gene polymorphisms on the folliculogenesis, embryogenesis and clinical outcomes of assisted reproduction programs.Materials and methods. The cross-sectional study included 300 patients without complications during the assisted reproduction cycle. The number of follicles, oocyte-cumulus complexes, mature and immature oocytes, the presence of oocyte dysmorphisms, number of zygotes, fertilization rate, and the number of obtained blastocysts were evaluated. The determination of three polymorphisms of the VDR gene (FokI – rs2228570, BsmI – rs1544410, TaqI – rs731236) was performed by real-time polymerase chain reaction.Results. The incidence of perivitelline space pathology of oocytes in FokI A/A genotype patients was 5.7%, in A/G genotype patients – 14.7%, in G/G genotype patients – 16.3% (p = 0.041 when comparing A/A genotype with A/G + G/G genotypes). Depending on the genotype, the pregnancy rate ranged from 38.5 to 52.8%, but no statistically significant differences were found.Conclusion. It was shown that the perivitelline space pathology of oocytes in the FokI A/G genotype patients is 2.6 times higher, and in the G/G genotype patients – 2.9 times higher than in the A/A genotype patients. However, there were no differences in pregnancy rate among patients with different genotypes of FokI, BsmI and TaqI gene polymorphisms of VDR.

Polycystic Ovary Syndrome Phenotype Does Not Have Impact on Oocyte Morphology

Purpose The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype.Methods We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)).Results A total of 1496 metaphase II oocytes (n=602 for phenotype A combining PCOM + HA + OA, n=462 oocytes for phenotype C: PCOM + HA, and n=432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5% to 39.1%), 25.8% (21.9% to 29.9%) and 34.0% (29.7% to 38.6%), respectively: adjusted p=0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype.Conclusion The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.

P–215 The degree of perivitelline space (PS) at the pronuclear stage affects subsequent embryonic development in human zygotes

Study question Was embryonic development affected by the degree of perivitelline space (PS) at the pronuclear stage in human zygotes? Summary answer Zygotes with a fully surrounding PS showed less cytoplasmic fragmentation and a higher blastocyst development rate (BDR) than zygotes with a partially surrounding PS. What is known already We previously used abnormally-fertilized oocytes (zygotes with three pronuclei; 3PN), donated by ART patients in our clinic who gave written consent for the research. The zona pellucida (ZP) was artificially removed from these oocytes at the pronuclear stage, termed ZP-free culture. The resultant ZP-free 3PN embryos showed less cytoplasmic fragmentation and a higher rate of good-quality embryos (GQE) compared with ZP-intact embryos. Furthermore, in our clinical setting, the rate of GQE and BDR of normally-fertilized embryos were clearly improved by ZP-free culture in patients with recurrent failure of ART treatments due to severe cytoplasmic fragmentation at the early cleavage stage. Study design, size, duration This study included 49 patients who gave written informed consent for our study and were treated with ART in our clinic between March and December 2020. Embryonic development was compared between zygotes with a fully surrounding PS [PS(+)] with those with a partially surrounding PS [PS(-)] at the pronuclear stage. Furthermore, the ZP of PS(-) embryos were artificially removed at the pronuclear stage, and the rate of GQE and BDR were compared with ZP-intact embryos. Participants/materials, setting, methods The degree of PS in 128 zygotes was confirmed by hypertonic preparation using 0.125M sucrose-containing HEPES medium. PS(+) and PS(-) embryos were both cultured as ZP-intact, and the rate of GQE was compared. Furthermore, 223 zygotes were divided into three groups: 1) PS(-)/ZP-intact, 2) PS(-)/ZP-free, and 3) PS(+)/ZP-intact, and cultured in an incubator equipped with time-lapse monitoring up to Day 7, and the rate of GQE, BDR and useable embryos were compared between each groups. Main results and the role of chance The degree of PS was confirmed by a hypertonic preparation (shrinkage of the ooplasm) in 128 normally-fertilized zygotes obtained from 44 cases. There were 86 PS(-) (67.2%) and 42 PS(+) (32.8%) zygotes. The mean maternal age was 35.9 in PS(-) and 40.5 in PS(+) (P < 0.01), and the rate of GQE was significantly higher in PS(+) [64.3% (27/42)] than in PS(-)[38.4% (33/86)] (P < 0.01). In addition, of 223 normally-fertilized zygotes obtained from 41 cases, there were 51 PS(-)/ZP-intact (Group 1), 132 PS(-)/ZP-free (Group 2) and 40 PS(+)/ZP-intact (Group 3) zygotes. The rate of GQE was significantly lower in Group 1 [29.4% (15/51)] compared with Group 2 [59.8% (79/132)] and Group 3 [62.5% (25/40)] (P < 0.01). BDR was also significantly lower in Group 1 [51.3% (10/39)] compared with Group 2 [75.0% (99/132)] and Group 3 [65.0% (13/20) (P < 0.01). Limitations, reasons for caution Although the artificial removal of ZP at the pronuclear stage (ZP-free culture) clearly increased the rate of GQE, embryonic development was not improved in all cases. It seems that this procedure is only effective in embryos with a viable ooplasm. Wider implications of the findings: The degree of PS at the pronuclear stage affects subsequent embryonic development in human zygotes. The artificial removal of ZP at the pronuclear stage (ZP-free culture) helps to suppress fragmentation and leads to an increase in GQE and BDR, and eventually, improves pregnancy rate in cases with severe fragmentation. Trial registration number non

