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Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development¹

Follicle Biology Laboratory,³ Free University of Brussels, 1090 Brussels, Belgium Fertility and IVF Unit,⁴ Assaf Harofeh Medical Center-Tel Aviv University, Tel Aviv 70300, Israel

ABSTRACT

Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6–12 mm) were retrieved 34–36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

cumulus cells, cyclic adenosine monophosphate, gamete biology, oocyte development, phosphodiesterases

INTRODUCTION

The competence of germinal vesicle (GV)-stage oocytes to undergo meiosis to the metaphase II stage and, on monospermic fertilization, to complete meiosis and ensure embryonic development generally is considered to be the key parameter of oocyte quality. In humans, GV-stage oocytes enclosed in antral follicles gradually acquire meiotic competence during the final phase of oocyte development. On retrieval from their follicles, fully grown, meiosis-competent, GV-stage oocytes spontaneously undergo nuclear maturation in vitro, progressing through the first meiotic division to reach the second metaphase. These oocytes, however, attain nuclear maturation before reaching optimal cytoplasmic maturity. Despite the fact that nuclear and cytoplasmic maturation of oocytes can proceed independently, both processes need to be coordinated to ensure developmental competence. The path to attain cytoplasmic competence includes buildup and storage of transcripts [1, 2], synthesis and accumulation of proteins [3], posttranslational modifications [4], and ultrastructural changes [5]. In primates, cytoplasmic maturation is acquired in fully grown oocytes during the later stages of follicle development [6].

From the results of assisted reproductive studies, it is known that in vitro-matured (IVM), GV-stage oocytes derived from unstimulated ovaries, or following controlled ovarian hyperstimulation (COH), can be fertilized by intracytoplasmic sperm injection (ICSI) [7–9]. These oocytes, however, progress to normal pre- and postimplantation embryonic development at lower rates compared with oocytes matured in vivo following ovarian stimulation [10, 11]. Therefore, it has been postulated that the impaired quality of these IVM oocytes is caused by incompletely developed cytoplasm.

Oocyte maturation is a complex process, and extrinsic factors strongly influence the intrinsic quality of the oocyte. Improvement of culture conditions may enhance ooplasm quality. Hypothetically, prematuration culture (PMC) of oocytes designed to retard nuclear maturation could allow time for ooplasmic maturation to catch up [12–14] and, therefore, better synchronize the nuclear and cytoplasmic compartments.

Interference of the cAMP pathway (specifically, maintenance of steady-state cAMP levels) provides an efficient means to retard nuclear maturation [15–17]. Cyclic AMP is synthesized by adenylate cyclases, and cAMP degradation to 5'-AMP is dependent on activation of phosphodiesterases (PDEs). Two of the 11 distinct families of PDE isoenzymes are differentially expressed in a cell-specific manner within the ovarian follicle (for review, see Conti [18]). Phosphodiesterase 4 is expressed in cumulus, mural granulosa (PDE4D), and thecal cells (PDE4B), and PDE3A is expressed in oocytes [14, 17, 19]. By using specific inhibitors of PDE3 isoenzyme, it has become clear that PDE3 plays an important role in regulating oocyte maturation in rodents [17, 20], cattle [21, 22], and primates [14, 23]. Consistent data on the reversibility of the PDE3 inhibition has been reported in animal species [21, 22, 24].

Phosphodiesterase 3-specific inhibitor (PDE3-I) has been added to oocyte culture media from animal species in an attempt to improve oocyte quality and embryonic development. The PMC for 24 h in medium containing PDE3-I improved oocyte developmental competence in mice [24]. In cattle, delaying nuclear maturation by applying PDE3-I during FSH-induced maturation enhanced the oocyte potential for embryonic development [25]. In humans, PDE3-I arrested meiosis in oocytes retrieved from small antral follicles. This artificial nuclear arrest was highly successful even when oocytes were collected 36 h post-hCG administration. Moreover, preliminary data suggest that reversal of this inhibition is feasible [14].

The present study tests the effects of nuclear arrest induced in vitro by PDE3-I on the developmental potential of immature human oocytes. By using oocytes collected from small antral follicles following COH, the oocyte capacity for nuclear maturation, fertilization, and embryonic development following the PDE3-I-induced meiotic arrest in vitro were investigated.

