|Year : 2015 | Volume
| Issue : 1 | Page : 7-13
A correlative study of oocytes morphology with fertilization, cleavage, embryo quality and implantation rates after intra cytoplasmic sperm injection
Emad Ghanem Qassem1, Kani Muhmmed Falah2, Ismaeel H.A Aghaways3, Tan Azad Salih4
1 Assistant Professor, Dept. of Anatomy, Al-Mostensrea University, Baghdad, Iraq
2 Lecturer, Human Embryology, Sulaimania University, Sulaimnia KRG, Iraq
3 Head of General Surgery Departmentat, Sulaimania University, Sulaimania KRG, Iraq
4 Demonstrator at Anatomy, Department of School of Medicine, University of Sulaimani, Sulaimnia KRG, Iraq
|Date of Web Publication||4-Jul-2017|
Emad Ghanem Qassem
Assistant Professor, Dept. of Anatomy, Al-Mostensrea University, Baghdad
Source of Support: None, Conflict of Interest: None
Introduction: Non-invasive selection of developmentally human oocytes may increase the overall efficiency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. The objective is to evaluate the influence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. The criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fine granularity, a round or ovoid first polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (first polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). There were no significant differences in fertilization, cleavage, and embryo quality between the groups but there was a highly significant difference in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not affect fertilization, cleavage, and embryo quality, but affecting implantation rate.
Keywords: Fertilization, Implantation, Oocytes morphology
|How to cite this article:|
Qassem EG, Falah KM, Aghaways IH, Salih TA. A correlative study of oocytes morphology with fertilization, cleavage, embryo quality and implantation rates after intra cytoplasmic sperm injection. Acta Med Int 2015;2:7-13
|How to cite this URL:|
Qassem EG, Falah KM, Aghaways IH, Salih TA. A correlative study of oocytes morphology with fertilization, cleavage, embryo quality and implantation rates after intra cytoplasmic sperm injection. Acta Med Int [serial online] 2015 [cited 2020 Sep 19];2:7-13. Available from: http://www.actamedicainternational.com/text.asp?2015/2/1/7/209453
| Introduction|| |
Infertility is defined as the inability of a couple to achieve conception or bring a pregnancy to term after 12 months or more of regular unprotected sexual intercourse. It is a growing concern affecting up to 15% of couples trying to conceive globally. Intra cytoplasmic sperm injection (ICSI) had been widely used to treat couples with infertility because of severely impaired sperm characteristics and for whom in-vitro fertilization (IVF) had failed. Little attention has been focused on oocyte morphology in standard IVF techniques, because it is often difficult to assess the cytoplasmic morphology of the oocyte and the exact stage of maturation as the oocytes are always surrounded by cumulus oopheros or corona cells at the time of collection. Following the removal of the cumulus–corona cells in preparation for intra cytoplasmic sperm injection (ICSI), oocyte evaluation is more accurate. Oocyte maturation is based on the nuclear maturation status, the morphology of the cytoplasm and on the appearance of the extra cytoplasmic structures. The presence of the first polar body (IPB) is normally considered to be a marker of oocyte nuclear maturity. However, recent studies using polarized light microscopy have shown that oocytes displaying a polar body may still be immature.
Only those displaying a meiotic spindle (MS) can in fact be considered as true, mature, Metaphase II (MII) stage oocytes. Nuclear maturity alone is, in fact, not enough to determine the quality of an oocyte. Nuclear and cytoplasmic maturation should be completed in a coordinated manner to ensure optimal conditions for subsequent fertilization. An ideal mature human oocyte, based on morphological characteristics, should have a ‘normal-looking’ cytoplasm, a single polar body, an appropriate zona pellucida (ZP) thickness and proper perivitelline space. However, the majority of the oocytes retrieved after ovarian hyper stimulation exhibit one or more variations in the described ‘ideal’ morphological criteria. This is also true for oocytes obtained from proven fertile donors. Oocytes quality and its influence on fertilization rate and embryo development is still a matter of controversy in ART cycles, reported oocyte morphology to be an important prognostic factor in successful treatment of ICSI cases. Also reported a higher rate of miscarriage in women with dysmorphic oocytes. On the other hand, some study reported that oocyte morphology does not correlate with fertilization rate and embryo quality after ICSI. To date, the extent to which the morphology of the oocyte at the light microscopy level correlates with the results of ICSC.
| Material and Method|| |
This study was carried out at Dwarozh-IVF center in sulaimani. A prospective analysis was performed on the data obtained from 132 patients undergoing ICSI cycle that had been performed between May 2013 and January 2014. Only male infertility factors were excluded.
