The effect of human follicular fluid on bovine oocyte developmental competence and embryo quality

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

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342 DISTINCT EFFECTS OF FOLLICULAR FLUID ON CUMULUS CELL APOPTOSIS AND PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab342 ◽

2007 ◽

Vol 19 (1) ◽

pp. 286

Author(s):

C. G. Grupen ◽

T. S. Hussein ◽

S. J. Schulz ◽

D. T. Armstrong

Keyword(s):

Oxidative Stress ◽

Follicular Fluid ◽

Cell Apoptosis ◽

Polar Body ◽

Cumulus Cell ◽

Developmental Competence ◽

Potent Inducer ◽

Oocyte Developmental Competence ◽

Cytoplasmic Maturation

Supplementing medium with follicular fluid (FF) during in vitro maturation (IVM) enhances the developmental competence of porcine oocytes, indicating that factors present in FF are beneficial to cytoplasmic maturation. Previous findings suggest that porcine FF contains high levels of superoxide dismutase activity and exerts a beneficial effect on cytoplasmic maturation by protecting oocytes from oxidative stress (Tatemoto et al. 2004 Biol. Reprod. 71, 1150–1157). Since oxidative stress is a potent inducer of apoptosis, the aim of the present study was to examine the temporal effects of FF during IVM on cumulus cell apoptosis and oocyte developmental competence. Ovaries of prepubertal pigs were collected from a local abattoir and antral follicles, 3 to 7 mm in diameter, were aspirated. Cumulus–oocyte complexes (COCs) with at least 3 uniform layers of compact cumulus cells (CCs) were recovered, washed, and transferred to maturation medium (MM) with or without 25% FF. At 22 h of IVM, COCs from each group were washed and transferred to fresh MM with or without 25% FF, forming 4 groups: -FF/-FF, -FF/+FF, +FF/-FF, and +FF/+FF. Cohorts of COCs were TUNEL stained at 22 and 44 h of IVM using the In Situ Cell Death Detection kit (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer's instructions, and apoptotic CCs were visualized using confocal microscopy. Oocytes denuded at 44 h, that had a polar body, were treated with ionomycin and 6-dimethylaminopurine to induce parthenogenetic development, and were cultured for 7 days in NCSU-23 medium at 38.5°C in 5&percnt; O2, 5&percnt; CO2, and 90&percnt; N2. Data were subjected to ANOVA and Tukey's post-hoc test. At 22 h of IVM, the presence of FF reduced the proportion of apoptotic CCs in COCs (2.1&percnt; vs. 4.6&percnt;). COCs matured with FF from 22 to 44 h of IVM had much lower proportions of apoptotic CCs (+FF/+FF: 0.9&percnt;; −FF/+FF: 2.6&percnt;) compared with those matured without FF (+FF/−FF: 10.3&percnt;; −FF/−FF: 17.8&percnt;). The rate of maturation to the metaphase-II stage was greater when oocytes were matured with FF from 0 to 22 h of IVM (−FF/−FF: 68.6&percnt;; −FF/+FF: 72.8&percnt;; +FF/−FF: 89.2&percnt;; +FF/+FF: 86.2&percnt;). Maturation without FF for the entire IVM interval reduced the proportion of activated oocytes that formed blastocysts compared with the other groups (−FF/−FF: 25.1&percnt;; −FF/+FF: 44.6&percnt;; +FF/−FF: 46.6&percnt;; +FF/+FF: 47.3&percnt;). Despite a 4-fold difference in the proportion of apoptotic CCs between COCs of the +FF/−FF and −FF/+FF groups, exposure to FF for the first or second half of IVM was as beneficial to oocyte developmental competence as exposure to FF for the entire IVM interval. This suggests that the protective effect of FF in reducing oxidative stress on oocytes during IVM is distinct from the effect on oocyte developmental competence.

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Trimethylamine-N-oxide is present in human follicular fluid and is a negative predictor of embryo quality

Human Reproduction ◽

10.1093/humrep/dez224 ◽

2020 ◽

Vol 35 (1) ◽

pp. 81-88 ◽

Cited By ~ 1

Author(s):

R A Nagy ◽

I Homminga ◽

C Jia ◽

F Liu ◽

J L C Anderson ◽

...

