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Regular Article |
a Infertility Center, Department of Obstetrics and Gynecology, Ghent University Hospital, B-9000 Ghent, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, follicle, in vitro fertilization, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experimental protocols and the use of animals were approved by the Animal Research Ethical Committee, Ghent University Hospital. C57bl/6j x CBA/Ca F1 female mice were used in this study. Early preantral mouse follicles were mechanically isolated from the ovaries of 14-day-old mice. The follicles with a diameter between 110 and 160 µm were selected using criteria described previously [4]. The follicle culture system was also described previously [6]. In the first set of experiments, we intended to find the optimal culture time for obtaining the highest percentage of oocytes that underwent germinal versicle breakdown and extrusion of a polar body. Preantral follicles were cultured for 8, 10, and 12 days and then given 2.5 IU/ml human chorionic gonadotropin (hCG; Pregnyl; Organon, Oss, The Netherlands) as an ovulatory stimulus. Mucified cumulus-oocyte complexes (COCs) were recovered 16–20 h later and oocytes were denuded from companion cumulus cells. Oocyte nuclear maturation was scored [4]. Based on the results of the first set of experiments, cultured follicles were stimulated with 2.5 IU/ml hCG at Day 10 and Day 12 of culture (designated as 10-day and 12-day groups, respectively) in the second set of experiments. Mucified COCs were collected 16–20 h later and fertilized by IVF or ICSI.
In Vivo-Matured Oocytes
In vivo-matured oocytes were obtained from 10- to 12-wk-old F1 female mice. Gonadotropin priming was performed with 5 IU eCG (Folligon; Intervet, Turnhout, Belgium) followed by 5 IU hCG (Chorulon; Intervet) given 48 h later. Cumulus-enclosed oocytes were collected 14 h after hCG injection and underwent fertilization.
Fertilization
Mucified COCs from in vitro-cultured follicles and COCs from superovulated mice were subjected to conventional IVF or ICSI after removal of their cumulus cells.
Sperm preparation and conventional IVF A concentration of about 2 x 105 sperm cells/ml in potassium simplex optimized medium (KSOM) supplemented with 4 mg/ml BSA was prepared, and conventional IVF was carried out as described previously [6].
ICSI procedure Before ICSI, the zona pellucida (ZP) of oocytes was dissected on 10%–15% of the circumference with a fine glass needle at a right angle to the spindle area. It took about 30 min to make a slit in the ZP of 60 oocytes. This manipulation was performed in Hepes-KSOM on a warm microscope stage at 37°C.
Five 3-µl drops of flushing holding medium (FHM) [14] were placed in line in the manipulation chamber (cover of 60-mm Petri dish; Falcon no. 3002, Beckon Dickinson, Merck-Eurolab, Leuven, Belgium) and covered with mineral oil. Spermatozoa were kept in the central drop until injection at room temperature. Immediately before injection, 3 µl of freshly thawed 10% polyvinylpyrrolidone solution (PVP; Sigma, Bornem, Belgium) was added to the drop with spermatozoa to facilitate handling and immobilization. Plain FHM was aspirated from marginal drops of the line and replaced with FHM supplemented with 20% (v/v) fetal bovine serum (FBS; Life Technologies, Merelbeke, Belgium). Oocytes were rinsed in 50-µl drops of FHM with 20% FBS prepared separately and transferred to the marginal drops of the same medium in the manipulation chamber. Between the drop with oocytes and the drop with sperm-PVP, there was an additional drop of plain FHM to remove excess PVP surrounding the injection needle when it was traveling between the drop with oocytes and the drop with sperm-PVP. Injections were performed at 16–18°C. For temperature reduction, the manipulation chamber with transferred oocytes was placed on a precooled inverted microscope stage under the control of a microscope thermal stage controller, MTS-1 (Wilten Instruments, Antwerp, Belgium). Equilibration of the temperature was allowed for 20 min before injections were started. The blunt injection pipettes with inside diameters of 7–8 µm were prepared immediately before use. To control the movement of the sperm head inside the pipette during injection smoothly, the pipettes were filled with a highly viscous inert fluid—dimethylpolysiloxane (catalog no. DMPS-12M, Sigma) and were attached through Teflon tubing (Omnilabo, Aartselaar, Belgium) to the microinjector.