Ovarian Stimulation and Oocyte Retrieval
In all patients short agonist protocols was used to stimulate follicular development. The short protocol includes pituitary desensitization with gonadotrphin releasing hormone agonist (Decapeptyl 0.1 mg, Ferring GmbH, Germany) at day 2 of the cycle and ovarian stimulation with follicle- stimulating hormone (fostimon, IBSA, Lugano 3, Suisse; Gonal- f, Laboroteries Serono S.A, Switzerland; Puregon, Schering-Plough, NV.Organon, Oss, Netherlands); or combined LH and FSH (Merional, IBSA, Lugano3, Suisse; Menegon, Ferring GmbH, Germany) starting at day 3 of the cycle. Follicular development was monitored by measuring serum E2 and performing trans-vaginal ultrasound. Ovulation and final maturation of the ova were induced with human chorionic gonadotrophin hCG (Choriomon, IBSA, Lugano 3, Suisse) as a single dose of 10 000 IU, when the leading follicle reached 18 mm in average diameter in addition to the presence of at least two other follicles of more than 16 mm in size and E2 > 500 pg/ml then the oocytes were retrieved. Oocytes were aspirated 34-36 hours after hCG administration. Oocyte retrieval was performed by Trans vaginal ultrasound guided puncture using 16 gage 35 cm double lumen aspiration needle (William A.Cook, Australia pty Ltd.) with negative pressure 20 mmHg.
Oocytes Denudation and Evaluation
Removal of the surrounding cumulus cells was accomplished by a combined enzymatic and mechanical treatment carried out under a stereoscopic dissecting microscope. Oocytes were denudated from cumulus oophorus by exposure to 80 IU/ml hyaluronidase enzyme in HEPES-buffered medium (Hyase, fertipro N.V, Belgium) followed by mechanical removal of the corona radiate with the use of plastic pipettes stripper tips (Ez-strip, Research Instruments Ltd, United Kingdom) with decreasing inner diameters of 290 and 135 μm.
Oocytes are assessed for their maturation and for their morphology under an inverted microscope (intgera Ti, R.I., Olympus, IX51/IX70, Tokyo, Japan) at 400X magnification. Metaphase II oocytes were separated from the immature oocytes (metaphase I oocytes and germinal vesicle) just before sperm injection (3-4 h after retrieval).
The MII oocytes used for micromanipulation were grouped as follows:
- Normal MII oocytes showed a clear cytoplasm with homogeneous fine granularity, a round or ovoid first polar body with smooth surface and size within a small perivitelline space, and a colorless zona pellucida with regular shape.
- Abnormal MII oocytes with cytoplasmic abnormalities such as (i) inclusion body (ii) Presence of vacuoles (the presence of sERC was not evaluated). (iii) Granular and dark cytoplasm.
- Abnormal MII oocytes with extra cytoplasmic abnormalities such as large polar body, fragmented polar body dark zona pellucida, wide perivitelline space and perivitelline debris.
- MII oocytes with combined cytoplasmic and extra cytoplasmic abnormalities. Shape abnormalities were not recorded separately.
Intra Cytoplasmic Sperm Injection
Intra cytoplasmic sperm injection was performed in MII oocytes according to the technique described by oocytes were transferred to the ICSI dish prepared with drops of IVF media (ferticult-IVF, Fertipro N.V, Belgium) covered by mineral oil.
Assessment of Fertilization, Embryo Cleavage and Embryo Transfer
Fertilization assessments were performed 17±1 hour post injection. Normally, fertilized oocytes should be spherical and have two polar bodies and two PNs. PNs should be juxtaposed, approximately the same size, centrally positioned in the cytoplasm with two distinctly clear, visible membranes.
Embryo quality was evaluated under an inverted microscope. The following parameters were recorded: (1) the number of blastomeres; (2) the fragmentation percentage; (3) variation in blastomere symmetry (4) defects in the zona pellucida and the cytoplasm.
High-quality (grade A) embryos were defined as those having all of the following characteristics: either 4-6 cells on day 2 or 8-10 cells on day 3 of development, less than 15% fragmentation, symmetric blastomeres, colourless cytoplasm with moderate granulation with no inclusions, absence of perivitelline space granularity and absence of zona pellucida dysmorphism. Embryos lacking any of the above characteristics were considered as low quality. For each couple, 1-4 embryos were transferred, depending on the embryo quality and the female's age. Embryo transfer was cancelled if no embryos were available. Embryo transfer was performed on day 2 or day 3 using a Gynetics catheter (Gynetics medical products N.V, Lommel, Belgium). Transfers were performed with trans-abdominal ultrasound guidance.