MATERIALS AND METHODS

Ethical Approval

The present research project was approved by the ethical committee and was in line with rules of the Declaration of Helsinki and Good Clinical Practice, project approval number 130/03. Written informed consent was obtained from patients to aspirate small follicles and to culture aspirated oocytes to test a new system for oocyte maturation.

Study Population and Patient Treatment

A prospective study was conducted that included all consecutive women subjected to in vitro fertilization (IVF)/ICSI treatment in the Assisted Reproductive Technology unit at the Assaf Harofeh Medical Center from November 2003 to March 2004. To minimize confounding factors, patients diagnosed with polycystic ovaries or poor response (n 4 mature follicles on ultrasound scan before oocyte pickup) were excluded from the study. Only one treatment cycle per patient was included in the analyses. Of 172 patients recruited (mean age ± SD, 31.8 ± 4.8 yr), 152 ranged from 21 to 35 yr of age, and 20 ranged from 36 to 42 yr of age.

Patient Treatment and Collection of Oocytes

Patients were treated by routine COH protocols using a combination of GnRH analogues (agonist: nafarelin acetate, 200 mg three times a day by intranasal spray, or triptoreline, 0.1 mg/day s.c. injection; antagonist: 0.25 mg of Cetrotide [Serono Laboratories] or Orgalutran [Organon]) and variable doses of gonadotropins (Menogen [Ferring], Gonal-F [Serono], or Puregon [Organon]) while being monitored carefully on the basis of serum hormone levels and vaginal ultrasound. An injection of 5000 IU of hCG (Chorigon; Teva) was administered when at least two lead follicles of 18 mm or greater were present. Oocytes were retrieved by transvaginal aspiration under ultrasound guidance 35–38 h after the hCG administration. Once all follicles greater than 12 mm were aspirated for routine IVF/ICSI treatment, CEOs were collected from small follicles (diameter, 6–12 mm) for the present study. Routine oocyte pickup from small follicles was performed using a 17-G oocyte aspiration needle (Avmed), applying an aspiration pressure of 80 mm Hg.

In Vivo-Matured Controls

Freshly collected, polar body (PB)-extruded oocytes, originating from follicles with a diameter from 7 to 12 mm, were used as in vivo-matured controls for IVM of oocytes, because they were retrieved from the same patients. Soon after retrieval, PB-extruded oocytes were stripped from their cumulus cells by chemical (40 IU/ml of hyaluronidase type VIII; Sigma) and mechanical means and then submitted to routine ICSI [26].

Culture of Oocytes

The GV-stage oocytes placed in culture consisted of two types of cumulus mass: those surrounded by a compact mass of three to five layers of corona cells (i.e., fully compacted cells), or those surrounded by a compact corona cell layer but with expanded layers of distal cumulus cells (i.e., moderate cell expansion).

The basal culture medium was minimum essential medium supplemented with 2 mM Glutamax-I, 0.4 mM glycine, 1 ng/ml of human insulin, 5 ng/ml of sodium selenite, and 5 µg/ml of human transferrin (all from Invitrogen-Rhenium) as well as 0.47 mM sodium pyruvate, 3.3 mM L-lactate, 100 ng/ml of Long R³ insulin growth factor-I, and 100 µM cysteamine (M-6500; all from Sigma-Aldrich), along with 10 mIU/ml of recombinant FSH (rFSH; Gonal-F; kindly donated by Ares Serono International) and 0.5% human serum albumin (10% HSA; HSA-solution; Vitrolife).

The IVM medium was constituted by the basal medium supplemented with 10 ng/ml of recombinant epidermal growth factor (EGF; Roche).

The PMC medium was composed of the basal medium plus Org9935, a specific PDE3-I (kindly provided by Organon). Org9935 was added at a 10 µM final concentration to the culture medium [14]. All cultures were carried out in a humidified atmosphere of 5% CO₂ in air at 37°C. All GV-stage oocytes were cultured singly in 20-µl droplets covered with mineral oil (Irvine Scientific, Zotal Ltd.).

Embryo Culture and ICSI

Sperm derived from a single donor were used for microinjection of all oocytes. Thereafter, injected oocytes were placed individually in separate droplets of G1 medium (Vitrolife) and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C. Fertilization was assessed 16–18 h postinjection. Embryonic cleavage and extent of fragmentation were assessed on Day 2 (42–46 h) and Day 3 (64–68 h) after ICSI.

Embryos containing any number of cells with blastomeres of equal or unequal size, with no apparent morphological abnormalities, and with less than 20% of total volume consisting of anucleated fragments were defined as "good morphology" embryos.

