1 Instituto Valenciano de Infertilidad and 2 Department of Paediatrics, Obstetrics andGynaecology, Valencia University School of Medicine, Valencia, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: blastocyst/embryo co-culture/immature oocyte/intracytoplasmic sperm injection/oocyte maturation in vitro
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The technique of maturation in vitro of human oocytes is an attractive option for the treatment of infertility. It would enable a decrease in the use of ovarian stimulation drugs in normo- and anovulatory patients, especially those with polycystic ovaries (PCO), who are at risk of developing ovarian hyperstimulation syndrome. Another possible application is related to cryopreservation. It is known that the freezing of oocytes in the metaphase II (MII) stage damages the genetic material. Oocytes in germinal vesicle (GV) stage have their DNA protected by the nuclear membrane, and animal studies have shown the protective effect of the surrounding cumulus cells (Pellicer et al., 1988). Thus, freezing immature oocytes may be an excellent alternative for patients who wish to postpone their reproductive function, for women undergoing treatment with chemotherapy or radiation for cancer, or for the creation of banks for ovum donation.
Although some work has already been carried out in humans, there has been no systematic analysis of the subject. Acceptable rates of maturation, fertilization, embryonic development and term pregnancies have been reported employing immature oocytes from stimulated cycles (Veeck et al., 1983; Nagy et al., 1996
; Jaroudi et al., 1997
). Similarly, pregnancies and newborn infants have been obtained after maturation in vitro of oocytes recovered following ovariectomies (Cha et al., 1991
; Cha and Chiang, 1998
), from PCO patients (Trounson et al., 1994b
; Barnes et al., 1995
), or from women undergoing intracytoplasmic sperm injection (ICSI) in natural cycles (Russell et al., 1996
). These results show that, if oocyte retrieval procedures are optimized and adequate techniques for maturation in vitro and fertilization are developed, it may be possible to consider the use of immature oocytes in a clinical setting.
Healthy follicles measuring 25 mmreferred to as selectable folliclesare observed at all stages of the cycle. Those present during the late luteal phase constitute the population from which the follicle destined for ovulation during the subsequent cycle will be selected (McNatty et al., 1983; Gougeon, 1996). The newly selected follicle belongs to the class of follicles measuring 58 mm, and its size increases greatly during the follicular phase by cellular multiplication and accumulation of fluid in the antrum. Thus, from a clinical standpoint it has been considered that the dominant follicle can be easily recognized by ultrasound when its diameter has reached 10 mm (Fauser and van Heusden, 1997
).
The intrafollicular steroid milieu of the follicle changes dramatically when selection has been completed and dominance begins. Healthy and atretic follicles have a similar milieu characterized by a low oestrogen/androgen ratio, although the concentration of androgens present in healthy follicles is low because there is only limited expression of enzymes such as P450scc and P45017 hydroxylase (Sasano et al., 1989
; Tamura et al., 1992
). When selection takes place, there is a shift in oestrogen/androgen ratio in the dominant follicle, while the other follicles remain androgenized and become atretic. Thus, although the androgen influence does not disappear from the non-dominant follicles, the period of exposure to androgens and the possible (unknown) increase in androgen concentrations during the follicular phase may cause a degree of oocyte damage.
In order to define the optimal conditions for the use of human immature oocytes in clinical practice, we have divided the technique into several distinct steps: oocyte retrieval, storage, maturation in vitro, fertilization and development, and implantation. This study explores the first step. Our hypothesis was that the time of ovum retrieval, whether before or after obvious follicular selection, may make a difference to the quality and developmental potential of human oocytes following in-vitro maturation. To test our hypothesis, a prospective study was designed which employed natural cycles from volunteer donors.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Follicle aspiration was performed by ultrasound-guided transvaginal puncture, employing regular IVF needles (Swemed Laboratories, Billdal, Sweden; 1.6x350 mm, 16 g) and 80 mmHg aspiration pressure. The system was previously washed with flushing heparinized medium (Medicult, Copenhagen, Denmark), and the follicular fluid collected in 2 ml of the same medium in sterile plastic tubes.
Oocytes were examined under the dissecting microscope and classified according to cumulus appearance as type 1, with few surrounding granulosa cells; type 2, with a moderate number of granulosa cells; and type 3, with several layers of granulosa cells surrounding each oocyte.