Results are expressed as mean ± standard deviation (SD) for numeric variables and percentage for categorical variables. Mean values are compared by students t-test, and proportions are compared by z-test. The results are considered to be significant at the (P <0.05). Data analysis is carried out using the statistical analysis program (Statistical package for social science SPSS; version 20).
| Results|| |
The mean age of patients was (33.27 ± 5.81) years. A total of 1200 oocytes were retrieved. Of these 1056 (88%) were mature in metaphase II stage and the remainder were immature oocytes either at metaphase I stage 84 (7%) or germinal vesicle stage 60 (5%). On assessment of metaphase II oocytes, 180 oocytes (17.04%) had normal MII oocytes morphology, 876 (82.95%) had at least one demonstrable morphological abnormality. Total injected MII oocytes were 954 of these 774 had abnormal oocytes morphology and 180 normal oocytes morphology the remaining 102 MII oocytes were not injected due to sever abnormalities. The number of embryo transferred was 363 with mean (2.77±1.40).
Incidence of the Different Oocyte Morphologic Features
From 876 abnormal MII oocytes morphology, Cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, presence of inclusion body and vacuoles) were seen in 516 (58.9%) of the abnormal MII oocytes. Extra cytoplasmic abnormalities (large/fragmented polar body, large and debris of perivitelline spaces) were seen in 104 (11.87%) while combined cytoplasmic and extra cytoplasmic abnormalities were responsible for the remaining 256 (29.22%).
Relationship between MII Oocytes Morphology and Implantation Rate
The result of this study show that there was a highly significant difference in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic,extra cytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, 4.34% respectively [Table 1],[Table 2],[Table 3],[Table 4], [Figure 1].
|Table 1: Fertilization, cleavage and embryo quality rates after injection of normal and morphologically abnormal MII oocytes|
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|Table 2: Fertilization, cleavage and embryo quality rates after injection of normal and MII oocyte with cytoplasmic abnormalities|
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|Table 3: Fertilization, cleavage and embryo quality rates after injection of normal and MII oocyte with extra cytoplasmic abnormalities|
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|Table 4: Fertilization, cleavage and embryo quality rates after injection of normal and MII oocyte with combined abnormalities|
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|Figure 1: (a) Normal MII oocyte morphology (b) Abnormal MII oocyte with inclusion body (c) Abnormal MII oocyte with vacuoles (d) Abnormal MII oocyte with central granulation (e) Abnormal MII oocyte with large polar body and small polar body (f) Abnormal MII oocyte with dark zona pellucida and wide PVS (g) Abnormal MII oocyte with PVS debris (h) Fertilized oocyte with 2 PN (i) Day 3 embryo|
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| Discussion|| |
As an attempt to improve the results of ICSI cycles, it is important to identify and utilize non-invasive parameters able to predict oocyte quality. Considering the vital role played by the oocyte in the developmental process, selection criteria involving the stage preceding fertilization would be extremely useful in selecting embryo for transfer., Nonetheless, previous reports are conflicting regarding the effects of oocyte morphological abnormalities on fertilization rate and embryo quality rate.,,
In this work, the presence of extra cytoplasmic and cytoplasmic oocytes dismorphisms and their influence on fertilization, embryo quality and implantation rates in a total of 1056 MII oocytes, was evaluated. So far as is known, this is the first study in this regard carried out in Kurdistan Region of Iraq.
The majority (60%-70%) of oocytes retrieved from stimulated cycles exhibited one or more abnormal morphological characteristics.,,,,
The present study shows that 876 (82.95%) out of 1056 MII oocytes had at least one abnormal morphological characteristics. It is also observed that abnormal oocytes morphology was much higher than normal oocytes morphology 180 (17.04%). Similar results, though lower, was observed by,, whose studies showed that >60% of oocytes retrieved had at least one morphological abnormality, while a study by found that 94.5% of all oocytes had at least one demonstrable anomaly. The best explanation for that was morphological variations of the oocyte may result from intrinsic factors such as age and genetic defects, or extrinsic factors such as, the controlled ovarian stimulation protocol and the ovarian response to controlled ovarian stimulation.,,
The Result of current study clearly indicate that normal MII oocyte and abnormal MII oocytes morphology does not unfavourably alter the fertilization, cleavage and high quality embryo rates. Similarly, other studies,,, demonstrated that the fertilization rate and embryo quality did not differ in the group of oocytes with no abnormality or one, two, or three morphological abnormalities. On the other hand studies by,, showed that oocytes dismorphisms is associated with a decreased fertilization, cleavage and embryo quality.
