Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

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130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab130 ◽

2008 ◽

Vol 20 (1) ◽

pp. 145

Author(s):

H. J. Kim ◽

S. R. Cho ◽

C. Y. Choe ◽

S. H. Choi ◽

D. S. Son ◽

...

Keyword(s):

In Vitro Maturation ◽

Polar Body ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Developmental Potential ◽

Non Invasive ◽

First Polar Body ◽

Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

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158 Predicting embryo development success with physical parameters of oocytes

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab158 ◽

2019 ◽

Vol 31 (1) ◽

pp. 204

Author(s):

C. L. Timlin ◽

A. Lynn ◽

R. R. White ◽

K. Lee ◽

V. R. G. Mercadante

Keyword(s):

Polar Body ◽

Digital Camera ◽

Blastocyst Stage ◽

Physical Parameters ◽

Oocyte Diameter ◽

Blastocyst Development ◽

Noninvasive Methods ◽

Developmental Potential ◽

Maturation Medium ◽

Blastocyst Rate

There is a continual search for reliable, noninvasive methods of selecting viable oocytes and embryos. Previous studies have indicated that the physical size of oocytes may reflect their developmental potential. The objective of this study was to observe the correlation between an oocyte’s diameter [including zona pellucida (ZP), cell area, and ZP thickness] and its ability to develop. Bovine cumulus-oocyte complexes were collected from abattoir-derived ovaries and incubated for 24h in TCM-199-based maturation medium. The cumulus-oocyte complexes were denuded by vortexing with hyaluronidase, and mature oocytes were selected based on presence of a visible polar body. Selected oocytes were artificially activated by incubating in 5 μM ionomycin for 5min followed by incubation in 2mM 6-DMAP for 3h (n=723). After activation, oocytes were placed in individual 5-μL culture droplets under oil and photographed using an inverted scope with digital camera. Oocytes were then group cultured in 50-μL droplets in a polyester micromesh for identification of individual oocytes. Development to the blastocyst stage was noted on Days 7 and 8 of culture. ImageJ was used to measure diameter, area, and ZP thickness from images. A logistic regression using the lme4 package in R was run with Day 7 or 8 blastocyst development as the response; diameter, area, and ZP thickness as predictors; and replicate and culture droplet as random effects. The residual estimates of area and ZP thickness from diameter were used to account for correlation between predictors. Only significant interactions were kept in the model. The ZP thickness ranged from 6.1 to 17.7μm with a mean of 12μm. There was a significant correlation between diameter and ZP thickness (P<0.01, R2=0.12) with a correlation coefficient of 0.35. Oocyte diameter had a significant effect on subsequent blastocyst development on Day 7 (P<0.01) and Day 8 (P<0.01), with larger oocytes more likely to develop on both days. Oocytes were also grouped into quantiles by diameter. Larger groups were more likely to develop to the blastocyst stage on Day 7 (P<0.001) and Day 8 (P<0.001). Blastocyst rate on Day 8 for oocytes with diameters <149.5μm was 24.2%, whereas blastocyst rate on Day 8 for those with diameters=159μm was 41.2%. In addition, ZP thickness also had an effect: oocytes with thinner ZP were more likely to develop to the blastocyst stage on Day 7 (P<0.001) and Day 8 (P<0.001). Blastocyst rate for oocytes with a ZP thickness <11μm was 37%, whereas the rate for those with a ZP thickness of 12.9μm was 27.6%. Area did not have an effect on blastocyst formation on Day 7 or 8 (P=0.21). Here we have demonstrated that the oocyte diameter, including the ZP and ZP thickness, affects its probability of development. Larger oocytes and those with thinner ZP are more likely to develop to the blastocyst stage. Differences in the size of the perivitelline space could explain why diameter had a significant effect on development and area did not. Further studies will focus on determining the relationship between these physical parameters of oocytes and embryo quality.

