In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection

A. De Vos1, H. Van de Velde, H. Joris and A. Van Steirteghem

Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected ~4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.

Key words: embryo cleavage/fertilization/intracytoplasmic sperm injection/metaphase-I oocyte/oocyte in-vitro maturation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The combination of gonadotrophin-releasing hormone agonists and human menopausal gonadotrophin (GnRHa/HMG) is a standardized ovarian stimulation protocol routinely used in many in-vitro fertilization (IVF) centres (Ubaldi et al., 1995Go; Grimbizis et al., 1998Go). A large number of oocytes can be retrieved after GnRHa/HMG superovulation. However, not only the number of oocytes, but also the maturity of the retrieved oocytes is important for the success of artificial reproductive techniques. Enzymatic and mechanical removal of the cumulus and corona cells prior to intracytoplasmic sperm injection (ICSI) reveals that a proportion of the retrieved oocytes in gonadotrophin-stimulated cycles are immature, either at the metaphase-I (absence of both a germinal vesicle and a first polar body) or at the germinal vesicle (GV) stage. Our experience is that ~4% of the retrieved oocytes are at the metaphase-I (MI) stage, ~11% are at the GV stage and ~85% are at the metaphase-II (MII) stage. Attempts have been made to in-vitro mature these MI and GV oocytes obtained from stimulated cycles [including the human chorionic gonadotrophin (HCG) stimulus for maturation in vivo] and to perform second-day ICSI on matured oocytes. Only two deliveries (Nagy et al., 1996Go; Edirisinghe et al., 1997Go) resulting from such immature oocytes have been reported up till now. Some pregnancies derived from immature oocytes harvested from gonadotrophin stimulated cycles in the absence of HCG (Jaroudi et al., 1997Go; Liu et al., 1997Go) need to be differentiated from the above mentioned work.

At present, the available data with regard to ICSI on matured MI oocytes retrieved from stimulated cycles are limited. A recent report has indicated a reduced fertilization rate for MI oocytes matured in vitro for 8 h compared to MII oocytes at the moment of oocyte retrieval (Bonada et al., 1996Go). No data are available on embryo cleavage and embryo quality resulting from such in-vitro matured MI oocytes. The present study includes 896 ICSI cycles and fertilization rate and developmental capacity of in-vitro matured MI oocytes are evaluated and compared to those of mature oocytes at the moment of oocyte denudation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
This study includes 896 ICSI cycles performed in our centre between January 1991 and December 1996, involving 8803 injected MII oocytes and 1210 injected MI oocytes. In each cycle at least one in-vitro matured MI oocyte was injected. These cycles were selected where the MI oocytes matured to MII within 4 h and could still be injected on the day of oocyte retrieval. ICSI was performed with spermatozoa from the ejaculate in 774 (86.4%) cycles. ICSI was performed with spermatozoa retrieved from the epididymis in 49 cycles (5.5%) and with testicular spermatozoa in 73 cycles (8.1%).

Ovarian stimulation and oocyte handling
Ovarian stimulation was performed with GnRHa, HMG and HCG as previously described (Ubaldi et al., 1995Go; Grimbizis et al., 1998Go). Thirty-six hours after HCG administration, oocyte retrieval was carried out by guided vaginal ultrasound. The procedure for the removal of surrounding cumulus and corona cells, including an enzymatic hyaluronidase step followed by mechanical denudation, has also been described previously (Van de Velde et al., 1997Go). Denuded oocytes were observed under an inverted microscope at x200 magnification. Observations include assessment of the zona pellucida and the oocyte, and the presence or absence of a germinal vesicle or a first polar body. Immature MI oocytes are defined as those oocytes containing no germinal vesicle but not yet having extruded the first polar body. From each patient, the oocytes that had extruded the first polar body at the moment of denudation were subjected to intracytoplasmic sperm injection, either immediately or later depending on the workload of the day. MI oocytes were left in 25 µl droplets of B2 medium covered with lightweight paraffin oil (M-8410, Sigma, St Louis, USA) for further maturation. They were kept at 37°C in an atmosphere of 5% O2, 5% CO2 and 90% N2. Those immature oocytes that matured to the MII stage within ~4 h were also subjected to ICSI on the day of oocyte retrieval.