Embryo Spreading

Embryos were fixed at 66 ± 2 h (Day 3) post-ICSI for evaluation of nuclear status. Acid tyrode dissolved zona pellucida, and then Ca²⁺/Mg²⁺-free medium (EB-10; Vitrolife) disaggregated blastomeres. Blastomeres were observed continuously during spreading under an inverted phase-contrast microscope (CK40 Olympus) at 40x, 100x, and 200x. Nuclei were fixed onto a slide (catalog no. S1308; Oncor) using HCl/Tween-20 (0.01 N/0.1%).

Experimental Setup/Design

Effect of PMC period on the maturation capacity of oocytes After retrieval, CEOs were evaluated and randomly distributed for each patient in four culture conditions (Fig. 1): In the Control-CEO group, intact CEOs were placed in IVM medium and cultured for up to 48 h. In the Control-DE group, CEOs were immediately denuded (DE) after collection and cultured in IVM medium for up to 48 h. In the 24-h PMC group, intact CEOs were placed in PMC for 24 h, and subsequently, PDE3-I was removed by denuding GV-stage oocytes from the remaining cumulus cells and placing the denuded (DE) oocytes in fresh IVM medium for up to 48 h. In the 48-h PMC group, intact CEO were placed in PMC for 48 h, followed by the procedure of PDE3-I removal as used in the 24-h PMC.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Schematic diagram of the study for the PMC and IVM

The efficacy and safety of the PDE3-I in arresting nuclear maturation at the GV stage (88% after 48 h of culture) has been determined previously [14]. In that study, we observed that a proportion of oocytes (65%) had lost contact with neighboring cumulus cells, leading to total or partial spontaneous denudation within 24–48 h of CEO culture. In the present study, we removed the corona/cumulus cells of the oocytes after the arrest period with PDE3-I to start IVM with all oocytes having the same type of morphology (i.e., completely denuded) for a more controlled study and, therefore, included a control group of DE oocytes (Control-DE group).

Intact oocytes were classified according to nuclear maturational stage as GV, GV breakdown (GVBD), or PB after PMC and/or after IVM. Evaluation during the IVM period was performed up to 30 h (from 24 to 30 h) and up to 48 h (from 31 to 48 h) in culture.

Effect of PMC on fertilization and embryo development Mature oocytes with a PB extruded at the time of collection (in vivo controls) or matured in vitro in controls or following culture in PDE3-I-containing media were microinjected with a single spermatozoon. The ICSI was performed between 2 and 4 h after the first PB had been visualized. Afterward, normal fertilization (presence of two pronuclei), and embryo developmental stages were evaluated. On Day 3 post-ICSI, embryos of good quality were spread. The number and appearance of the nucleus/nuclei (intact or fragmented) for each blastomere were recorded.

Statistical Analysis

Differences in oocyte maturational stages, incidence of fertilization, cleavage and cleavage rate, embryo fragmentation degree, and embryo nuclear status were assessed by chi-square test (contingency table) using Fisher exact as a posttest. Differences in mean number of blastomeres were calculated using ordinary, one-way ANOVA, followed by Tukey post-hoc test. A level of P < 0.05 were considered to be statistically significant.

RESULTS

Effect of PMC Period on Maturation Capacity of Oocytes

Cumulus-enclosed oocytes (Control-CEO) had a higher maturation rate following IVM compared to DE oocytes (Control-DE; P > 0.05). The PDE3-inhibited oocytes (24-h and 48-h PMC groups), although having been denuded before undergoing IVM, also achieved a higher maturation rate compared to the Control-DE group, but this trend was not statistically significant. To obtain sufficient statistical power, we limited the number of conditions, thus increasing the number of subjects in two groups: 24-h PMC, and Control-DE.

Results in Table 1 represent data collected during the entire experimental period. A total of 251 GV-stage oocytes were distributed in the four culture conditions. Of oocytes exposed to PDE3-I, 99% and 97% had an intact GV at 24 and 48 h of culture, respectively. Following withdrawal from PDE3-I culture and denudation, oocytes underwent IVM. After 30 h, the proportions of GV and GVBD stages in the inhibitor groups did not differ significantly from those in controls. At this time, a PMC period for CEOs before IVM of DE oocytes improved the oocyte's ability to undergo nuclear maturation compared to immediate IVM of the Control-DE group. Extrusion of PB occurred in 67% of oocytes from the 24-h and 48-h PMC groups. In the Control-CEO group, 63% extruded the PB, compared to only 46% of the Control-DE group (24-h PMC group vs. Control-DE group; P = 0.01).