The injection pipette was used to touch and press the neck of the spermatozoon right behind the head while being moved slightly aside and forward, leading to separation of the sperm head from the tail. In order to facilitate the process, usually 2–3 sperm heads were aspirated at some distance in the pipette. The pipette was moved to the drop with oocytes suspended in injection medium, and 1 sperm head was injected into 1 oocyte. The injection pipette was inserted deep into the oocyte through the predissected slit in the ZP, the sperm head was brought to the very tip of the injection pipette, and oocyte cytoplasm was slowly aspirated until breakage of the cellular membrane was clearly noted. The sperm head was then injected into the oocyte cytoplasm with the smallest amount of medium possible. It took approximately 25–30 min to inject a group of 20 oocytes. After injection oocytes were allowed to recover for 15 min at reduced temperature on the cooled microscope stage followed by 10–15 min at room temperature.
After IVF and ICSI, the oocytes were washed 5 times in fresh KSOM + 0.4% BSA (w/v) medium. Then, 10 oocytes per drop of the same medium under oil were cultured for 24–30 h in an atmosphere of 6% CO2, 6% O2 in air. Embryos that cleaved to the 2-cell stage were collected and counted and cultured in the same medium until the blastocyst stage at Day 5 after fertilization.
Cytogenetic Analysis
Uncleaved mature oocytes (first polar body visible) from the 10-day IVF group were fixed 24 h after insemination, according to a method described elsewhere [15], to analyze their chromosome constitution. Briefly, the oocytes were completely washed to remove the attached sperm on the surface of the zona; oocytes were transferred into 75 mM KCl solution for a 2- to 3-min hypotonic treatment, then placed into precooled (-25°C) fixative (1–2 ml; methanol:acetic acid = 3:1) in a watchglass for 5–20 sec. After fixation, the oocytes were transferred to a glass slide with a small amount of fixative. Air-dried slides were stained in 20% Giemsa solution (Sigma) for 20 min.
Blastocyst Cell Number Counts
Blastocyst-stage embryos obtained at 144 h post-hCG from IVF, ICSI 10-day groups, and IVF 12-day groups were fixed for cell number determination. Fixation and staining procedures were performed as described above. Blastocyst cell numbers were counted under an Olympus microscope (Model CHS; Olympus Optical Co., Ltd., Hamburg, Germany) (100x).
Statistics
Percentages of oocytes at various stages of development were calculated from 3 independent replicate experiments. Percentages were compared between groups by contingency-table analysis followed by chi-square subtests. Blastocyst cell numbers in different groups, presented as the mean ± SEM, were compared using ANOVA followed by Newman-Keuls post-t-tests. When P 
0.05, the difference was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preantral follicles were cultured for 8 days (n = 44), 10 days (n = 39), and 12 days (n = 52) before hCG stimulation in 3 independent replicate experiments. There was no difference in the percentages of oocytes that were competent to undergo germinal vesicle breakdown (GVBD) (92.3% and 90.1%, respectively) and extrude the polar body (MII stage; 80.6% and 81.2%, respectively) in 10-day and 12-day groups (Fig. 1). In the 8-day group, maturation was clearly delayed; about 60% had undergone GVBD and only 40% had reached the MII stage. Maximum meiotic maturation potential was obtained after a 10-day culture period with preantral follicles between 110 and 160 µm in diameter. On the basis of these experiments, it was decided to restrict further investigations to preantral follicles kept in culture for 10 and 12 days prior to hCG stimulation.
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Cleavage and Blastocyst Formation after IVF or ICSI (Second Set of Experiments)
In the conventional IVF group, a total of 180, 99, and 108 MII oocytes were collected and inseminated in 10-day, 12-day, and control groups, respectively (Table 1). There was no difference in the percentages of MII oocytes undergoing fertilization and cleavage to the two-cell stage (56.1% and 54.5%, respectively) in 10-day and 12-day groups. However, further development to the blastocyst stage was significantly compromised in the 12-day group (40.7% and 62.4% in 12-day and 10-day groups, respectively; P = 0.016). In vivo-matured oocytes had higher cleavage and blastocyst formation rates (89.8% and 91.8%, respectively) than the in vitro-matured oocytes from both experimental groups (P < 0.001).