ICSI procedure and the evaluation of injected oocytes
The detailed procedure for ICSI has been described previously (De Vos et al., 1997Go; Joris et al., 1998Go; Van Steirteghem et al., 1998Go). After the injection procedure, the oocytes were washed and transferred into 25 µl microdroplets of B2 medium covered with lightweight paraffin oil and kept at 37°C in an atmosphere of 5% O2, 5% CO2 and 90% N2. The injected oocytes were examined for fertilization ~16–18 h after ICSI (Nagy et al., 1994Go). Normal fertilization was considered to have occurred when two clearly distinct pronuclei (2PN) were present. The number of oocytes showing one pronucleus (1PN) or abnormal fertilization, i.e. appearance of three pronuclei (3PN), was also recorded. Neither type of embryo resulting from 1PN or 3PN oocytes were transferred to the patients. After another 24 h in-vitro culture, the cleavage characteristics of the fertilized oocytes were evaluated: numbers and size of blastomeres and the presence of anucleate cytoplasmic fragments were recorded (Albano et al., 1998Go). Type A, or excellent quality, embryos were defined as embryos in which all blastomeres were of an equal size or, if of non-equal size, were without anuclear fragments. Type B, or good quality, embryos had blastomeres of equal or unequal size and a maximum of 20% of the volume of the embryo filled with anucleate fragments. In the third category, type C or fair quality embryos, anucleate fragments were present in 20–50% of the embryo. Type D, or poor quality, embryos had anucleate fragments present in >50% of the volume of the embryo. If embryos of sufficient morphological quality (type A, type B and type C) were available, two or three embryos were replaced into the uterine cavity ~48 h after the injection. Extra embryos of good morphological quality (type A and type B) were cryopreserved (Van der Elst et al., 1997Go; Joris et al., 1998Go).

Statistical analysis
A paired Student's t-test was performed to analyse the results of fertilization and cleavage at the 5% level of significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Between January 1991 and December 1996, 2804 ICSI cycles with at least one MI oocyte at the moment of oocyte denudation were carried out. Of these, 74.3% contained a MII oocyte (i.e. a mean of 11.2/cycle) and 11.2% contained a MI oocyte (i.e. a mean of 1.7/cycle).

In only 896 cycles, maturation of at least one MI oocyte to the MII stage within 4 h of in-vitro culture was achieved and sibling MII ooyctes could be injected in parallel. In all, 1260 out of 4716 MI oocytes (or 26.7% of all MI oocytes) extruded the first polar body within 4 h of in-vitro culture. Of these in-vitro matured oocytes, 1210 were injected. Within the same ICSI cycle, 8803 sibling oocytes, mature at the moment of oocyte denudation, were injected. Table IGo represents the comparison of fertilization and embryonic development after ICSI in the two groups of oocytes. Both groups of oocytes survived microinjection equally well: 90.5 and 89.3% survival for matured MI oocytes and MII oocytes respectively. Approximately 16 h after microinjection, a clear difference in fertilization rate was observed. The fertilization rate of in-vitro matured oocytes was significantly lower (52.7%) than that of mature MII oocytes (70.8%, P < 0.001). For the two groups of oocytes, no statistical differences were observed in the numbers of 1PN oocytes or abnormally fertilized oocytes.


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Table I. Comparison of fertilization and embryonic development of mature oocytes at the moment of oocyte denudation and in-vitro matured metaphase-I oocytes. Data represent mean values (%) ± SEM
 
When normal fertilization ensued, the percentages of cleavage into embryos of transferable quality were similar for both groups of oocytes: 82.5% of the in-vitro matured oocytes cleaved with <50% fragmentation and 83.4% of the mature oocytes. The cleavage quality was comparable for both groups. Only a slight difference in the numbers of good quality embryos was observed: 47.7% good quality (type B) embryos were obtained in the in-vitro matured oocyte group as opposed to 52.8% in the MII oocyte group (P < 0.05). The numbers of excellent (5.7 and 7.0%) and fair quality (17.6 and 15.3%) embryos were not significantly different for the two groups.