Of oocytes evaluated 48 h after IVM, those in the Control-DE group had the lowest maturation rates. This group had a significantly higher percentage of oocytes still blocked at the GV stage, which was almost twofold that which had undergone PMC (P = 0.005 vs. 24-h PMC) (Table 1). The capacity of the oocytes to resume meiosis in the PMC groups was comparable to that in the Control-CEO group, which was higher than that in the Control-DE group. At 48 h of IVM, the PMC and Control-CEO groups had at least 70% PB-extruded oocytes, whereas only 54% of oocytes in the Control-DE group had extruded their PBs. A PMC period by PDE3-I was beneficial to the nuclear maturation capacity of oocytes, because a significant difference existed between the 24-h PMC and Control-DE groups (74% vs. 54%, respectively; P = 0.01), in which oocytes underwent IVM stripped of somatic cells.

Oocyte Maturation in Relation to Cumulus-Corona Morphology

A comparison was performed to evaluate differences in maturation capacity of oocytes at the end of IVM in relation to cumulus-corona morphology at retrieval, classified either as fully compacted CEOs (Fig. 2a) or as CEOs with moderate cell expansion (Fig. 2h) (Table 2). At the end of the control culture, CEOs that presented moderate cell expansion at retrieval resulted in a higher proportion of PB-extruded oocytes (83%) compared to CEOs comprised of fully compacted cell layers (46%; P < 0.05). An equivalent result was observed even when oocytes with these two morphological classes were denuded before IVM in controls (Control-DE: 60% for fully compacted vs. 31% for moderately expanded; P < 0.05). In contrast, PB extrusion was proportionally similar between oocytes derived from both cumulus morphology classes that expanded when IVM was preceded by a PMC period (Table 2).

View larger version (59K): [in this window] [in a new window]   FIG. 2. Photomicrographs representing the developmental stages of oocytes derived from CEOs of distinct morphologies. a–c) CEO with fully compact cell layers at retrieval (a), at 24 h (b), and at 48 h (c) of PMC in the presence of PDE3-I. d) Oocyte with PB (arrow) visualized at 30 h after denudation and PDE3-I withdrawal. e) 2PN fertilization achieved after ICSI. f and g) Embryos developed to the 4-cell (f) and 6-cell (g) stages on Day 2 (f) and Day 3 (g) post-ICSI. h–m) CEO with moderate cell expansion is shown at retrieval (h) and at 24 h of PMC (i), when the oocyte became spontaneously denuded. Also shown is an oocyte with PB (arrow) visualized at 30 h following PDE3-I withdrawal (j). After ICSI, fertilization (k) was achieved, and embryos were developed to the 6-cell (l) and 8-cell (m) stages on Day 2 (l) and Day 3 (m) post-ICSI. Bar = 50 µm

The maturation capacity of oocytes derived from fully compacted CEOs was significantly improved in the 24-h and 48-h PMC groups compared to those in the Control-DE group (P < 0.05).

Effect of PMC on Fertilization and Embryo Development

The ICSI was performed on oocytes from the different treatment groups and embryos cultured to Day 3 (Fig. 2). A group of in vivo-matured oocytes from the same patients was considered to be an in vivo control to evaluate the quality of IVM oocytes.

A prematuration period of 24 or 48 h resulted in PB oocytes that were fertilized at similar rates to in vivo (67%) and in vitro CEO and DE controls (60% and 52%, respectively) (Table 3). Because of a higher maturation rate, however, the 24- and 48-h prematured oocytes yielded more fertilizable oocytes compared to conventional IVM of DE oocytes (36% and 47% of 2-pronuclei (2PN), respectively, over the total number of oocytes that underwent IVM vs. 27% of Control-DE; P > 0.05).

No significant difference was found in embryonic cleavage rates among IVM oocytes. Embryonic cleavage was higher (96%) in the in vivo-matured oocytes compared to all the IVM groups (range, 79–90%), but this difference did not reach statistical significance (Table 3). On Day 2 of embryo evaluation, the mean number of blastomeres was similar among all groups. On Day 3 of culture, however, embryos derived from in vivo-matured oocytes had higher numbers of blastomeres compared to all IVM groups, which was significant only when compared to the 48-h PMC group (P < 0.05).

