Oocyte Morphology Research Articles

Influence of meiotic stages during in vitro maturation on the post thaw morphology of buffaloo oocytes

The present experiment has been conducted to study the post thaw morphology of buffalo oocytes vitrified at different stages of in vitro maturation (IVM). Cumulus oocyte complexes (COCs) obtained from slaughter house ovaries were randomly divided into 6 different groups:control (non- vitrified oocytes were matured for 24 h in maturation medium (MM) consists of TCM-199 supplemented with 10% w/v fetal calf serum (FCS) at 38±1 0 C and 5% CO 2 in a humidified atmosphere), 0 h (vitrified before the onset of maturation), 6,12,18 and 24 h groups (vitrified at 6, 12, 18 and 24 h, respectively, after the onset of maturation). Oocytes were exposed to vitrification solution (VS) consists of 40% w/ v propylene glycol and 0.25 M trehalose in phosphate buffered saline (PBS) supplemented with 4% w/ v bovine serum albumin (BSA) for 3 min at 20-25 0 C. Oocytes in VS were loaded into 0.25 ml French mini straw with 1M sucrose solution separated by two airspace on either side of VS. The straws were sealed with hot forceps and plunged directly into liquid nitrogen (LIS; -196 0 C). The straws were thawed after storage period of atleast 7 days by transferring them into a water bath at 37 0 C for 30 sec. The cryoprotectant was removed by exposing the oocytes to 1 M sucrose solution. Oocytes in 0, 6, 12, 18 and 24 h groups were further matured for additional 24, 18, 12, 6 and 0 h, respectively, to complete a total of 24 h maturation period.A sum of 495, 432, 457, 416 and 420 oocytes were vitrified in 0, 6, 12, 18 and 24 h groups, respectively. After thawing, 444 (89.70%), 384 (88.89%),418 (91.47%), 381 (91-59%) and 387 (92.14%) oocytes were recovered in 0, 6, 12, 18 and 24 h groups, respectively. Recovered oocytes were examined to assess the post thaw morphology, of which, 87.16 (387), 84.38 (324), 86.60 (362), 87.40 (333) and 90.96 (352) % of oocytes vitrified at 0, 6, 12, 18 and 24 h groups, respectively, were found morphologically normal. From this study, it is clear that meiotic stages of oocytes does not influence the post thaw morphology in different vitrification groups.

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre- and Post-Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre- and post-activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre- or post-electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1min pre- and 3min post-activation showed significantly higher blastocyst development than 0min pre- and 0min post-activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0min pre- and 0min post-activation. In conclusion, exposure of oocytes to mannitol for 1min pre- and 3min post-activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1min pre- and 3min post-activation.

Influence of follicle size, methods of retrieval on oocytes yield and morphology in Egyptian Jennies ovaries with special reference to maturation rate in vitro

This work was designed to evaluate the ovarian follicular development, oocytes morphology, methods of oocytes reterival, and the effect of different in vitro maturation (IVM) media on cumulus cell expansion and nuclear maturation of Jennies oocytes. Experiment 1, the number of small (<6 mm), medium (6 to 9 mm) and large size (>10 mm) ovarian follicles was recorded. Cumulus-oocyte-complexes (COCs) were reterived and classified into 4 Grades based on their cumulus-cells investment and the homogenous of the ooplasm. In Experiment 2, COCs were recovered by using 18-G, 20-G needle or slicing and scraping of ovarian follicles to determine the number and morphology of the recovered COCs. In Experiment 3, Grade A and B COCs were IVM in DMEM-HG, DMEM-LG, DMEM-F12, TCM199, TCM199-F12 or CR1aa media supplemented with 10% FCS+10 μg FSH/mL+10 IU hCG/mL+50 μg/mL gentamicin. Maturation was performed for 36 h at 38.5 °C under 5% CO2 in humidified air. After IVM, cumulus cell expansion and oocytes nuclear canfiguration were determined. An average of 6.40±0.26 follicles was recorded per Jenny ovary, representing 3.37±0.46, 1.89±0.14 and 1.14±0.16, for the small, medium and large size follicles, respectively. Oocyte recovery was higher (P<0.05) in large and medium size follicle than in the small one (62%, 60% and 45.1%, respectively). Small size follicles produced higher (P<0.05) percentage of Grade A COCs than large or medium size follicles. A higher number of oocytes was recovered by slincing and scraping of follicles (4.86±0.67), then aspiration of follicles using 18-G needle (3.14±0.36 COCs/ovary, P<0.05). Aspiration using 18-G needle or slicing and scraping of follicles using produced a significantly higher (P<0.05) percentage of Grade A COCs compared to aspiration of follicles using 20-G needle (56.6%, 46.7% and 32.0%, respectively, P<0.05). IVM of COCs in CR1aa and TCM 199-F12 media significantly increased (P<0.05) Grade 3 cumulus-cell expansion compared with TCM199, DMEM-F12, DMEM-LG and DMEM-HG (65.5% and 64.0%, 52.8%, 32.1%, 0.0% and 7.4%, respectively). The proportion of IVM oocytes reaching the M II stage was significantly higher (P<0.05) for oocytes matured in TCM199-F12 or CR1aa media than TCM199, DMEM-HG, DMEM-LG, DMEM-F12 (69.1% and 62.2%, 55.7%, 45.8%, 39.0% and 40.7%, respectively). The proportion of degenerated oocytes IVM in TCM199-F12 (10.3%), CR1aa (11.3%) or TCM199 (13.1%) was lower (P<0.05) than that matured in DMEM-HG, DMEM-LG or DMEM-F12 media (23.7%, 29.3% and 22.9%, respectively). Slicing and scraping or aspiration of follicles using 18-G needle increased the number and percentage of Grade A Jennies oocytes. TCM199-F12, CR1aa and TCM199 medi are more suitable for IVM of Jenny oocytes by promoting cumulus cells expansion and nuclear maturation to M II stage.

Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P<0.001), averaging 2.1±0.1 when compared to 1.2±0.1 in wild-type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P<0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P<0.001), averaging 1.61±0.07 in heterozygotes and 1.29±0.03 in wild-type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise.

Effects of postmortem interval on mouse ovary oocyte survival and maturation.

To study the time- and temperature-dependent survival of ovarian oocytes collected from postmortem carcass, ICR mice were killed and placed for different periods (0, 1, 2, 4, 6, 8 and 10 h) at different temperatures (25°C, 4°C and 37°C). After preservation, oocyte morphology, germinal vesicle (GV) oocyte number, oocyte meiotic maturation percentage, mitochondrial distribution and intracellular glutathione (GSH) level were evaluated. The results showed no surviving oocytes could be collected by 2h, 6h, and 12 h after carcass preservation at 37°C, 25°C and 4°C, respectively. The number of collected GV oocytes in the ovary deceased as the preservation time lasted at the same temperature. Meanwhile at the same point in time, the ratio of germinal vesicle breakdown (GVBD) and the first polar body emission (PBE) gradually reduced as preservation temperature increased. In addition, the percentage of abnormal mitochondrial distribution in the preserved oocytes was obviously higher than that in the control oocytes, while GSH level was not altered in collected oocytes. Unexpectedly, neither chromosome arrangement nor spindle organization was affected as long as the oocytes from preserved carcasses could complete maturation. These data are helpful for proper use of ovary oocytes from postmortem carcass of valuable individuals.

The effects of cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression in mice.

Cigarette smoking can harm fertility, but the existing research has targeted primarily on ovarian follicles, embryos or sex hormone. In this study, we tested cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression associated with inhibition of oxidative stress using C57BL/6 mice. Mice in the experimental group were administered a cigarette smoke extract (CSE) solution (2 mg/ml) orally daily, while the blank control group was given dimethylsulfoxide (DMSO). A positive control group (menadione) was used that received an intraperitoneal injection of 15 mg/kg menadione in oil solution daily. We found that the CSE group manifested a reduced diameter of zona pellucida-free oocyte (ZP-free OD) and a morphologically misshapen first polar body (PB). Our results suggest that CSE exposure is associated with a shrink size and poor quality of oocytes. Quitting smoking is a wise choice to ensure good fertility.

Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes.

Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects. The spindle assembly checkpoint (SAC) guarantees proper cell cycle progression. Highly Expressed in Cancer protein 1 (Hec1, also called Ndc80) is the core component of the Ndc80 complex and is involved in regulating both kinetochore-microtubule interactions and the SAC during mitosis in multiple cell types. However, its involvement in pig oocyte meiotic maturation remains uncertain. Thus, we investigated Hec1 expression, localization, and possible functions during porcine oocyte meiosis. Immunofluorescent staining showed that Hec1 was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages. Disrupting Hec1 function with its inhibitor INH1 resulted in polar body extrusion defects in porcine oocytes. Moreover, inhibiting Hec1 activity also resulted in severe chromosome misalignments and aberrant spindle morphology. Our results showed a unique localization pattern for Hec1 in porcine oocytes and suggested that Hec1 was required for chromosome alignment and spindle organization. Thus, Hec1 might regulate spindle checkpoint activity during mammalian oocyte meiosis.

