203 EFFECT OF FRUCTOSE ON THE DEVELOPMENT OF BOVINE SEXED EMBRYOS IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 199
Author(s):  
S. A. Chaubal ◽  
J. Xu ◽  
X. Yang ◽  
F. Du
Keyword(s):  
Embryo Transfer ◽  
Culture Medium ◽  
Subsequent Development ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Humidified Air ◽  
Vitro Development

It is important to optimize the condition of culture to promote the development of embryos fertilized by sex-sorted sperm. Fructose is a monosaccharide hexose that can be used as a substrate for glycolysis. Recently, there has been interest in exploring the embryotrophic properties of fructose during bovine embryo culture. The present study was undertaken to ascertain the effect of supplementation of fructose in culture on the development of sexed embryos in vitro. A total of 2310 slaughterhouse dairy oocytes, spread over 3 experimental replicates, were subjected to routine in vitro maturation (IVM) for 22 h. The maturation medium was M-199 with Earl’s salts, L-glutamine, 26.19 mm sodium bicarbonate, and 25 mm HEPES plus 10% fetal bovine serum. Fertilization of IVM oocytes was performed in Brackett and Oliphant’s medium by using frozen and sorted X-sperm from a single bull. Groups of 50 matured oocytes were added to a final volume of 100 μL of fertilization medium, with a final concentration of 0.3 × 106 spermatozoa mL–1. The oocytes were incubated with X-sperm for 6 h in 5% CO2 humidified air at 39°C. Presumptive zygotes were randomly allocated to culture either in a control medium (6 mg mL–1 of BSA CR1aa medium) or with 4.5 mm fructose added to CR1aa culture medium for 7 days in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C. Embryo cleavage, and development to the morulae and blastocyst stages were evaluated on Days 2, 5, and 7 of culture, respectively. Embryos were graded according to International Embryo Transfer Society standards. The total number of blastocysts recorded was the sum of grade C1 and C2 embryos. Data were analyzed by using the General Linear Model (SPSS 11.0, SPSS Inc., Chicago, IL, USA). As shown in Table 1, addition of fructose to the culture medium affected neither the rate of cleavage nor subsequent development to the morula stage or total blastocyst development. However, the proportion of C1-grade embryos of the total blastocysts was significantly (P < 0.05) improved. This study suggests that addition of fructose to the culture medium can promote embryo quality and yields that are suitable for embryo transfer. Table 1.Effect of addition of fructose on in vitro development of bovine sexed embryos

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

261 EVALUATION OF TWO GAS MIXTURES, ADDITION OF N-ACETYLCYSTEINE, AND LIPOPEROXIDATION LEVELS IN PORCINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 287
Author(s):  
A. L. Alvarez ◽  
Y. C. Ducolomb ◽  
H. Vera ◽  
A. Villa-Godoy ◽  
J. F. De la Torre ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Thiobarbituric Acid ◽  
Gas Mixtures ◽  
Embryo Viability ◽  
Morula Stage ◽  
Antioxidant Agent ◽  
Humidified Air ◽  
532 Nm

