Biological factors affecting subzonal sperm microinjection results

1994 ◽  
Vol 6 (1) ◽  
pp. 63
Author(s):  
L Gianaroli ◽  
MC Magli ◽  
AP Ferraretti ◽  
D Fortini ◽  
E Feliciani ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Embryo Development ◽  
Fertilization Rate ◽  
Early Embryo ◽  
Male Factor Infertility ◽  
Biological Factors ◽  
Male Factor ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Factors Affecting

One hundred and sixteen couples with severe male factor infertility underwent 139 subzonal sperm microinjection cycles. In total, 1343 oocytes were microinjected, resulting in a fertilization rate of 24%, followed by a cleavage rate of 65%. In 26% of the zygotes, fertilization was delayed and embryos derived from these zygotes demonstrated a poor capacity for further growth and implantation. In 102 of 139 cycles (73%) embryo transfer was performed, resulting in 9 pregnancies. This study followed the fate of injected oocytes and early embryo development to investigate biological factors that influence the results of subzonal injection.

Injection of multiple sperm into the perivitelline space as a treatment of male infertility

1994 ◽  
Vol 6 (1) ◽  
pp. 51 ◽  
Author(s):  
C O'Neill ◽  
JP Ryan ◽  
JW Catt ◽  
IL Pike ◽  
UB Krzyminska
Keyword(s):  
Embryo Transfer ◽  
Fertilization Rate ◽  
Development Rate ◽  
Male Factor Infertility ◽  
Multiple Injection ◽  
Perivitelline Space ◽  
Male Factor ◽  
Factors Affecting ◽  
The Relationship ◽  
Normal Fertilization

This paper reports the outcome of 274 treatment cycles using multiple injection of sperm into the perivitelline space as a treatment of male factor infertility. A total of 170 couples underwent this form of treatment; 59.1% of cycles had at least one oocyte normally fertilized with an overall normal fertilization rate of 17.2%. The development rate of normally fertilized embryos was high (98.5%) and resulted in a pregnancy rate (positive human chorionic gonadotrophin 18 days after embryo transfer) of 21.4% per embryo transfer procedure (a maximum of 3 embryos were transferred per procedure). The relationship between the number of sperm injected and the fertilization rate and other factors affecting the outcome are discussed.

scholarly journals Role of ghrelin in fertilization, early embryo development, and implantation periods

Reproduction ◽  
2014 ◽  
Vol 148 (2) ◽  
pp. 159-167 ◽  
Author(s):  
Eugenia Mercedes Luque ◽  
Pedro Javier Torres ◽  
Nicolás de Loredo ◽  
Laura María Vincenti ◽  
Graciela Stutz ◽  
...  
Keyword(s):  
Embryo Development ◽  
Ovulation Induction ◽  
Physiological Role ◽  
Fertilization Rate ◽  
Early Embryo ◽  
Corpora Lutea ◽  
Negative Effects ◽  
Early Embryo Development ◽  
And Control

In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4–71.4% and antagonist, 75.0–62.5% vs ghrelin 2, 46.2−15.4% and control, 10–0.0%;n=7–13 females/group;P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%;n=82–102 embryos/treatment;P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in ‘adequate’ concentrations) has a physiological role in early gestational events.

112 EFFECTS OF GUAIAZULENE ON IN VITRO CULTURE OF BOVINE ZYGOTES, AND ON mRNA TRANSCRIPTS RELATED TO EMBRYO QUALITY

2009 ◽  
Vol 21 (1) ◽  
pp. 156
Author(s):  
E. Dovolou ◽  
M. Clemente ◽  
G. S. Amiridis ◽  
I. Messinis ◽  
A. Kalitsaris ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Early Embryo Development ◽  
Mrna Transcripts ◽  
Oviductal Fluid ◽  
Maximum Humidity