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P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Á Martíne. Moro ◽

I Lamas-Toranzo ◽

L González-Brusi ◽

A Pérez-Gómez ◽

P Bermejo-Álvarez

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Early Embryo ◽

Developmental Competence ◽

Success Rates ◽

Developmental Potential ◽

Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab324 ◽

2004 ◽

Vol 16 (2) ◽

pp. 282 ◽

Cited By ~ 1

Author(s):

Z. Roth ◽

P.J. Hansen

Keyword(s):

Thermal Stress ◽

Heat Shock ◽

Blastocyst Stage ◽

Caspase Activity ◽

Blastocyst Development ◽

Cleavage Rate ◽

Developmental Potential ◽

Oocyte Competence ◽

Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P<0.01) and the number of oocytes developing to the blastocyst stage (P<0.04). There was a temperature x S1P interaction for cleavage rate (P<0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

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Strain-specific differences in mouse oocytes and their contributions to epigenetic inheritance

Development ◽

10.1242/dev.120.12.3419 ◽

1994 ◽

Vol 120 (12) ◽

pp. 3419-3426 ◽

Cited By ~ 1

Author(s):

K.E. Latham

Keyword(s):

Strain Difference ◽

Epigenetic Inheritance ◽

Blastocyst Stage ◽

Female Progeny ◽

Protective Function ◽

F1 Hybrid ◽

Developmental Potential ◽

Protein Gel ◽

The Difference ◽

Strain Dependent

Previous experiments revealed a strain-dependent effect of egg cytoplasm on the developmental potential of androgenetic (two paternal genomes) mouse embryos. Eggs obtained from C57BL/6 mice supported androgenone development to the blastocyst stage at a much higher frequency than eggs from DBA/2 mice. Transient exposure of paternal pronuclei to DBA/2 egg cytoplasm also compromised development, indicating that the DBA/2 egg cytoplasm negatively affected the ability of paternal pronuclei to support blastocyst formation. An essential first step toward understanding the molecular mechanism by which egg modifier factors influence gene expression is to determine the number of loci that are responsible for the strain difference. To do this, (B6D2)F1 hybrid females were backcrossed to DBA/2 males and the eggs from individual female progeny assayed for their ability to support androgenetic development. Approximately one fourth of the backcross females produced eggs that failed to support androgenone development, indicating that two independently segregating genetic loci are most likely responsible for the difference between DBA/2 and C57BL/6 egg phenotypes. Comparison of DBA/2 and C57BL/6 oocytes by two-dimensional protein gel electrophoresis revealed at least 17 proteins that exhibited significant, reproducible, quantitative differences in rates of synthesis. All of these proteins were synthesized in (B6D2)F1 oocytes. These data, combined with the previous observation that the C57BL/6 egg phenotype is dominant, are consistent with a model in which a C57BL/6 allele at either locus provides a protective function, either by antagonizing the actions of the DBA/2 alleles or by providing, through partial or complete redundancy, a function not provided by the DBA/2 alleles.

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P–080 Higher sperm DNA fragmentation reduces the proportion of good quality embryos at day 5 on IVF and ICSI cycles from unselected males

Human Reproduction ◽

10.1093/humrep/deab130.079 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

I Hervá. Herrero ◽

A Pacheco ◽

R Rivera-Egea ◽

M Gi. Julia ◽

A Navarro-Gomezlechon ◽

...

Keyword(s):