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In the ICSI group, a total of 144, 144, and 74 MII oocytes were injected with a single sperm head in 10-day, 12-day, and control groups, respectively (Table 1). After ICSI, a remarkably low survival rate was found in the in vitro-matured oocytes (37%–47%) compared with in vivo-matured oocytes (77%, P < 0.001). Oocytes from the 10-day group showed a higher, although not statistically significant, survival capacity than oocytes from the 12-day group (47.2% and 37.5%, respectively; P = 0.09). With respect to the competence of cleavage to 2-cell stage embryos and blastocyst formation, results were similar to those in the IVF group. Oocytes matured for 10 days had a similar cleavage rate but a higher competence to develop to the blastocyst stage than oocytes matured for 12 days (cleavage: 72.1% and 74.1%, respectively; blastocyst formation: 57.1% and 32.5%, respectively, P = 0.035). In contrast, in vivo-matured oocytes had a significantly higher cleavage (87.7%) and blastocyst development rate (88%) after fertilization with ICSI than in vitro-matured oocytes from both experimental groups.
When comparing the outcomes in conventional IVF and ICSI experiments, there was no difference between IVF control and ICSI control groups in terms of 2-cell and blastocyst formation. In vitro-matured MII oocytes in ICSI groups showed significantly higher cleavage rates than their counterparts in IVF groups both in 10-day (72.1% versus 56.1%, P = 0.03) and 12-day cultures (74.1% versus 54.5%, P = 0.028) (Table 1). There was no difference in blastocyst formation between the IVF and ICSI groups.
Cytogenetic Analysis of Uncleaved IVF Oocytes
A total of 74 uncleaved MII oocytes characterized by polar body extrusion from the 10-day IVF group was used for chromosome preparations. Fifty-eight of them were successfully fixed on glass slides and analyzed under the microscope (Table 2). Of these, 17 oocytes (29.3%) showed meiotic MII plates of oocyte chromosomal complement with no sign of sperm penetration (Fig. 2b). Seven oocytes (12.1%) showed a maternal set of chromosomes at metaphase II and prematurely condensed sperm chromosomes (PCC) (Fig. 2c). Thirty-four oocytes showed signs of incomplete activation with respect to the formation of pronuclei (PN) or further development to the mitotic metaphase of first cleavage. Of these 34 oocytes, 25 oocytes (43.1%) showed blockage at 2PN stage in the cytoplasm (Fig. 2a). Four oocytes (6.9%) were blocked at the metaphase of the first mitotic division (Fig. 2, d and e), and 5 (8.6%) showed uniprounclear or polypronuclear development (Fig. 2, f and g).
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Blastocyst Cell Number
The total cell numbers found in blastocysts obtained from IVF and ICSI 10-day groups were as follows: IVF, 43.1 ± 2.63, n = 38; ICSI, 46.7 ± 4.07, n = 22. The mean numbers of cells in both groups were not significantly different. Blastocyst cell numbers from IVF and ICSI 10-day groups were significantly lower compared with blastocysts from in vivo-matured oocytes fertilized by IVF (110.5 ± 5.65, n = 28, P < 0.001) or by ICSI (97.3 ± 5.69, n = 24, P < 0.001) (Fig. 3). The total cell number of blastocysts obtained from the IVF 12-day group was 28.4 ± 2.69, n = 20. It was significantly lower than the numbers from 10-day groups (P < 0.001) (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 2-cell formation rate of in vitro-matured oocytes fertilized by IVF was significantly lower than after ICSI in the present study, although the same set of in vitro-matured oocytes and sperm cells was used for IVF and ICSI experiments. The cytogenetic-cytological analysis of uncleaved MII oocytes following IVF has revealed three predominant anomalies: 1) blockage at the 2PN stage (43.1%), 2) oocyte chromosomes still at meiosis MII plate with no sign of sperm penetration (29.3%), and 3) maternal MII chromosomes plus PCC of the sperm nucleus (12.1%). In ICSI, the zona and oolemma barrier (sperm receptor) are surpassed, while in IVF, sperm penetration can fail. Zona hardening may occur before fertilization in mouse oocytes matured in culture and thereby suppress penetration [16–18]. The observation of a paternal set of PCC in seven cases indicates that sperm penetration had occurred in some oocytes but these oocytes did not activate and remain arrested at metaphase II. Previous studies indeed suggested that the formation of PCC is associated with oocyte immaturity [19, 20]. In those arrested MII oocytes, cytoplasmic chromosome condensation factors remain active, leading to the induction of PCC in the sperm nucleus [21]. The formation of 2PN without further development indicates sperm penetration but incompetence for complete activation of oocytes. A complete oocyte activation consists of a sequence of morphological and molecular events that include cortical granule exocytosis [22], resumption of meiosis, extrusion of the second polar body, formation of pronuclei, DNA synthesis, and mitotic division [23, 24]. The incompetence of complete activation of in vitro-matured oocytes may be a sign of cytoplasmic immaturity.