Data from this study showed that there was a significant difference in implantation rate which was higher in the group with normal oocyte morphology 11.11% than the group with abnormal oocyte morphology 7.33% so our results agreed with results in Studies by, which established a significant relationship between MII oocytes morphology and implantation potential. Whereas Studies by,,, demonstrated that outcomes (implantation rate, pregnancy, take home baby and multiple pregnancy rates) were similar when all embryos were derived from intact oocytes, or all from morphologically “handicapped” oocytes.
In this study, Cytoplasmic and extra cytoplasmic abnormalities were observed in 58.9% and 11.87% respectively, indicating that among the abnormal oocytes morphology group, cytoplasmic abnormalities was much higher than extra cytoplasmic abnormalities. Similarly, a study by,, revealed that extra cytoplasmic abnormalities were relatively lower than cytoplasmic abnormalities. In a study by extra cytoplasmic abnormalities were seen in 8.7% and cytoplasmic abnormalities were observed in 62%. The explanation for these cytoplasmic morphological abnormalities are probably multifactorial. Excessive ovarian response, characterized by an increased number of aspirated follicles and retrieved oocytes, has a detrimental effect on oocyte quality, resulting in a higher incidence of intra cytoplasmic defects. Deficient cytoplasmic maturity has been postulated to be reflected by cytoplasmic abnormalities. The oocytes retrieved from stimulated cycles may be derived from slower-developing follicles, and therefore, the cytoplasm of these oocytes would be at different maturation stages upon the resumption of meiosis.,
The result generated from our ICSI cycles demonstrated that cytoplasmic abnormalities had no effect on fertilization, cleavage, and embryo quality rates. Similarly results obtained from studies carried out by,,, show that cytoplasmic abnormalities did not affect the fertilization rate, embryo quality and clinical outcomes while The presence of cytoplasmic features was correlated with impaired fertilization and embryo quality in studies.,,,
In this study, implantation rate was statistically significant which was higher in the group with normal oocyte morphology 11.11% than the group with cytoplasmic abnormalities 9.03% similarly, results were obtained in studies,, showed that the presence of cytoplasmic features was correlated with lower implantation rate while studies by,, have used a cumulative evaluation for cytoplasmic features of oocytes and the presence of these features did not influence pregnancy and implantation rates.
In the present study, fertilization, cleavage and embryo quality was statistically not significant between the group of normal oocytes morphology and the group of oocytes with extra cytoplasmic abnormalities similarly studies,,,, showed that extra cytoplasmic abnormalities of the oocyte did not affect fertilization, embryo quality and clinical outcomes.
According to the Istanbul consensus workshop on embryo assessment extra cytoplasmic anomalies (PBI morphology, PVS size, the appearance of the ZP) are simply phenotypic variations often related to in vitro culture and/or oocyte aging.
Studies,,,, however, suggested that the presence of extra cytoplasmic abnormalities of oocyte at MII stage (large and degenerated polar body in addition to large perivitelline space) are associated with a decreased potential of the cell to fertilize, cleave, and/or develop into a viable embryo. It has been suggested that a degenerated IPB may reflect an asynchrony between nuclear and cytoplasmic maturation which would explain the reduced ability of the cell to support pronuclear formation after ICSI. It has been postulated that the emission of an abnormally large IPB is due to the dislocation of the meiotic spindle. Large PVS may be ascribed to an over maturity of these oocytes at the time of ICSI., In this study implantation rate was statistically significant which was higher in the group with normal oocytes morphology 11.11% than the group of oocytes with extra cytoplasmic abnormalities 2.38%. But, in studies by,, demonstrated that extra cytoplasmic abnormalities had no effect on implantation and clinical pregnancy rates. We conclude from this study that the fertilization, cleavage and high quality embryos rates were not affected by these high percentages of abnormal oocytes morphology. Implantation rate was higher in the group with normal oocytes morphology than the group with abnormal oocyte morphology. So evaluation of oocyte morphology before ICSI is strongly recommended in all scoring system applied in IVF laboratories.
| Acknowledgments|| |
I would like to show my gratitude to Dwarozh-IVF center in Sulaimania, KRG, Iraq for their assistance so I am grateful for all.
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[Table 1], [Table 2], [Table 3], [Table 4]