In vitro embryo development can be affected by many different factors, such as incubation atmosphere, which can cause oxidative stress. In this study the effect of 2 gas mixtures and the addition of the antioxidant agent N-acetylcysteine (NAC) on porcine embryos produced in vitro and lipoperoxidation (LPO) were evaluated. Ovaries collected from slaughtered gilts were aspirated to obtain oocytes, to be matured and fertilized in vitro. In experiment 1 (E1), putative zygotes were cultured in NCSU-23 medium at 38.5°C in humidified air and randomly allocated to each of the following atmospheres: conventional atmosphere (CA: 5% CO2 in air) and modified atmosphere (MA: 5% CO2, 5% O2, 90% N2). In experiment 2 (E2), zygotes were incubated in CA or MA and cultured in NCSU-23 with 2.5 mM NAC. In experiment 3 (E3), embryos produced in E1 and E2 were used to evaluate LPO with the TBARS method: embryos were sonicated, then 300 μL of distilled water and 680 μL of thiobarbituric acid were added and heated at 95°C for 20 min in a water bath. The resulting reaction was read by spectrophotometry at 532 nm. Final concentration was expressed in micromoles of TBARS/embryo (μMTE). Five days after IVF, embryos were evaluated to determine their development to the morula stage. Data from each experiment were transformed using arcsine square root function before analysis, and means were compared using ANOVA. For E1, 954 oocytes were used, 475 for CA and 479 for MA, in 5 replicates. The percentage of cleaved embryos was significantly higher in MA (300/479, 63%) compared with CA (245/475, 52%) (P < 0.05). The percentage of morulae obtained was higher in MA (127/479, 27%) than in CA (80/475, 17%) (P < 0.05). In E2, 936 oocytes were used, 470 for CA NAC and 466 for MA NAC, in 5 replicates. There were no differences in the percentages of cleaved embryos in MA NAC (250/466, 54%) compared with CA NAC (243/470, 52%), and morulae in MA NAC (86/466, 18%) compared with CA NAC (81/470, 17%) (P > 0.05). In E3, during the first 24 h of culture the embryos in CA NAC had lower LPO concentrations (0.029μMTE) (P < 0.05) than in CA (0.059μMTE), MA (0.057μMTE), and MA NAC (0.054μMTE). In morulae, a higher concentration of LPO was observed in the CA group (0.103μMTE) compared with MA (0.083μMTE), CA NAC (0.085μMTE), and MA NAC (0.081μMTE). A correlation analysis between morulae development and LPO in CA and MA, with or without NAC, did not show a significant relationship (r = 0.20; P > 0.05). It is concluded that MA improves the development of in vitro-derived porcine embryos; that NAC treatment did not cause differences in any stage of embryo development, probably meaning that the embryo has its own defensive mechanisms against LPO, and therefore that NAC is not a good antioxidant for porcine embryos, or that it does not work well at the dosage and/or in the culture medium used; that the embryos exposed to CA and MA showed higher LPO that the groups with NAC, indicating that the addition of NAC tends to decrease LPO; and that LPO could be a good indicator of embryo viability. We thank CONACYT Mexico for the graduate student’s scholarship.

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 111-115 ◽  
Author(s):  
Gamal Mohamed Kamel Mehaisen ◽  
Ayman Moustafa Saeed
Keyword(s):  
Culture Medium ◽  
Control Culture ◽  
M 10 ◽  
Morula Stage ◽  
High Level ◽  
Vitro Development ◽  
And Control ◽  
Different Levels ◽  
Rabbit Embryos

SummaryThis study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2–4 cells, 8–16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10−3 M, 10−6 M or 10−9 M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10−3 M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10−3 M versus 10−9 M, 10−6 M and control, respectively, P< 0.05). At the morula stage, melatonin at 10−3 M significantly increased the in vitro development of embryos (92% for 10−3 M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16–30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10−6 M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10−3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

2012 ◽  
Vol 24 (3) ◽  
pp. 443 ◽  
Author(s):  
Tomomi Mito ◽  
Koji Yoshioka ◽  
Shoko Yamashita ◽  
Chie Suzuki ◽  
Michiko Noguchi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Survival Rates ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Atp Content ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Defined Culture ◽  
Vitro Development

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0 = IVF) porcine blastocysts were determined. The addition of 2.5–10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

scholarly journals Influence of the uterine environment on the development of in vitro-produced equine embryos

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 173-181 ◽  
Author(s):  
Katrien Smits ◽  
Jan Govaere ◽  
Luc J Peelman ◽  
Karen Goossens ◽  
Dirk C de Graaf ◽  
...  
Keyword(s):  
Culture Medium ◽  
Subsequent Development ◽  
Cleavage Stage ◽  
Morula Stage ◽  
Contrast Exposure ◽  
Uterine Environment ◽  
Early Interaction ◽  
Suitable Environment

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

1990 ◽  
Vol 68 (11) ◽  
pp. 1457-1460 ◽  
Author(s):  
Subhash C. Juneja ◽  
Melvin G. Dodson
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
In Vitro Development ◽  
Stage Embryo ◽  
Morula Stage ◽  
Vitro Development

17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)estra-4,9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 °C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 μg/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 μg/mL culture medium (p < 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 μg/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 μg/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.Key words: RU486, mouse, preimplantation embryos, embryo culture, postcoital contraceptive.