We have previously shown that follicular and oviductal fluid provide greater total protection against lipid peroxidation than the respective media used for the in vitro embryo production. Reactive oxygen species (ROS) production has been implicated as a major cause for the reduced in vitro bovine embryo production; it is believed that they participate in meiotic arrest of oocytes, embryonic block and cell death. The aim of this study was to determine whether guaiazulene (G), an exogenous antioxidant, added in the post fertilization culture medium would affect the early embryo development and the quality of the produced blastocysts in terms of mRNA expression of several important genes. In a previous study we had shown that media modified with 0.01 mm of G provided the same antioxidant protection as the respective in vivo environments (i.e. the follicular and the oviductal fluid). Bovine cumulus–oocyte complexes (COC) were aspirated from ovaries derived from slaughtered cows and matured in groups of 50 in 500 μL in TCM199 with 10% fetal calf serum and 10 ng mL–1 Epidermal Growth factor at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Twenty-four hours later matured oocytes were inseminated with frozen/thawed bull semen and co-incubated in the same conditions as maturation. Presumptive zygotes were divided into 4 groups and cultured in groups of 25 in 25 μL of SOF with 5% FCS (Control–, n = 355), supplemented with 0.01 mm of G (n = 344) or 0.1 mm of G (n = 345) or 0.05% DMSO – the G diluent–(Control+, n = 347) at 39°C in an atmosphere of 5% CO2, 5% O2 and maximum humidity. Blastocyst yield was recorded on Days 6, 7, 8 and 9; Day 7 blastocysts from each group were snap frozen and stored at –80°C for mRNA extraction. Quantification of transcripts for aldose reductase mRNA (AKRIBI), prostaglandin G/H synthase-2 (PGHS-2, COX-2), glyceraldehyde 3-phosphate dehydrogenase (GADPH), facilitated glucose/fructose transporter, member 5 (GLUT-5) genes related to metabolism, glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD) antioxidant enzymes and placenta-specific 8 (PLAC8) related to implantation was carried out by real-time quantitative RT-PCR. Data for embryo development and on transcript abundance were analyzed by chi square and ANOVA, respectively. Cleavage rate tended to be higher in 0.01 mm group than in Control– (77.87% v. 71.41%, P = 0.07). Barring that, no other differences were detected in cleavage rate (Control+: 71.32%; 0.1 mm: 72.75%) or in the overall blastocyst yield on Day 9 (Control–: 25.50%; Control+: 26.71%; 0.1 mm: 25.75%; 0.01 mm: 29.58%). The relative abundance of genes studied varied among groups, but these differences were not significant. We infer that under the current culture conditions, G as an antioxidant has no serious direct effect on early embryo development or on embryo quality at least on the mRNA transcripts studied. Further studies using the same antioxidant in different atmospheric conditions are planed. ED and GSA were sponsored by COST (FAO702) and OECD fellowships, respectively.

The choice of the most appropriate microfertilization technique for human male factor infertility

1994 ◽  
Vol 6 (1) ◽  
pp. 37 ◽  
Author(s):  
AO Trounson
Keyword(s):  
Semen Quality ◽  
Fertilization Rate ◽  
World Health ◽  
Gamete Intrafallopian Transfer ◽  
Male Factor Infertility ◽  
Motile Sperm ◽  
Male Factor ◽  
Cleavage Rate ◽  
Pronuclear Formation

Comparisons were made among techniques used to treat male factor infertility. Patients with semen quality below that recognized by World Health Organization criteria as normal had a better success rate when treated by gamete intrafallopian transfer than by in vitro fertilization (25% v. 7% pregnancy rate per patient). When < 2 x 10(6) motile sperm were recovered, the fertilization rate and embryo cleavage rate were higher for microdrop insemination than for conventional insemination. When 7000-370,000 motile sperm were recovered, microdrop insemination resulted in a higher fertilization rate (46%) and a higher incidence of pregnancies (23% of patients treated) than subzonal sperm microinjection (SUSM). However, for patients with 5000-50,000 motile sperm, the immediate transfer of SUSM oocytes to the Fallopian tube increased pregnancy rates for this technique to 24% of patients treated. Direct microinjection of epididymal sperm from azoospermic men into the cytoplasm of oocytes resulted in pronuclear formation in 27% of oocytes; in comparison, pronuclear formation occurred in 5% of SUSM oocytes. These data led to formulation of a logical treatment programme for male factor infertility.