When comparing cleavage rates among groups, it appeared that all IVM oocytes generated few embryos with optimal rates of cleavage (i.e., reaching the 4-cell stage on Day 2 and the subsequent 8-cell stage on Day 3). Although oocytes derived after PMC had been 24 or 48 h longer in culture (during the PMC) than oocytes from IVM controls, they were able to cleave further on Day 3 post-ICSI. None of these embryos, however, had optimal cleavage rates. Only 5% of the total number of embryos from control groups (Control-CEO and Control-DE) exhibited optimal cleavage, whereas in vivo-matured control oocytes generated 18% of embryos with optimal development rate (4-cell stage on Day 2 and 8-cell stage on Day 3) at that time.

The proportion of good-quality embryos (i.e., no apparent morphological abnormalities and few anucleated fragments) was similarly high in all groups (80%) on Day 2 post-ICSI except when oocytes had been in culture for at least 72 h before injection (64% in the 48-h PMC group) (Fig. 3). The 24-h PMC preceding IVM did not influence embryo quality on Day 2. This prematuration period, however, seemed to have exerted a beneficial effect on embryo quality on Day 3 of development (88% in the 24-h PMC group), which was comparable to in vivo-matured oocytes (83%) (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Embryos of good morphology on Days 2 and 3 post-ICSI of oocytes that underwent PMC and/or IVM or in vivo maturation. No differences were observed on Day 2 of evaluation. Different letters denote significant differences on Day 3 of evaluation (P < 0.05, chi-square analysis). Good morphology embryos refer to embryos of any cell number with blastomeres of equal or unequal size, with no apparent morphological abnormalities, and less than 20% of total volume consisting of anucleated fragments

Nuclear Constitution of Embryos

Blastomeres of embryos from all IVM groups and those derived from in vivo-matured control oocytes were spread to determine their nuclear constitution on Day 3 after ICSI. Overall, no significant differences were observed among IVM groups for the different parameters (Table 4). In all IVM groups, significantly fewer blastomeres had interphase nuclei compared to the in vivo-matured group (P < 0.01). From these interphasic blastomeres, the proportion of mononucleation, binucleation, and multinucleation did not differ among IVM groups. The difference was significant for the levels of mononucleation and multinucleation only when compared to the in vivo-matured groups (P < 0.05). A similar proportion of binuclear blastomeres existed among all the groups.

Fewer embryos contained at least 50% mononuclear blastomeres in all IVM groups compared to embryos from in vivo-matured controls (P < 0.05) (Table 5). Few embryos in each of the IVM groups had all mononuclear blastomeres: one 4-cell embryo in the 24-h PMC group (4%), none in the 48-h PMC group, one 7-cell embryo in the Control-CEO group (7%), and two embryos of 8- and 6-cells in the Control-DE group (12%). In contrast, 62% of embryos derived from in vivo-matured oocytes had all mononuclear blastomeres; of those, one was at the 4-cell stage, six at the 6-cell stage, three at the 8-cell stage, and one at the 9-cell stage.

DISCUSSION

Many questions remain about the mechanisms governing the final process of oocyte development. Therefore, how to devise IVM systems is not obvious on a rational basis. When oocytes are matured in vitro, quality is compromised, leading to defective embryonic development. In human IVF clinics, implantation rates from IVM cycles are decreased by half compared to those from IVF cycles, and IVM cycles present an increased incidence of early pregnancy loss. Studies of human and nonhuman primate oocytes ascribe some of the causes of failed embryonic development to faulty microtubular and chromatin organization and stability as well as to abnormal onset of embryonic genomic activation [27, 28]. It has been recognized that the deficient IVM outcome is mostly a result of the asynchrony between nuclear and cytoplasmic maturation. Supposedly, extending the period of GV-stage arrest in culture (i.e., PMC) might alleviate this asynchrony by allowing time for completion of cytoplasmic maturity [12, 24, 25, 29–34]. Inhibition of meiosis by kinase inhibitors has been used as an attempt to improve oocyte developmental competence; however, inconsistent results have been obtained in large mammalian oocytes [29–34] and proved not to be successful in human oocytes [12]. To date, PDE3-I has been applied successfully to promote in vitro oocyte cytoplasmic maturation in animal species [24, 25], but similar studies have not been conducted using human oocytes.