O-209 The impact of sperm DNA fragmentation on pregnancy outcomes depends on oocyte dimorphisms

Study question Does the impact of sperm DNA fragmentation (SDF) on Intracytoplasmic sperm injection (ICSI) outcomes depend on the presence of oocyte dimorphisms? Summary answer There is a significant influence of oocyte quality on the impact of SDF on pregnancy outcomes. What is known already Sperm DNA fragmentation has been associated with ICSI outcomes. DNA damage is commonly encountered in human spermatozoa and it has been widely accepted that the oocyte assumes responsibility for the repair and remodelling of both the maternal and paternal genomes during the oocyte-embryo transition. Indeed, spermatozoa with DNA damage can fertilise oocytes and still lead to embryo development due to the oocyte DNA repair capacity. Considering the vital role played by the oocyte in the developmental process, it could be hypostatised that the oocyte quality, translated as oocyte morphology, would influence the machinery responsible for sperm DNA repair after fertilization. Study design, size, duration This cohort study included 3,035 oocytes from 525 patients undergoing ICSI cycles in a university-affiliated IVF-center, between June/2016 and July/2019. Oocytes were split into groups according to the SDF index of the sample used for ICSI: low-fragmentation (<30% SDF, n = 2,277) and high-fragmentation (≥30% SDF, n = 758). Oocytes were evaluated before sperm injection and the dimorphisms were recorded. The influence of SDF index on ICSI outcomes, depending on the presence of oocytes dimorphisms was evaluated. Participants/materials, setting, methods Data was evaluated using generalized linear models (GZLM) followed by Bonferroni post hoc. The results are expressed as mean ± standard error for continuous variables or percentages for dichotomous variables, and p-values. The sample size calculation suggested that a sample of at least 504 subjects had 95% power to detect a 20% effect with a significance level of 5% (α). The study was performed in a private university–affiliated in vitro fertilization (IVF) center. Main results and the role of chance The association of both factors: the presence of oocyte dimorphisms (dark cytoplasm, vacuoles in the ooplasm, and resistant membrane) and high SDF index resulted in the lowest fertilization rate among groups, while oocytes free of these dimorphisms injected with samples with <30% SDF had the highest fertilization rate (p = 0.05, p < 0.01 and p < 0.01 for dark cytoplasm, vacuoles in the ooplasm and resistant membrane respectively). The impact of SDF index on high quality embryos rate on cleavage stage was also influence by the presence smooth endoplasmic reticulum clusters and resistant membrane oocytes (p = 0.013 and p = 0.018). As for the clinical outcomes, the impact of SDF index on the implantation rate was influenced by the presence of vacuoles in the ooplasm (p < 0.01), smooth endoplasmic reticulum clusters (p < 0.01), large perivitelline space (p < 0.01), resistant membrane (p < 0.01), and non-resistant membrane (p < 0.01), while the influence of SDF index on the pregnancy rate was influenced by the presence large perivitelline space (p < 0.01), resistant membrane (p = 0.018) and non-resistant membrane (p < 0.01). The effect of SDF on the miscarriage rate was also increased in the presence of large perivitelline space (p = 0.045), non-resistant membrane (0.037) and centrally located cytoplasmic granular area (p = 0.025). Limitations, reasons for caution The retrospective nature is a limitation. It could be argued that using samples with high SDF index does not necessarily mean that a sperm cell with a fragmented DNA was injected, however, the higher the SDF index, the higher the chance of selecting one with fragmented DNA. Wider implications of the findings The findings presented here highlight the crucial role of male and female factors when facing assisted reproduction. The association of low oocyte quality and high SDF index may lead to impaired results. As the oocyte defect cannot be modified, in vivo upgrading of spermatozoa before the treatment should be encouraged. Trial registration number Not applicable

Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 μm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 μm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 μm (n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.

Cellular Fragments in the Perivitelline Space Are Not a Predictor of Expanded Blastocyst Quality

The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of in vitro produced (IVP) embryos. However, this parameter is difficult to assess after blastocyst expansion. In this study, we used mechanical hatching to confirm the presence of cellular fragments in the perivitelline space of bovine IVP blastocysts. We further looked for associations between possible apoptosis within extruded cells/ cellular fragments and the quality of bovine blastocysts using quantitative RT-PCR and immunofluorescence. Surprisingly, more than 42% of expanded blastocysts had cellular fragments in the perivitelline space; however, more than 37% of extruded cells were TUNEL negative. We observed no significant difference in embryo quality between expanded blastocysts with and without cellular fragments in the perivitelline space. Overall, our data suggest that embryos extrude abnormal cells to maintain their developmental potential. The presence of fragmented cells is not an indicator of embryo quality.

46 Use of time-lapse imaging technology to assess relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos with their developmental competence invitro

The assessment of morphology and digital image opacity may provide valuable information on embryo viability because such traits are linked to embryonic gene expression, metabolism and ultrastructure. Time-lapse imaging has been used in research to monitor the dynamic nature of the developing pre-implantation embryo, which includes capturing alterations in various morphological parameters over time. The present study examined the effectiveness of time-lapse technology in assessing several morphometric and phototextural parameters for predicting the developmental potential of ovine embryos. The development of 37 long wool sheep embryos from IVF to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Image-Pro Plus software was then used to measure zona pellucida thickness, embryo diameter, cellular grey-scale pixel intensity and heterogeneity, and total area of the perivitelline space. A one-way analysis of variance (ANOVA) was done using SigmaPlot® 11.0 for all attributes at various time points during embryo development [i.e. presumptive zygote stage, t(0); first cleavage, t(2) or t(3); second cleavage, t(4) or t(6); and third cleavage, t(7) or t(8)]. Our results indicate that most parameters analysed did not differ among embryos varying in their developmental fate, with the exception of the perivitelline space area, which was greater (P<0.05) for non-dividing embryos than for future blastocysts at the presumptive zygote stage (4040±4137 vs. 857±642µm2, respectively; mean±s.d.). Consequently, the measurement of perivitelline space at t(0) could be used to predict developmental potential of invitro-produced ovine embryos, but further investigation is required.