The role of ionizing radiaton on ovulation rate and oocyte morphology in mouse

We investigated the effects of ionizing radiation on maturation ability and radiosensitivity of oocytes enclosed in preantral and antral follicles. Balb/c female mice received total body single dose gamma radiation (7.2 Gy) at the diestrous to proestrous transition period. In the first experiment, spontaneously ovulated oocytes were collected from irradiated animals. In the second experiment, irradiated animals were allowed to superovulate to assess the ovarian function. The spontaneous ovulation rate of the follicles exposed at antral stage was significantly lower than the sham-irradiated mice (p < 0.01), and most of the oocytes were found at the metaphase I stage. Oocyte morphology and the ovulation rate of the follicles exposed at preantral stage were similar to the sham-irradiated group. Minimal morphological abnormalities were observed in the oocytes and the polar body as well. The superovulation response of all the irradiated animals was lower than the respective control animals. The superovulation rate was significantly lower in the first ovulation after irradiation (p < 0.01). In conclusion, our findings indicate that total body gamma irradiation, on a basis of estrous cycle stages, leads to ovulation failure in the antral stage while causes abnormal oocyte morphology in the preantral stage follicles in mice.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer.

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter ('small' ≤110µm; 'medium' >110µm; 'large' ≥120µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Caption: Oocyte morphology after 24 h and 48 h of incubation with phosphodiesterase 3A inhibitor. (See 147–153 by AM Taiyeb, et al.)

Cover Image: Use Fig.4 from CEP 12193 Clinical tests for intestinal permeability: ‘Sweet’ transition from clinical to basic physiology Gut permeability to saccharidic probes Pioglitazone and irritable bowel syndrome Muscarinic receptor interaction

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes)

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Characterization of immature sheep oocytes and zona pellucida morphology using scanning electron microscope (SEM)

The aim of the present work was to study the differences among different categories of immature sheep oocytes. Oocytes were divided into three categories: excellent, good and fair oocytes. Results showed that cumulus cells were close to each other and zona pellucida in all the three oocytes categories. All oocytes categories with cumulus cells exhibited an irregular shape, mucous material inter-granular cells, low number of small droplets and minutes tapes on the cell surface. Good oocytes category was characterized by the presence of a belt surrounding the oocytes. Moreover, there are large numbers of small holes on the ZP surface in all categories. The good oocytes also contained a large number of big holes. In contrast, fair and excellent oocytes have low number of large holes in the ZP surface. In conclusion, there are several differences between oocytes categories in the morphology of oocytes and ZP characteristics. This study recommends using excellent and good oocytes in the in vitro embryo production for quality of the outer surface and integrity of zona pellucida than fair oocytes.

One/Two Days Preservation of goat oocytes at refrigerator and their maturation in vitro

This study was conduct to evaluate the feasibility of one/two days preserving immature goat oocytes without freezing. Cumulus oocyte complexes (COCs) were obtained from ovaries of slaughtered goats. Selected COCs were kept in tissue culture medium (TCM-199) and 10% Fetal Calf Serum (FCS) and stored at 4°C for 1 or 2 days. After preservation, oocyte morphology and viability were evaluated. Post-warmed stored and Non-stored oocytes were cultured in TCM-199 and 10% FCS in CO2 incubator for 30 h. Oocyte maturation was indicated by cumulus expansion after in vitro culture. Data analysis revealed a high percentage of morphologically normal and viable oocytes after preservation either for 1 or 2 days (70, 60 vs. 60, 50%, respectively) with low percentage of damaged oocytes. There was no significant difference in the maturation of stored oocytes for 1 or 2 days in comparison to fresh oocytes although the value of the fresh oocytes was higher (55, 50 vs. 65%, respectively). Thus, we have developed a simple method for preserving immature goat oocytes by refrigeration at 4°C for two days without evident damage of oocyte and keeping oocyte developmental competence.

Anomalies in ovarian condition of manybar goatfish Parupeneus multifasciatus (Mullidae) from the coastal zone of south Central Vietnam

We describe anomalies in oocyte structure of manybar goatfish Parupeneus multifasciatus from the coastal zone of southern Central Vietnam. The anomalies are registered in the majority of cells (>80%) from ovarian fragments of several individuals. The abnormal cells are at the periods of maturation, vitellogenesis, and, sometimes, previtellogenesis. Possible effect of pollutants on oocyte morphology is discussed.

