Maturation of human oocytes in vitro and their developmental competence.

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro.

The response of murine, bovine and human oocytes to pure recombinant preparations of human follicle stimulating hormone (rFSH) and luteinizing hormone (rLH) for meiotic maturation and subsequent developmental competence in vitro were examined in the present experiments. Maturation of immature bovine oocytes to the metaphase II stage was significantly increased by the addition of 1 IU/ml of rFSH in combination with either 1 IU/ml rLH or 10 IU/ml rLH. Similarly, embryonic development to the blastocyst stage was improved in bovine oocytes treated with a 1:10 combination of rFSH:rLH. However, no significant difference was observed in the number of inner cell mass or trophectoderm cells of the resulting blastocysts. Although the increased maturation to metaphase II was not significant, human embryonic developmental competence was improved by maturing oocytes in the presence of a 1:10 ratio of rFSH:rLH as only those oocytes exposed to a 1:10 ratio of rFSH: rLH during maturation showed normal cleavage patterns beyond day 2. In addition, 1 IU/ml rFSH and 1 IU/ml rLH increased the expression of oocyte proteins in human oocytes. The inclusion of recombinant gonadotrophins, either singly or in combination, had no significant effect on the maturation, fertilization or embryonic development of in-vitro matured mouse oocytes. These data provide support for the responsiveness of human and bovine oocytes to gonadotrophins in vitro and the need to consider variations in the relative concentrations for optimization of oocyte developmental competence.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.

Oocyte maturation.

Primary oocytes recovered from small and growing follicles of > or = 3 mm in the ovaries of untreated women, can be matured in vitro, will fertilize and develop in vitro, and when transferred to the patient, develop to term. However, the implantation rate of cleaved embryos has been disappointingly low and when embryos are allowed to develop beyond the 4-cell in vitro, retardation of development and blockage is frequently observed, with relatively few embryos developing to blastocysts. We have devised new culture systems for human embryos to enable high rates of development of in-vivo matured oocytes to blastocysts within 5-6 days of culture, and high implantation rates of these blastocysts when they are transferred to the patients' uterus. These culture systems are now being used for in-vitro matured oocytes. In order to determine whether embryo developmental competence could be improved, a number of factors were examined. Treatment of patients with pure follicle stimulating hormone (FSH) early in the follicular phase, or treatment with oestrogen prior to oocyte recovery, had no apparent effect on any parameters of oocyte developmental competence. There was no indication that a medium made specifically for human oocyte maturation improved oocyte developmental competence. Nuclear and cytoplasmic changes in oocytes matured in vitro appear to be similar to that in vivo, although some lack of synchronization in completing maturation is evident. It is possible that follicles of < 10 mm diameter in the human contain developmentally-incompetent oocytes. However, the development to term and birth of normal babies from germinal vesicle stage oocytes recovered from small follicles and matured in vitro, suggests that further research will identify the factors necessary to improve embryo developmental competence. The application of immature oocyte collection (IOC) and in vitro maturation (IVM) as an alternative to ovulation stimulation with high doses of gonadotrophins for in-vitro fertilization (IVF), remains a priority for research in human medicine.

Functional integrity of granulosa cells from polycystic ovaries.

OBJECTIVE: The polycystic ovarian syndrome is frequently associated with human infertility and is a partially characterized syndrome of unknown aetiology. The aim of this study was to describe the functional integrity of granulosa cells from polycystic ovaries. PATIENTS: Follicular aspirates were collected from polycystic ovaries of ovulatory (n = 24) and anovulatory (n = 7) patients. Follicular aspirates were also collected from normal ovaries of untreated (n = 24) and superovulated (n = 10) subjects. All patients were enrolled for the recovery of their oocytes for in vitro maturation and fertilization. MEASUREMENTS: FSH receptors and apoptosis were measured in the granulosa cells of the different patients. FSH-stimulated oestradiol and LH-stimulated progesterone production by granulosa cells of the different patients were also measured. RESULTS: The binding of 125I-labelled human recombinant FSH to granulosa cells from anovulatory subjects with polycystic ovaries was significantly higher than that found in granulosa cells from normal (180%) and superovulated (163%) ovaries. However, the ligand binding to granulosa cells from ovulatory subjects with polycystic ovaries was not significantly higher than that found in normal granulosa cells. Also, granulosa cells obtained from anovulatory subjects with polycystic ovaries cultured with FSH produced more oestradiol than normal granulosa cells but oestradiol production was similar to that of granulosa cells from superovulated ovaries (mean +/- SEM, 224.94 +/- 22.02, 24.23 +/- 2.92, 211.87 +/- 50.39 nmol/l/24 h, respectively). Flow cytometric analysis showed that the proportions of viable and apoptotic granulosa cells (mean +/- SDM, 70 +/- 5 and 7 +/- 1%, respectively) were similar in normal subjects and in those with polycystic ovaries. CONCLUSION: We conclude that most of the granulosa cells of polycystic ovaries are healthy and non-apoptotic, expressing high levels of FSH receptors and highly responsive to this hormone in culture. These data provide direct evidence that most of the follicles of polycystic ovaries are not atretic.

The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro.

A dose-dependent inhibition of meiotic maturation and embryonic development was observed in both cumulus-enclosed and cumulus-denuded murine oocytes following incubation in the presence of 10, 20, 40, 60 and 80 microM testosterone for 18 h in vitro. Maturation to metaphase II was enhanced in cumulus-enclosed oocytes following maturation in the presence of human pre-ovulatory mural granulosa cells. However, maturation of cumulus-denuded oocytes was enhanced only when oocytes were cultured on a monolayer of human polycystic ovarian granulosa cells. The presence of cumulus cells had a significantly positive effect on both oocyte maturation (P = 0.002) and embryonic development (P < 0.001). In addition, the presence of follicular cells during maturation improved the number of fertilized oocytes reaching the blastocyst stage. These data indicate that the exposure of immature murine oocytes to testosterone during maturation significantly reduces their ability to mature and undergo normal embryonic development.