Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 197 ◽  
Author(s):  
SP Flaherty ◽  
D Payne ◽  
NJ Swann ◽  
CD Matthews
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Sperm Head ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Metaphase Chromosomes ◽  
Polar Bodies ◽  
Abnormal Fertilization ◽  
Second Polar Body ◽  
Sperm Chromosomes

The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 245-256 ◽  
Author(s):  
Tetsuo Ono ◽  
Eiji Mizutani ◽  
Chong Li ◽  
Teruhiko Wakayama
Keyword(s):  
Room Temperature ◽  
Polar Body ◽  
Methylation Status ◽  
Cost Effective ◽  
Oocyte Activation ◽  
Male Gametes ◽  
Effective Manner ◽  
Cell Embryo ◽  
Cauda Epididymidis ◽  
Second Polar Body

SummaryThe development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

Experiences with intracytoplasmic sperm injection

1995 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Susan E Lanzendorf
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Meiotic Division ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Cortical Granules ◽  
Specific Sequence ◽  
Mammalian Fertilization ◽  
Pass Through

Mammalian fertilization, whether it takes place within the female reproductive tract or within a laboratory dish, is comprised of many processes which must follow a specific sequence. The spermatozoon must bind to and pass through the zona pellucida, fuse with the oolemma and become incorporated into the cytoplasm of the oocyte. Fusion of the two gametes triggers oocyte activation, resulting in exocytosis of the cortical granules and completion of the second meiotic division of the oocyte. A block in one or more of these processes, due either to abnormalities in the spermatozoon or oocyte, may result in fertilization failure.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 69-76 ◽  
Author(s):  
S.A. Ock ◽  
J.S. Bhak ◽  
S. Balasubramanian ◽  
H.J. Lee ◽  
S.Y. Choe ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chromosomal Abnormality ◽  
Polar Body ◽  
Ultrastructural Changes ◽  
Time Interval ◽  
Oocyte Activation ◽  
Group 4 ◽  
Group 5 ◽  
Bovine Oocytes

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Xin Tan ◽  
An Peng ◽  
Yong-Chao Wang ◽  
Yue Wang ◽  
Qing-Yuan Sun
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Second Polar Body

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte–second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

The localization of LAP2β during pronuclear formation in bovine oocytes after fertilization or activation

Zygote ◽  
2006 ◽  
Vol 14 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Mamiko Isaji ◽  
Hisataka Iwata ◽  
Hiroshi Harayama ◽  
Masashi Miyake
Keyword(s):  
Chromatin Condensation ◽  
Polar Body ◽  
Somatic Cells ◽  
Histone H3 ◽  
Cumulus Cells ◽  
Polar Bodies ◽  
Second Polar Body ◽  
Pronuclear Formation ◽  
Bovine Oocytes

SummaryWe have shown that the assembly of lamin-associated polypeptide (LAP) 2β was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2β assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2β assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2β assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2β assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2β did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2β assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2β assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2β assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2β assembly around oCh but not histone H3 dephosphorylation.

56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121 ◽  
Author(s):  
Y. M. Toishibekov ◽  
R. K. Tursunova ◽  
M. Sh. Yermekova
Keyword(s):  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Maturation Medium ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques ◽  
Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Intracytoplasmic sperm injection in dysmorphic human oocytes

Zygote ◽  
1995 ◽  
Vol 3 (4) ◽  
pp. 283-288 ◽  
Author(s):  
Mina Alikani ◽  
Gianpiero Palermo ◽  
Alexis Adler ◽  
Massimo Bertoil ◽  
Marlena Blake ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Smooth Endoplasmic Reticulum ◽  
Polar Body ◽  
Light Microscopic Level ◽  
Human Oocytes ◽  
Fertilisation Rate ◽  
Polar Bodies ◽  
First Polar Body ◽  
Multiple Abnormalities ◽  
Further Development

SummaryFertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes – most probably a product of controlled ovarian stimulation – are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.

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