Membrane lipid changes in mouse blastocysts induced by ovarian stimulation, IVF and oocyte vitrification.

RESEARCH QUESTION: Is the membrane lipid profile of mice blastocysts affected by ovarian stimulation, IVF and oocyte vitrification? Could supplementation of vitrification media with L-carnitine and fatty acids prevent membrane phospholipid changes in blastocysts from vitrified oocytes? DESIGN: Experimental study comparing the lipid profile of murine blastocysts produced from natural mating, superovulated cycles or after IVF submitted or not to vitrification. For in-vitro experiments, 562 oocytes from superovulated females were randomly divided into four groups: fresh oocytes fertilized in vitro and vitrified groups: Irvine Scientific (IRV); Tvitri-4 (T4) or T4 supplemented with L-carnitine and fatty acids (T4-LC/FA). Fresh or vitrified-warmed oocytes were inseminated and cultured for 96 h or 120 h. The lipid profile of nine of the best quality blastocysts from each experimental group was assessed by multiple reaction monitoring profiling method. Significantly different lipids or transitions between groups were found using univariate statistics (P < 0.05; fold change = 1.5) and multivariate statistical methods. RESULTS: A total of 125 lipids in blastocysts were profiled. Statistical analysis revealed several classes of phospholipids affected in the blastocysts by ovarian stimulation, IVF, oocyte vitrification, or all. L-carnitine and fatty acid supplements prevented, to a certain extent, changes in phospholipid and sphingolipid contents in the blastocysts. CONCLUSION: Ovarian stimulation alone, or in association with IVF, promoted changes in phospholipid profile and abundance of blastocysts. A short exposure time to the lipid-based solutions during oocyte vitrification was sufficient to induce changes in the lipid profile that were sustained until the blastocyst stage.

Equilibration solution composition and extended exposure to equilibration phase affect embryo development and lipid profile of mouse oocytes.

RESEARCH QUESTION: Can exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile? DESIGN: Experimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified-warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions. RESULTS: Longer equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (P = 0.00223; impact =1) and alpha-Linolenic acid metabolism (P = 0.00084; impact = 0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (P = 0.0167; impact = 0.25) CONCLUSION: A longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.

The Impact of Oocyte Vitrification on Offspring: a Systematic Review.

Oocyte vitrification is a widespread and well-established assisted reproduction technique that has enabled some patient groups to obtain clinical results equivalent to those using fresh oocytes. However, as the number of babies born from vitrified oocytes has increased, so has the discussion regarding the method's safety for the offspring. Cryogenic oocyte damage caused by chemical, mechanical, and thermal stress has raised concern. In this systematic review, we asked the question of whether oocyte vitrification impacts offspring health. From 2007 to 2021, 13 studies were included in the analysis. All studies were observational and presented neonatal outcomes. A total of 4,159 babies were analyzed. Data from these studies were used to assess the following outcomes: multiple pregnancies, cesarean section, gestational age at delivery, the number of live births, birth weight, Apgar scores, congenital anomalies, and baby health. The most extended follow-ups evaluated children until 1, 2, and 6 years of age. According to the evidence appraised in this systematic review, vitrification seems to be a safe method for oocyte cryopreservation and child health, at least in the short term. Nevertheless, there is an urgent need for additional long-term data results from big databases and also for randomized controlled trials to improve the levels of evidence.

Risk of Contamination of Gametes and Embryos during Cryopreservation and Measures to Prevent Cross-Contamination.

The introduction and widespread application of vitrification are one of the most important achievements in human assisted reproduction techniques (ART) of the past decade despite controversy and unclarified issues, mostly related to concerns about disease transmission. Guidance documents published by US Food and Drug Administration, which focused on the safety of tissue/organ donations during Zika virus spread in 2016, as well as some reports of virus, bacteria, and fungi survival to cryogenic temperatures, highlighted the need for a review of the way how potentially infectious material is handled and stored in ART-related procedures. It was experimentally demonstrated that cross-contamination between liquid nitrogen (LN2) and embryos may occur when infectious agents are present in LN2 and oocytes/embryos are not protected by a hermetically sealed device. Thus, this review summarizes pertinent data and opinions regarding the potential hazard of infectious transmission through cryopreserved and banked reproductive cells and tissues in LN2. Special attention is given to the survival of pathogens in LN2, the risk of cross-contamination, vitrification methods, sterility of LN2, and the risks associated with the use of straws, cryovials, and storage dewars.

Dataset on lipid profile of bovine oocytes exposed to Lα-phosphatidylcholine during in vitro maturation investigated by MALDI mass spectrometry and gas chromatography-flame ionization detection.

Data presented in this article are related with the research article entitled "Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro" [1]. This article describes the differences in the relative abundance of the lipid ions detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in control and Lα-phosphatidylcholine-treated oocytes. In addition, the fatty acids (FA) content in pure Lα-phosphatidylcholine supplement and oocytes was analyzed by gas chromatography-flame ionization detection (GC-FID). The dataset provides information and inputs for further studies aiming to optimize in vitro maturation conditions and cryotolerance of mammalian oocytes.

MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes.

The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro.

The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100µM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100µM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the development of novel strategies to modulate oocyte membrane dynamics and structure.

Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model.

This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus-oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions.

BACKGROUND: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. METHODS: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. RESULTS: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose-response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone production was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. CONCLUSIONS: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.