Keyword(s):

Follicular Fluid ◽

Embryo Quality ◽

Conflicts Of Interest ◽

Medical Center ◽

Academic Medical Center ◽

Gut Bacteria ◽

Human Follicular Fluid ◽

Cross Sectional ◽

Oocyte Fertilization ◽

Negative Predictor

Abstract STUDY QUESTION Are levels of trimethylamine-N-oxide (TMAO) in human follicular fluid (FF) related to IVF outcomes? SUMMARY ANSWER Higher levels of TMAO are a negative predictor of oocyte fertilization and embryo quality. WHAT IS KNOWN ALREADY TMAO is a metabolic product of dietary choline and l-carnitine produced via subsequent enzymatic modifications by the intestinal microbiota and hepatocytes. TMAO promotes inflammatory and oxidative stress pathways and has been characterized as a causative biomarker for the development of cardiometabolic disease. STUDY DESIGN, SIZE, DURATION For the present cross-sectional study, samples (FF and plasma) from 431 modified natural cycle (MNC)-IVF cycles of 132 patients were collected prospectively between October 2014 and March 2018 in a single academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS TMAO and its precursors (choline, l-carnitine and gamma-butyrobetaine) were measured by ultra-high-performance liquid chromatography/mass spectrometry in (i) matched FF and plasma from 63 MNC-IVF cycles, in order to compare metabolite levels in the two matrices and (ii) FF from 232 MNC-IVF cycles in which only one oocyte was retrieved at follicular puncture. The association between metabolite levels and oocyte fertilization, embryo fragmentation percentage, embryo quality and the occurrence of pregnancy was analyzed using multilevel generalized estimating equations with adjustment for patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE The level of choline was higher in FF as compared to matched plasma (P < 0.001). Conversely, the levels of TMAO and gamma-butyrobetaine were lower in FF as compared to plasma (P = 0.001 and P = 0.075, respectively). For all metabolites, there was a positive correlation between FF and plasma levels. Finally, levels of TMAO and its gut-derived precursor gamma-butyrobetaine were lower in FF from oocytes that underwent normal fertilization (TMAO: odds ratio [OR] 0.66 [0.49–0.90], P = 0.008 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.77 [0.60–1.00], P = 0.047 per 0.1-μmol/L increase) and developed into top-quality embryos (TMAO: OR 0.56 [0.42–0.76], P < 0.001 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.79 [0.62–1.00], P = 0.050 per 0.1-μmol/L increase) than in FF from oocytes of suboptimal development. LIMITATIONS, REASONS FOR CAUTION The individual contributions of diet, gut bacteria and liver to the metabolite pools have not been quantified in this analysis. WIDER IMPLICATIONS OF THE FINDINGS More research on the contribution of diet and the effect of gut bacteria on FF TMAO is warranted. Since TMAO integrates diet, microbiota and genetic setup of the person, our results indicate potential important clinical implications for its use as biomarker for lifestyle interventions to improve fertility. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received for this project. The Department of Obstetrics and Gynecology of the University Medical Center Groningen received an unrestricted educational grant of Ferring Pharmaceutical BV, the Netherlands. The authors have no other conflicts of interest. TRIAL REGISTRATION NUMBER Netherlands Trial Register number NTR4409.

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Oocytes recovered from cows treated with retinol become unviable as blastocysts produced in vitro

Reproduction ◽

10.1530/rep.1.00548 ◽

2005 ◽

Vol 129 (4) ◽

pp. 411-421 ◽

Cited By ~ 21

Author(s):

C Hidalgo ◽

C Díez ◽

P Duque ◽

J M Prendes ◽

A Rodríguez ◽

...

Keyword(s):

Retinoic Acid ◽

Follicular Fluid ◽

Teratogenic Effect ◽

Developmental Competence ◽

Bovine Oocyte ◽

Blastocyst Development ◽

Time Limits ◽

Oocyte Donors ◽

First Session

Retinoids have been shown to enhance developmental competence of the oocyte in cattle, sheep and pigs. In this study we investigated whether exogenous retinol stimulates the bovine oocyte during its intrafollicular growth and the time limits of exposure to exogenous retinol. In addition, we also determined the efficiency of ovum pick-up techniques in combination with retinol treatment and the viability of embryos after transfer to recipients. In Experiment 1, heifers were injected with retinol or vehicle, and concentrations of retinol in the blood were analysed on Day 0 (prior to injection), Day 1 and, together with follicular fluid, Day 4. Blood retinol increased by Day 1 and cleared on Day 4, but retinol remained higher within the follicle. In Experiment 2, oocyte donors were injected weekly with retinol or vehicle four times during a twice-per-week cycle of eight recovery sessions (starting 4 days before the first session), followed by a second eight-session cycle without treatment. Oocytes recovered were fertilized and culturedin vitro.Retinol treatment yielded higher numbers of low-quality oocytes throughout, although retinol measured during cycles did not change. Total oocytes, and morulae and blastocyst rates, increased during the first five sessions following treatment with retinol. As previously shown with oocytes from slaughterhouse ovaries, retinoic acid stimulated blastocyst development. Following transfer to recipients, blastocysts from oocytes exposed to retinol were unable to establish pregnancy. Our study confirms the existence of an effect of retinol on the intrafollicular oocyte in the cow and provides evidence regarding the teratogenic effect of retinol.

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