The difficulty in acquiring human oocytes for research has hindered the development of a reproducible IVM technology. A valuable source of oocytes for such research purposes is the GV-stage oocyte, retrieved from small antral follicles after COH, because these oocytes are easily accessible from assisted reproductive technology cycles. We used these oocytes to evaluate the effects of PMC with PDE3-I on oocyte health, maturation, and potential for development.

Applying the same culture conditions to CEO and DE oocytes can exert distinct effects on their maturation capacity, being inhibitory to one and stimulatory to the other, because of metabolic differences between these models [35, 36]. Besides, the regulation of oocyte maturation in CEO and DE oocytes is strongly affected by the balance between amino acids and substrates composing the media [37]. The culture media used in the present study apparently served CEO metabolism well during the maturation process, because these oocytes acquired higher maturation than DE oocytes derived from similar follicles.

Surprisingly, when CEOs were meiotically arrested in vitro by PDE3-I and denuded following withdrawal of the inhibitor to allow progression of meiosis, these oocytes achieved higher maturation rates compared to DE oocytes without PMC, reaching maturation rates similar to those achieved by CEOs. The precise mechanisms causing this beneficial effect are unclear. Among the numerous substances supplied by cumulus cells to oocytes (i.e., amino acids, energy substrates, purines, etc.), which could have been accumulated by the oocyte during arrest, cAMP is especially crucial. In cattle, intraoocyte cAMP levels are higher when CEOs, instead of DE, are cultured with PDE3-I in combination with forskolin, an adenylate cyclase stimulator [22]. The FSH is a physiologic stimulator of adenylate cyclase in follicular cells, and it increases cAMP concentrations, which might be propagated to the oocyte [38]. Taking into account that, in the present experimental conditions, rFSH was added in combination with the PDE3-I to the PMC and that human oocytes are more sensitive to the inhibitory effects of PDE3-I compared with bovine oocytes (<30% of oocytes inhibited), we speculate that intraoocyte cAMP levels might have increased during the PMC of CEOs.

In the present study, two morphological classes of retrieved CEOs were cultured: CEOs with moderate cell expansion, and CEOs with fully compacted cell mass. In controls, when CEOs with moderate cell expansion were immediately placed in culture after retrieval, the capacity of the oocytes for maturation was significantly increased compared to that of oocytes from fully compacted CEOs. It is assumed that CEOs with moderate cell expansion might have originated from more developed follicles, which acquired a higher level of responsiveness to the in vivo hCG signaling. Immature oocytes collected post-LH/hCG administration in vivo acquired higher maturation rates in vitro compared with oocytes retrieved before hCG administration [14, 39]. The LH/hCG effect is strongly associated with a rise in cAMP levels in somatic cells of the follicle [15]. It might be that cAMP production was elevated in the expanded CEOs in response to hCG, providing the oocyte with a rise in the level of this nucleotide. Perhaps the CEOs with expanded cells had already been sensitized by paracrine factors emanating from mural granulosa cells within the follicles in response to LH. This is supported by the fact that LH/hCG indirectly causes cumulus expansion and oocyte maturation within preovulatory follicles, partly by inducing expression of EGF-like growth factors in mural granulosa cells [40, 41]. The present findings confer conditional support to the premise that the in vivo effect of hCG on small antral follicles influences oocyte maturation in vitro. It would be interesting to acquire information regarding the correlation between the morphology of the immature CEO at retrieval and subsequent embryonic development, but our sample was too small to allow any significant conclusions on this matter.

A PMC period significantly benefited the CEO of fully compacted cell mass, and these oocytes attained higher maturation rates compared to those in controls. This contributed to an increased number of fertilizable oocytes. The prematuration group resulted in at least 10% more fertilized oocytes (over the total oocytes that underwent IVM) compared to the conventional IVM of DE oocytes. This suggests that a PMC brought about positive changes in factors in those CEOs related to oocyte nuclear maturation mechanisms. Whether this is caused by the increase in cAMP accumulation in the oocyte by the presence of PDE3-I or by cytoplasmic changes induced through a PMC of CEOs needs further research.

To investigate whether PMC period with PDE3-I could interfere with oocyte quality following IVM, ICSI was performed and embryos cultured for up to 3 days. It was shown that, although a period of arrest improved maturation rates, it did not significantly improve fertilization rates of human oocytes, in contrast to the results of an earlier study performed in mouse oocytes using the same PDE3-I [24]. Several biological differences in oocyte development between the species might account for the discrepant results. These include differences in follicular environment and temporal differences between the species to build up cellular landmarks for acquisition of meiotic, zygotic, and embryonic competencies.