Dna Fragmentation ◽

Embryo Quality ◽

Blastocyst Stage ◽

Dna Integrity ◽

Embryo Morphology ◽

Sperm Dna Fragmentation ◽

Cleavage Stage ◽

Sperm Dna ◽

Embryo Fragmentation ◽

The Impact

Abstract Study question Does sperm DNA fragmentation (SDF) reduce the ratio of good-quality embryos in day 3 (D3) and day 5 (D5) of embryonic development? Summary answer High sperm DNA fragmentation (SDF >15%) is associated with poor embryo quality at blastocyst-stage per cycle in unselected patients undergoing IVF and ICSI. What is known already It has been shown that the proportion of spermatozoa with DNA fragmentation is higher in infertile men than in semen from fertile men. However, the controversy regarding the impact that sperm genome damage can have on IVF or ICSI treatments is evident in the published literature. The effects of SDF would become evident after activation of the embryonic genome at 8-cell stage, compromising not only the quality of the embryos obtained, but also the reproductive outcomes, as reduced implantation rates, higher miscarriages rates and thus, a decreased chance of pregnancy. Study design, size, duration This multicentric observational retrospective study included 1339 couples who underwent 2759 IVF-ICSI cycles using autologous oocytes from January 2000 to March 2019. All men have an SDF test in their ejaculated spermatozoa by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The subjects were divided into two groups according to their sperm DNA integrity: low (≤15%) (n = 2287 cycles) or high (>15%) (n = 472) SDF. Participants/materials, setting, methods Embryo quality was assessed complying morphological standards at cleavage-stage on D3 and at blastocyst-stage on D5 (inner cell mass (ICM) and trophectoderm (TE) grade (A, B, C or D)) in according to ASEBIR’s embryo selection criteria, being embryos of good quality those categorized as A+B. The outcomes were calculated in relation to the total number of zygotes obtained. The results were compared by Student t test; p value <0.05 was considered significant. Main results and the role of chance The SDF average of the low group was 5.8% (95% CI 5.6–5.9) whereas in high group was 23.7% (95% CI 23.0–24.4). The female age was equal, 37.1 years (95%CI 37.0–37.2) and 37.1 years (95% CI 36.8–37.4) respectively. A total of 9796 embryos were evaluated. The optimal cleavage-stage embryo rate per cycle was 25.0% (95% CI 21.7–28.3) (8.0 average cells number, 1.5 embryo fragmentation average, symmetry 1, mononucleated cells) versus 26.7% (95%CI 19.1–34.2) (7.9 average cells number, 1.8 embryo fragmentation average, symmetry 1, mononucleated cells) when comparing between groups (p < 0.001). Blastocyst-stage arrival rate (number of embryos at D5) per cycle was 55.8% (95% CI 54.3–57.2) in ≤ 15% SDF group (embryo quality score was ICM A:12.1%, B:69.5%, C:8.8%, D:4.5%; TE A: 7.5%, B:42.2%, C:42.2%, D:8.1%) and 55.9% (95% CI 52.8–59.1) in the >15% SDF group (ICM A:12.0%, B:68.7%, C:10.6%, D: 5.2%; TE A:9.1%, B:44.8%, C:37.8%, D:8.3%) (p < 0.001). The good quality blastocyst rate per cycle was significantly higher in the group with SDF ≤15%, 27.7% (95%CI 26.5–29.0) versus SDF >15% (27.4% (95%CI 24.6–30.2)). Of the total number of blastocysts, the proportion of A+B blastocyst was 60.5% (95% CI 58.3–62.7) and 64.2% (95% CI 59.2–69.2) (p < 0.001), respectively. Limitations, reasons for caution The retrospective and multicenter nature of this study leads to uncontrolled biases derived from the clinical practice. Although the results were not adjusted for female’s age, it was not statistically different between groups. Embryo morphology evaluation was performed by senior embryologists, it still remains a subjective evaluation, though. Wider implications of the findings: In this study, a higher amount of data was compiled so that a large number of embryos were analyzed. The DNA integrity of the sperm may be an important consideration when poor quality embryos were obtained in the previous cycle when deciding on the next clinical strategy to apply. Trial registration number NA

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PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

Journal of Animal Science ◽

10.1093/jas/skab235.602 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 327-328

Author(s):

Galina Singina

Keyword(s):

Growth Factor ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Control Group ◽

Total Cell Number ◽

Developmental Potential ◽

Maturation Medium ◽

Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P<0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P<0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

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P–030 A sperm chromatin damage >15% negatively impacts on the quality of embryos obtained from ovum donation ICSI cycles of unselected couples

Human Reproduction ◽

10.1093/humrep/deab130.029 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Pachec. Castro ◽

I Hervas ◽

R Rivera-Egea ◽

M Gi. Julia ◽

A Navarro-Gomezlechón ◽

...