This line of evidence for incomplete cytoplasmic maturity was further supported by the rates of blastocyst formation of in vitro-matured oocytes in both IVF and ICSI groups. Cytoplasmic maturity is necessary to support cell cleavages and the formation of the blastocyst. The results of this study demonstrated that blastomere cell numbers in the mouse blastocyst derived from in vitro-matured oocytes was only half that observed in the in vivo-matured control groups. The decrease in cell number may be attributed either to retarded cell proliferation or cell death by apoptosis [25, 26]. The regulation of cell proliferation and cell death in the blastocyst is likely to be critical for later development because a critical number of blastomeres is required for normal postimplantation development. This may account for the low efficiency of animal production after embryo transfer.
Recent studies have shown that the quality of oocytes may be adversely affected by extended culture [3, 27]. In a study by Cortvrindt et al. [3] using a follicle range from 100–130 µm in diameter, a 12-day culture period provided the highest rate of MII oocytes; but there was no evidence of embryo preimplantation developmental capacity after fertilization of these 12-day cultured MII oocytes in that study. The present study was performed to determine whether there are differences in developmental capacities between oocytes that reach the MII stage after 10- and 12-day culture of preantral follicles with a diameter of 110–160 µm. The results from the first set of experiments showed that the nuclear maturation rates were similar between 10- and 12-day cultured oocytes. Furthermore, the cleavage rates did not show any differences between 10- and 12-day cultured oocytes either after IVF (56.1% versus 54.5%) or ICSI (72.1% versus 74.1%). However, the capacity for further development to the blastocyst was severely decreased in 12-day cultured oocytes compared with 10-day cultured ones (IVF: 40.7% versus 62.4%, respectively, P = 0.016; ICSI: 32.5% versus 57.1%, respectively, P = 0.035). Moreover, the blastocysts derived from 10-day cultured oocytes had higher numbers of blastomeres than those from 12-day cultured oocytes. These results show that the quality of oocytes may be adversely affected by extended culture in vitro, probably due to oocyte aging. Besides, a higher survival rate after ICSI, in 10-day cultured oocytes than in 12-day cultured oocytes, indicates that oocyte membrane properties change in vitro-matured oocytes. Indeed, the membrane became more difficult to heal after sperm injection into oocytes from a 12-day culture.
Considering the data presented in this study, we can conclude that in vitro-matured oocytes from mouse early preantral follicles can be fertilized by IVF or ICSI and that preimplantation development is possible. However, the preimplantation developmental competence in terms of capacity of cleavage and blastocyst formation was impaired in the in vitro-matured oocytes starting from preantral follicles. Fertilization by ICSI did improve the proportion of 2-cell formation compared with IVF. Ten-day culture of the preantral follicles with the size of 110–160 µm can give better developmental capacity to the blastocyst than a longer 12-day culture period. However, it was still significantly lower than in vivo-matured oocytes. These results suggest that the preimplantation developmental capacity of in vitro-matured oocytes from preantral mouse follicles under the conditions of the current culture system was not fully achieved, even though they developed to the MII stage in terms of nuclear maturation. Further studies should explore factors that are able to improve oocyte maturation and embryo preimplantation development in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by research grant BOF01112199 from the Bijzonder Onderzoeksfonds of the Ghent University, Belgium. 
2 Correspondence: Jun Liu, Infertility Center, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. FAX: 32 9 240 4972; jun.liu{at}rug.ac.be
Accepted: February 7, 2002.
Received: August 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1997; 56:1088-1096[Abstract]This article has been cited by other articles:
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