On Day 2 of development after ICSI, a high percentage of good-quality embryos derived from IVM oocytes was observed. On Day 3, however, it seemed that the 24-h PMC resulted in a high proportion of embryos with less fragmentation, similar to the in vivo-matured group and superior to the other in vitro groups. The embryonic cleavage rate was not improved by the prematuration period. Although the number of embryos in each group was small, the results indicate that improvement of the PMC and IVM culture media holds promise. The culture media formulated for the present experiment had similar supplements throughout all oocyte culture steps except the addition of EGF in the IVM medium. The PMC composition likely needs supplements specifically to sustain the development of the immature oocyte that are distinct from supplements needed for the oocyte nuclear maturation process. Additionally, adjustment of the PMC to sustain oocyte-granulosa cell interactions up to and during IVM might improve the outcome. Morphological and functional analyses have indicated that high oocyte developmental competence is accompanied by persistence of junctional communications between cumulus cells and oocyte [25, 42]. Although the present study did not specifically analyze gap junctional communication, a proportion of the CEOs clearly had lost cumulus-oocyte contact by the end of the 24-h PMC, and this proportion increased after the 48-h PMC. In some CEOs, although the cells were still surrounding the oocytes, they were easily removed during mechanical denudation. It seems that FSH stimulation results in a retraction of transzonal projections in vivo [43] and does not effectively prevent disruption of gap junctions in vitro [25]. Modifications of our culture media, such as performing coculture with mural granulosa cells and substituting the rFSH by forskolin [44] or invasive adenylate cyclase, could prevent further disruption of gap junctions between oocyte and cumulus-corona cells. Furthermore, in the present experiment, as a first step to evaluate our system, we studied oocytes collected post-hCG administration. The endocrine environment of these oocytes differs from unstimulated or stimulated follicles before hCG induction of ovulation. Applying the PMC system to oocytes collected before hCG administration might improve oocyte-cell connections and oocyte developmental potential.

Presumably, to mimic the in vivo environment more closely, conditions could be created to overcome gradually the inhibitory effect of the arrester rather than removing it abruptly. At present, it is unknown which stimulatory substances can gradually overcome the potent nuclear arrest by this specific PDE3-I in human oocytes. In the bovine IVM model, oocytes are less sensitive to the action of PDE3-I, so FSH can override the effects of the PDE3-I, delay the maturation process, and increase the blastocyst rate as well as blastocyst cell number [22].

In the present study, all oocytes were collected from follicles of 12 mm or less in diameter. The differences in the intrinsic quality of embryos generated from the in vivo-matured and IVM oocytes from the small follicles (diameter, 12 mm) were significant. The latter had embryos that more frequently demonstrated disturbed karyokinesis. It is uncertain whether the impaired quality of these oocytes is inherent or also caused by poor in vitro conditions. The fact that modifying culture conditions in the present experiment could enhance the maturation rate of IVM DE oocytes is encouraging. The next step is to evaluate further how this beneficial effect can be improved and translated into better embryonic development if the interactions between somatic cells and oocyte are maintained during IVM.

In conclusion, the present results provide a proof of concept for a novel culture approach in human IVM, and add new insights regarding the importance of the in vitro conditions for oocyte maturation. Overall, PMC with PDE3-I was beneficial to the maturation capacity of human oocytes, without affecting subsequent embryonic development. Using a specific inhibitor to control PDE3 activity may be a significant approach to adapt culture conditions further and, thus, to improve cytoplasmic maturation of immature human oocytes.

ACKNOWLEDGMENTS

The authors wish to thank the embryologists and medical staff at the Infertility Unit, Assaf Harofeh Medical Center-Tel Aviv University, for their kind collaboration to this project and Dr. David Keefe for critical reading of this manuscript.

FOOTNOTES

¹ Supported in part by grants from Novo Nordisk A/S.

² Correspondence: D. Nogueira, Follicle Biology Laboratory, Free University of Brussels (Vrije Universiteit Brussel), Laarbeeklaan, 101, Brussels 1090, Belgium. FAX: 32 2 477 50 60; daniela_nogueira{at}yahoo.com

Received: 1 February 2005.

First decision: 21 February 2005.

Accepted: 28 September 2005.

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