For embryo transfer, priority was always given to embryos derived from mature MII oocytes. Forty-five percent of the embryos in this group were transferred, while only 35.2% (P < 0.001) of the embryos derived from in-vitro matured oocytes were transferred. On the other hand, similar numbers of supernumerary embryos were frozen in both groups (22.2 and 21.3%). The majority of transfers involving embryos derived from MI oocytes were mixed transfers with embryos derived from MII oocytes. Fifteen transfers involved only embryos derived from in-vitro matured oocytes. These were 11 single embryo transfers and four transfers of two embryos. Of the latter, one resulted in a singleton pregnancy and the birth of a healthy baby.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present retrospective study, fertilization and embryo cleavage of microinjected in-vitro matured MI oocytes was compared to those of sibling mature MII oocytes. Indeed, after controlled ovarian stimulation, oocytes are collected at various stages of meiotic maturity. In-vitro culture allows some immature oocytes to resume meiosis so that they become available for microinjection. It is known, however, that in conventional IVF fertilization rates drop when MI oocytes require a longer incubation time in order to reach the MII stage (Veeck, 1988Go). For this reason, we selected only those MI oocytes that reached maturity in 4 h of in-vitro culture. An additional advantage is that microinjection can then be performed with fresh semen instead of 1-day-old semen, possibly favouring better fertilization results. Of all the MI oocytes, 26.7% matured to MII within 4 h of in-vitro culture in B2 medium. This figure correlates well with a previous report in which 24.5% of MI oocytes matured to MII within 4 h (Junca et al., 1995Go).

This study involves a large series of 896 ICSI cycles, including 1210 injected matured MI oocytes and 8803 sibling MII oocytes. In-vitro matured MI oocytes displayed lower fertilization rates than oocytes already mature at the moment of oocyte denudation. The fertilization rates observed in this study (52.7 and 70.8% respectively) correlate well with the ones obtained in a previous report on ICSI fertilization rates of MI oocytes matured in vitro for 8 h (Bonada et al., 1996Go), also indicating reduced fertilization rates for these oocytes as compared with oocytes that were at the MII stage at the moment of oocyte retrieval (42.9 and 67.4% respectively, report on 145 ICSI cycles, including only 87 injected matured MI oocytes and 1154 injected mature MII oocytes) a low fertilization rate for in-vitro matured MI oocytes has been reported in only seven out of 29 matured MI oocytes fertilized, i.e. 28% (Junca et al., 1995Go). No difference in fertilization rate between matured MI oocytes and mature MII oocytes was observed (Bergère et al., 1997Go). However, from these small numbers of oocytes (22 initially MI oocytes and 27 MII oocytes) no definite conclusions can be drawn.

The reduced fertilization rate of in-vitro matured oocytes can be explained by the cytoplasmic immaturity of these oocytes in contrast to their nuclear maturation. Nuclear maturation involves processes reinitiating meiosis from prophase-I arrested oocytes and driving meiotic division up to the MII stage, at which point meiosis is again arrested until fertilization. Nuclear maturity of oocytes can easily be evaluated by the presence of the first polar body. Oocytes reaching the MII stage may require an additional period of maturation in order to be activated promptly by the fertilizing spermatozoon (Lopata and Leung, 1988Go). This process of cytoplasmic maturation is much less well understood and at present no markers are available to evaluate the maturity of the cytoplasm of the oocyte. As a result, no direct evidence exists with regard to the time period needed for cytoplasmic maturation after the extrusion of the first polar body. However, complete cytoplasmic maturation seems to be required for egg activation, fertilization and further embryonic development. Indeed, it has been demonstrated that when insemination of in-vitro matured bovine oocytes was delayed for 8 h, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after MII arrest (Dominko and First, 1997Go). In the present study, only MI oocytes that matured within 4 h after denudation or oocyte retrieval were included. If nuclear maturity was observed within this time interval, the oocytes were injected immediately. No exact time recordings of complete nuclear maturation were made. The in-vitro matured MI oocytes were therefore injected at different unknown time points after extrusion of the first polar body. Some oocytes might have completed cytoplasmic maturity, others might not have reached completion of this process. The latter oocytes might be responsible for the reduced fertilization rates observed in in-vitro matured MI oocytes. The additional period of maturation needed by oocytes reaching the MII stage in order to be promptly activated by the fertilizing spermatozoa remains unknown. Exact timing of polar body extrusion and injection at different time points after this process should reveal on optimal injection time, resulting in improved fertilization rates, possibly correlated with completed cytoplasmic maturation. However, this type of experiment would be very time-consuming.