156 IN VITRO DEVELOPMENT OF BOVINE MORULAE PRODUCED AND/OR CULTURED WITH ACTIVIN

2010 ◽  
Vol 22 (1) ◽  
pp. 236 ◽  
Author(s):  
B. Trigal ◽  
E. Gómez ◽  
C. Diez ◽  
J. N. Caamaño ◽  
I. Molina ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Morula Stage ◽  
Vitro Development ◽  
And Control

We reported that the presence of activin during in vitro culture improves embryo development without changing the cell distribution in the blastocyst (Díez et al. 2009 AETE in press). In the present work, we aimed to analyze the morula stage as a putative milestone to activin exert differential effects. Day -5 morulae were produced with IVMFC oocytes from abattoir ovaries, using SOF with amino acids, myo-inositol, and 3 g L-1 of BSA as a culture medium. Embryo culture contained 10 ng mL-1 or 0 ng mL-1 of activin from Day -3 to Day -5. Early morulae (n = 543 out of 1099 cultured oocytes) were selected and subsequently cultured with or without 10 ng mL-1 of activin up to Day -8. Embryo development was daily monitored and cells differentially counted in Day -8 expanded blastocysts. (Thouas et al. 2001 Reprod. Biomed. 2001 3, 25-29). Data were analyzed by general linear model and presented as least squares means ± SEM. Activin from Days 3 to 5 did not change Day -5 morulae rates (P > 0.8). In morulae produced without activin (Days 5 to 8 and control), a treatment with activin from Days 5 to 8 improved total blastocyst rates v. controls, both in Day -7 and Day -8 (50.9 ± 3.6 v. 32.6 ± 3.6 and 60.8 ± 2.9 v. 42.3 ± 2.9, respectively; P < 0.01). Similarly, Day -7 expansion rates with activin (Days 5 to 8) were higher than controls (14.6 ± 1.8 v. 8.6 ± 1.8; P < 0.03). However, the above effects were not the same as those observed in morulae produced with activin (Days 3 to 5 and Days 3 to 8), where blastocyst development between activin treatment and controls only significantly differed in expansion rates on Day -7 (14.9 ± 1.8 v. 5.8 ± 1.8, respectively; P < 0.03). Morulae treated with activin (Days 5 to 8) yielded Day -7, total and expanded blastocyst rates, higher than morulae produced with activin (Days 3 to 5) (50.9 ± 3.6 v. 37.4 ± 3.6 and 14.6 ± 5.8 v. 5.8 ± 1.8, respectively; P < 0.03). Expansion rates on Day -8 were numerically higher within morulae produced and/or treated with activin (Days 3 to 8, Days 5 to 8, and Days 3 to 5) (values between 26.7 ± 2.6 and 27.4 ± 2.6) than in controls without activin at any time (19.2 ± 2.6) (P > 0.05). Trophectoderm (TE) cell numbers were reduced in embryos produced and/or treated with activin (Days 3 to 8, Days 3 to 5, and Days 5 to 8) (values between 109.4 ± 7.6 and 115.3 ± 7.9) as compared with untreated controls (141.2 ± 10.1) (P < 0.05). In morulae produced without activin, total cell counts were lower with activin being present from Day -5 to Day -8 (154.0 ± 8.8 v. 128.4 ± 7.2; P < 0.05). Inner cell mass (ICM) and ICM/total cell ratio were not affected by the presence of activin (P > 0.05). Activin did not change Day -5 morulae rates, although subsequent blastocyst development was in part affected by the presence of activin before the morula stage. Interestingly, improvements in blastocyst development, including expansion rates, triggered by activin led to reduced TE and unaltered ICM cell counts, suggesting that activin inhibits TE differentiation. Support: Cajastur (B. Trigal). MCINN: M. Muñoz (RYC08-03454); D. Martín (PTA2007-0268-I); INIA (I. Molina); Project HF2007-0126.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Du ◽  
R Li ◽  
Q Zhang ◽  
W Wang
Keyword(s):  
Escherichia Coli ◽  
In Vitro Culture ◽  
Clinical Outcomes ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Culture System ◽  
Microbial Contamination ◽  
Culture Medium ◽  
Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

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