Keyword(s):

Clinical Practice ◽

Dna Fragmentation ◽

Embryo Quality ◽

Blastocyst Stage ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Cleavage Stage ◽

Ovum Donation ◽

Embryo Fragmentation

Abstract Study question Is embryo quality downgraded in couples with elevated sperm DNA fragmentation (SDF) in the ejaculated semen of male partner using donated eggs? Summary answer The rate of good quality embryos at day 3 and blastocyst-stage is statistically inferior in males with SDF>15% undergoing ICSI cycles with donated oocytes. What is known already The effect of a damaged paternal chromatin will be shown from the 8-cell stage of embryo development, a time which the genome of the embryo is transcriptionally active. Fertilization with a spermatozoon with fragmented DNA may impair the quality of the embryos obtained per cycle, and therefore reduce the chances of pregnancy. The use of donated oocytes is an ideal model to evaluate the real effect of SDF on embryo quality by standardizing the female factor. In addition, we have a large cohort of ovum donation cases in our history, which allows a more proper evaluation of the effect. Study design, size, duration Retrospective multicentric study including the clinical data of 864 couples of ovum donation program who underwent 1903 ICSI cycles between January 2000 and March 2019. The DNA fragmentation of their ejaculated spermatozoa was measured by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Two study groups were created according to the SDF level: ≤15% (low) (n = 1626) or > 15% (high) (n = 277). Participants/materials, setting, methods Embryos were evaluated throughout embryonic development according to classical morphological parameters at day 3 (D3), on cleavage-stage, and at day 5 (D5), on blastocyst-stage (trophectoderm (TE) and inner cell mass (ICM)), following ASEBIR guidelines, categorized from A to D. Embryos scored as A and B were considered to be good quality. The proportion of embryos was calculated according to the total number of correctly fertilized oocytes or zygotes. A p < 0.05 was considered significant. Main results and the role of chance A total of 6130 embryos were evaluated. The SDF average of ≤ 15% group was 5.9% (95%CI 5.7–6.1) and 24.3% (95%CI 23.2–25.3) in the >15% group. The cycle-related characteristics and the seminal parameters were comparable. The proportion of optimal cleavage-stage embryo (number of A+B embryos at D3) per cycle was 21.7% (95%CI 19.0–24.5) (8.1 average cells number, 0.8 embryo fragmentation average, symmetry 1, mononucleated cells) in ≤ 15% SDF group versus 21.1% (95%CI 13.9–28.3) (8.2 cells number average, 1.3 embryo fragmentation average, symmetry 1, mononucleated cells) (p < 0.001). The blastocyst-stage arrival rate (number of embryos at D5) per cycle was higher in the >15% SDF group (p < 0.001), 53.4% (95%CI 48.8–58.1) (TE quality A:20.5%, B:42.5%, C:22.7%, D:14.8%, and the ICM quality A:26.1%, B:52.1%, C:13.2%, D:6.2%) versus 49.9% (95%CI 48.1–51.6) (TE quality A:21.1%, B:42.8%, C:21.85, D:14.1% and ICM A:26.6%, B:55.5%, C:11.1%, D:4.7%) in the low SDF group. The rate of good quality blastocyst (number of quality A+B embryos in D5) per cycle was significantly higher in the couples with low SDF (24.8% (95%CI 23.6–25.9)) than in those with elevated SDF (23.5% (95%CI20.9–26.2)) (p < 0.001). Accordingly, the A+B blastocyst rate divided by the total number of blastocysts was 59.1% (95%CI 56.7–61.4) versus 55.9% (95%CI 49.9–62.0) (p < 0.001), respectively. Limitations, reasons for caution The main limitation is that retrospective design of the study may not eliminate the potential unaccounted-for bias derived from the clinical practice of multiple centers even though both groups were statistically comparable. Also, the assessment of embryo quality is still remaining highly subjective to embryologists. Wider implications of the findings: Although the effect size is small, it may be useful in clinical practice when an ICSI cycle yields no good-quality embryos, as one of the underlying causes of that fact. Knowing the SDF level can be a helpful tool in making subsequent clinical decisions aimed at improving outcomes for couples. Trial registration number Not applicable

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MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

International Journal of Molecular Sciences ◽

10.3390/ijms21238888 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8888

Author(s):

Bárbara Melo-Baez ◽

Yat S. Wong ◽

Constanza J. Aguilera ◽

Joel Cabezas ◽

Ana C. F. Mançanares ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fatty Acid Biosynthesis ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Target Cells ◽

Bovine Embryos ◽

Developmental Potential ◽

Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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