Immature human oocytes retrieved from stimulated cycles are capable of in-vitro maturation and subsequent fertilization can be achieved by means of conventional IVF (Veeck et al., 1983) or ICSI (Nagy et al., 1996Go; Edirisinghe et al., 1997Go). However, the capacity for normal development of fertilized concepti from these oocytes has not been well documented, either for GV stage oocytes or for MI oocytes. The present study indicates that in-vitro matured MI oocytes, if fertilized, do not display higher rates of cleavage arrest than their sibling MII oocytes (82.5 and 83.4% total cleavage per 2PN oocyte respectively). In a previous study (Junca et al., 1995Go), fertilized in-vitro matured MI oocytes (n = 7) entered normal cleavage, also indicating that having the potential to be fertilized, in-vitro matured MI oocytes do not differ from original MII oocytes in their capacity for embryonic development. Also, the resulting embryo quality observed in this study does not really differ between the two groups of oocytes.

It is important to mention that the in-vitro matured MI oocytes studied in this report need to be differentiated from `true' in-vitro maturation where oocytes have been recovered from small antral follicles without the benefit of gonadotrophin stimulation (Trounson et al., 1994Go; Russell, 1997Go). In stimulated cycles, the maturation process is initiated in vivo and completed in vitro, whereas in unstimulated cycles the whole maturation process takes place in vitro. There seems to be an important difference between the capabilities of immature oocytes that have been recovered following an in-vivo stimulus for maturation and those that have truly been matured in vitro. Live births have been obtained from `true' in-vitro matured oocytes (Cha et al., 1991Go; Trounson et al., 1994Go; Barnes et al., 1995Go; Russell et al., 1997Go) but the developmental competence of these embryos is severely retarded when compared to embryos resulting from in-vivo maturation (Trounson et al., 1994Go, 1997Go; Barnes et al., 1996Go). Moreover, endometrial priming is a significant aspect of the clinical application of immature oocyte retrieval in combination with in-vitro maturation and transfer of the resulting embryos within the same cycle (Russell, 1997Go).

Because similar embryo quality was obtained with in-vitro matured MI oocytes and mature MII oocytes retrieved from the stimulated cycles reported here, the injection of matured MI oocytes might be important, especially when few oocytes are available, in order to increase the number of embryos available for transfer or cryopreservation. It must be added however that in this study the implantation rate of embryos resulting from in-vitro matured MI oocytes is rather low (5.3%) compared to the overall implantation rates obtained with embryos resulting from mature MII oocytes (~20%). It is unknown whether this phenomenon is intrinsic to the embryos or whether the receptivity of the endometrium is different depending on the percentage of immature oocytes retrieved during the cycle. Moreover, concern about the safety of in-vitro oocyte maturation followed by embryo transfer should be voiced. Gonadotrophin stimulation is known to result in a higher incidence of gross meiotic aberrations than is the case for unstimulated cycles (Gras et al., 1992Go; Munné et al., 1993Go; Delhanty et al., 1997Go). However, in-vitro maturation of oocytes from unstimulated cycles leads to an incidence of meiotic aberrations similar to that for in-vivo matured oocytes (Racowski and Kaufman, 1992). The combined effect of ovarian stimulation and in-vitro maturation of immature oocytes retrieved from these stimulated cycles on meiotic aberrations remains elusive. In the present report, the only pregnancy obtained resulted in the birth of a healthy baby when embryos derived from in-vitro matured MI oocytes are replaced. No definite conclusions on the incidence of pregnancy loss can be drawn from just this one case.


    Acknowledgments
 
The authors wish to thank the clinical, paramedical and laboratory staff at the Centre for Reproductive Medicine, and especially colleagues at the micro-injection laboratory. Furthermore, we are grateful to Frank Winter of the Language Education Centre at our University for correcting the manuscript and to Marie-Paule Derde from the Biostatistics Department for statistical advise. This work was supported by grants from the Belgian Fund for Medical Research.


    Notes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 26, 1998; accepted on March 18, 1999.