181 Equine androgenic embryos: ability of the equine sperm to develop in a heterospecific ooplasm

2019 ◽  
Vol 31 (1) ◽  
pp. 215
Author(s):  
M. B. Rodriguez ◽  
A. Gambini ◽  
D. F. Salamone
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Uv Light ◽  
Early Embryo ◽  
Control Treatment ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Sperm Dna ◽  
Haploid Embryos ◽  
Androgenic Embryos

Androgenic haploid embryos were originally produced for the study of certain aspects of early embryo development. The generation of androgenic haploid embryos allows us to better understand the complementary parental contribution to embryonic development, and to examine the effects of haploid development on gene expression. Because mare oocytes for research are scarce, the generation of heterospecific androgenic embryos could be useful to study aspects of the biology of early embryo development, or to identify genes and their variations or mutations that are responsible for reproduction-related problems in mares and stallions, which is of interest for the breeding industry. Therefore, the aim of this work was to study the capability of equine sperm to induce embryonic development after injection into an enucleated oocyte from a different species. Porcine cumulus-oocyte complexes (COC) were obtained from abattoir ovaries and placed in 100-µL drops in vitro maturation (IVM) medium for 42h. Cumulus cells were removed with hyaluronidase and vortexing. Then, mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) with stallion frozen-thawed semen (according to Rodriguez et al. 2015). Immediately after the last injection, the zona pellucida of injected oocytes was removed with protease treatment, the oocytes were treated with cytochalasin B, and the metaphase II enucleated with a 20-µm micropipette. Finally, embryos were placed in culture medium (SOF) in plates with the well-of-the-well (WOW) system. As control treatment, non-enucleated pig oocytes were injected with stallion (CE) and boar (CC) semen. At Day 4, embryos were evaluated for cleavage and number of blastomeres, and stained with Hoechst 33342 to verify the presence of DNA in each blastomere under the UV light. Embryos were stored for future PCR studies to validate the presence of equine DNA. Data were analysed by chi-squared test to compare the cleavage of both controls with the androgenic embryos. From a total of 53 androgenic haploid embryos, the cleavage rate was 62% (33/53). Embryos were cleaved in 2 to 4 cells in 72.7%, 5 to 8 cells in 18.2%, and 9+ cells in 9.1% at Day 4. Presence of DNA in all blastomeres was observed in 60.6% (20/33) of the androgenic haploid embryos, while 21.2% (7/33) of the embryos had 10 to 50% of blastomeres with DNA, and 18.6% (6/33) of the embryos did not have DNA in their blastomeres. The ICSI control embryos cleaved in 45.3% (34/75) and 64.9% (98/151) for groups CC and CE, respectively. Cleavage rates in control CE were significantly higher than those in control CC (P&lt;0.004). No statistical difference was observed in the control groups versus androgenic embryos. This preliminary results showed that a heterospecific ooplasm can be successfully used to allow an equine sperm DNA to decondense and to develop, even in absence of the female counterpart. Using this method, copies of a single sperm DNA can be produced to potentially evaluate individual aspects of early embryo development concerning the male contribution. This is the first report of successful androgenic embryos using a heterospecific oocyte to create copies of a horse sperm DNA.