Characteristic cytoplasmic morphology of oocytes in endometriosis patients and its effect on the outcome of assisted reproduction treatments cycles

Introduction: Endometriosis is a clinical disease that is associated with poor outcomes in in vitro fertilization programs with a decrease in oocyte retrieval, oocyte quality, implantation and pregnancy rates.Based on the observations that the majority of the oocytes obtained in patients diagnosed with endometriosis have some characteristics, we conducted a retrospective study to reveal a more definite picture and to establish a simple way of prediction of the outcome. Materials and Methods: The patients were triaged in two groups based on the diagnosis of endometriosis. After denudation, the oocytes were examined by inverted microscope with ×200 magnification just before intracytoplasmic sperm injection. Oocytes with a centrally dark and granular cytoplasm were diagnosed to have degenerative signs. Results: Data of 792 patients were reviewed retrospectively. The percentage of oocytes with degenerative signs is significantly higher in endometriosis (27.4%) group compared with control group (7.9%) ( P < 0.001). Although, there is a tendency to lower pregnancy rate in endometriosis group (38.7%) the difference is not statistically significant (50.4%) ( P = 0.077). Conclusion: The oocytes have a characteristic appearance of dark and granular ooplasm in endometriosis cases. This finding can be used as a clue in cases in which the presumptive diagnosis has not verified by laparoscopy. The typical morphology also has an adverse effect on clinical outcome.

Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.

Multifactorial Analysis of the Follicular Environment is Predictive of Oocyte Morphology in Cattle

Numerous attempts have been recently made in the search for a reliable, fast and noninvasive assay for selection of oocytes suitable for in vitro embryo production. Potential markers have been described in the follicle such as follicular fluid (FF) or cumulus cells (CCs). However, the reported findings are contradictory, which may reflect the complexity of metabolism of the ovarian follicle. In the present experiment, a data set from individual follicles of known diameter was obtained: cumulus-oocyte complex (COC) morphology, fatty acid composition and glucose concentration in FF as well as apoptotic index in CCs. The obtained data was statistically analyzed either separately (univariate analysis) or simultaneously (multivariate analysis) to examine its predictive value in morphology assessment of bovine COCs. Although the univariate analysis yielded a complex relation system of the selected parameters, no clear outcome could be established. In multivariate analysis, the concentration of the four fatty acids (C16:0, C16:1, C18:1cis9, C22:5n3) and Δ9-desaturase (16) as well as elongase activities were selected as covariates. This allowed prediction of the morphology of a COC with an accuracy of 72%, which is the most interesting finding of the experiment. The present study indicates that the multifactorial model comprising of selected parameters related to the follicle appeared more effective in predicting the morphology of a bovine COC, which may improve the effectiveness of in vitro production systems.

Ovary histology and quantification of hemolymph proteins of Rhipicephalus (Boophilus)microplus treated with Melia azedarach

This study aimed to analyze ovary histology and quantify total protein in the hemolymph of Rhipicephalus (Boophilus) microplus females treated with hexane extracts from green fruits of Melia azedarach. Eight engorged females were immersed in the extract at 0.25% concentration, and eight in water containing 5% acetone (control). The females were dissected 72 hours after treatment, and the ovaries were weighed and subjected to standard histological techniques. The total protein concentration was measured in the hemolymph of 200 females, of which 100 were treated as described above and 100 served as a control. In the treated group, ovary weight reduction and predominance of immature oocytes were observed. In addition, there were decreases in the diameters of the cytoplasm and germ vesicle of the oocytes in the treated group, compared with the controls. The protein concentration in the hemolymph was higher in the treated group than in the controls. The morphological changes observed in the treated ovaries included: presence of vacuolization; alteration of oocyte morphology, which changed from rounded to elongated; deformation of the chorion; and disorganization of the yolk granules. These results demonstrate the action of M. azedarach fruit extracts on R. (B.) microplus oogenesis.

Does oocyte morphology abnormalities affect the pregnancy rates of singe embryo transfer on day 5 or day 3?

Does oocyte morphology abnormalities affect the pregnancy rates of singe embryo transfer on day 5 or day 3?

Evaluation of the oocyte morphology in the patients undergoing IVF-ICSI cycles receiving GnRH agonists and antagonists protocols: a retrospective study

Maternal age, genetic defects and controlled ovarian stimulation protocols may cause extra and/or intracytoplasmic morphological alterations in the oocytes which in turn may result in fertilization failure or developmental impairments. The data in the literature concerning the impact of GnRH agonist and antagonist protocols on oocyte morphology still remains controversial. Our aim is to investigate the effect of agonist and antagonist protocols on oocyte dysmorphism.