Quantitative analysis of sperm mRNA in the pig: relationship with early embryo development and capacitation

2013 ◽  
Vol 25 (5) ◽  
pp. 807 ◽  
Author(s):  
Jae Yeon Hwang ◽  
Brendan P. Mulligan ◽  
Hyung-Min Kim ◽  
Byoung-Chul Yang ◽  
Chang-Kyu Lee
Keyword(s):  
Embryo Development ◽  
Early Embryo ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Specific Mrnas ◽  
Porcine Spermatozoa ◽  
Mammalian Spermatozoa

Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n = 20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n = 10 high cleavage group semen samples) than in the low cleavage group (n = 10; P < 0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n = 12; P < 0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

124 CRYOPRESERVED BOVINE OVIDUCTAL EPITHELIAL CELLS SURVIVAL, GROWTH, AND ABILITY TO SUPPORT EARLY EMBRYO DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 154
Author(s):  
E. Corbin ◽  
A. Cordova ◽  
J. Grosbois ◽  
P. Mermillod
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Embryo Development ◽  
Culture Medium ◽  
Early Embryo ◽  
Cell Culture Medium ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Blastocyst Rate

Previous experiments demonstrated that co-culture of bovine embryos with bovine oviducal epithelial cells (BOEC) improved blastocyst rate and quality (Cordova et al. 2014). However, the use of primary cell support for improving embryo development in vitro may introduce a higher variability of the results between different BOEC batches used, as well as sanitary risks. The use of well-controlled large batches of frozen BOEC may help to solve these problems. Therefore, the aim of the present study was to characterise the survival and functionality of frozen-thawed BOEC. Bovine oviducts attached to ovaries showing recent ovulation were collected at a local slaughterhouse during 4 replicates (3 oviducts per replicate). Epithelial cells were expelled by gentle squeezing and washed 3 times. Half of the cell pellet was diluted 100-fold in culture medium (TCM199 + 10% FCS) for culture of fresh cells. The other half was diluted 10-fold in cell freezing medium (TCM199 + 20% FCS + 10% dimethyl sulfoxide), allowed to equilibrate in this medium for 10 min, and frozen at –80°C in a container filled with isopropyl alcohol. After 4 h, the tubes were transferred into LN for at least 1 h. The tubes were then thawed (5 min in 37°C water bath), diluted 1 : 1 in cell culture medium, and centrifuged for 10 min at 100 × g. The pellet was then diluted 100× in cell culture medium. Fresh or frozen-thawed cells were seeded in 4-well NUNC plates for 7 days at 38.8°C in a humidified atmosphere with 5% CO2 in air. The medium was renewed every 48 h, and the viability of cells was assessed by calcein-AM and ethidium homodimer labelling. After 7 days of culture, the medium was replaced by SOF medium + 5% FCS, and bovine in vitro-produced zygotes were added the day after and co-cultured for 8 days at 38.8°C in a humidified atmosphere with 5% CO2 in air to evaluate embryo development. Half of the medium was renewed every 48 h. Frozen-thawed cells showed the same viability than fresh ones at Days 0 and 7 of culture and reached confluence at the same time (Day 7). Development results are shown in Table 1. Frozen and fresh cells support early embryo development at the same rate. In conclusion, the present study showed that BOEC frozen on the day of collection are equivalent to fresh BOEC in regards to their survival and proliferation and their ability to support early embryo development. At collection, the cells may face stresses that are just as considerable as freezing/thawing (temperature shock, scrapping, change of environment). This may explain why they are not affected by freezing than at collection. The differentiation status of these cells is now under analysis by immunocytochemistry. Table 1.Cleavage rate and blastocyst rate in 3 different types of culture systems

scholarly journals Effects of Activation Timing on the Fertilization Rate and Early Embryo Development in Porcine ROSI Procedure

2004 ◽  
Vol 21 (9) ◽  
pp. 329-334 ◽  
Author(s):  
Jong Yeob Choi ◽  
Eun Young Lee ◽  
Hee Tae Cheong ◽  
Byung Koo Yoon ◽  
Duk Soo Bae ◽  
...  
Keyword(s):  
Embryo Development ◽  
Fertilization Rate ◽  
Early Embryo ◽  
Early Embryo Development
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