Oocytes were cultured in M-199 medium (Sigma, St Louis, MO, USA), supplemented with 0.4% HSA and 0.33 mM pyruvate, at 37°C in a 5% CO2 atmosphere. Hormones were added as follows: 0.075 IU/ml recombinant follicle stimulating hormone (FSH) (Gonal-F; Serono Laboratories, Madrid, Spain); 2 ng/ml recombinant endothelial growth factor (EGF) (Sigma); 0.5 IU/ml human chorionic gonadotrophin (HCG) (Profasi; Serono); and 1 µg/ml oestradiol (Sigma). After 2436 h, cumulus cells were removed by pipetting, and oocyte nuclear maturation was evaluated from the presence of the first polar body.
Fertilization and embryo development
Oocytes reaching MII stage were donated to women on the waiting list of our oocyte donation programme. Each was microinjected by ICSI with husband's spermatozoa that were normozoospermic in all cases. The ICSI procedure has been described in detail elsewhere (Gil-Salom et al., 1995). Fertilization was evaluated after 18 h of culture under standard conditions (37°C, 5% CO2) from the presence of two pronuclei and two polar bodies. Embryos at the 24-cell stage were co-cultured with human autologous endometrial epithelial cells as described previously (Simon et al., 1997
, 1999
). Embryo development was evaluated as far as the blastocyst stage, and those reaching this stage of development were cryopreserved.
Oestradiol concentrations
Serum oestradiol concentrations were measured using commercially available radio-immunoassay kits (bioMérieux, Charbonniéres les Bains, France). The inter- and intra-assay variability for oestradiol at a concentration of <40 pg/ml were 2.8% and 4.3% respectively.
Statistical analysis
Results are expressed as mean ± SEM. Comparative analysis of quantitative variables such as age, puncture day and serum oestradiol concentrations was made by ANOVA. Categorical and or dichotomised variables, i.e. follicular diameter, maturation, fertilization and development to blastocyst, were analysed by 2 test and Fisher's exact test. To compare the number of oocytes in each group, the binomial test was performed. In the comparative analysis of the ordinal variable `type of cumulus', the MannWhitney U-test was used. Three dichotic variables (maturation, fertilization and development to blastocyst) were grouped in one single ordinal variable named `oocyte development degree' with the purpose of adding more power to the statistical calculations. Spearman's correlation coefficient (r) was employed to study the relationship between the two ordinal variables `type of cumulus' and `oocyte development degree'. Significance was defined as P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table I shows the results obtained in the two groups defined according to follicular diameter. Follicular size was significantly higher in group 2 (P < 0.05), in which ovum aspiration occurred two days later than in group 1. The retrieval rate was 70.8% in group 1 and 50.5% in group 2 (P < 0.05). Serum oestradiol concentrations were significantly (P < 0.05) higher in group 2 than in group 1, as an indication of a more advanced stage of folliculogenesis.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although aromatase activity is initially low in follicles from the cohort before selection, in-vitro production of oestradiol can be rapidly stimulated by adding FSH to the culture medium (Hillier et al., 1980; Mason et al., 1994
). Oestradiol may in turn act upon the oocyte, as detection of oestradiol receptor mRNA in the human oocyte has been confirmed by Southern blot analysis (Wu et al., 1993
). In addition, it has been shown that the addition of oestradiol to oocyte maturation medium can directly influence the quality of the maturing oocyte (Tesarik and Mendoza, 1995
). Other hormones and growth factors may also play a role in the process of maturation in vitro. We have shown in rodents (Pellicer et al., 1989
) and humans (Gómez et al., 1993
) that EGF induces oocyte maturation in vitro, and this growth factor was also included in our culture system.
Thus, we believe that the hormones added to the culture medium in group 1 oocytes exerted a positive effect on their meiotic and cytoplasmic competence. Evidence suggests that once the dominant follicle is selected, granulosa cells become less receptive to FSH (Fauser and van Heusden, 1997) and perhaps other hormones, and this may reflect the situation in group 2 oocytes.
Maturation rates did not differ between groups. This observation is not surprising because it has long been recognized that oocytes removed from their follicles can undergo nuclear maturation with extrusion of the first polar body (Pincus and Enzmann, 1935; Pincus and Saunders, 1939
; Edwards, 1965
). This is also true for atretic follicles, both in vitro and in vivo (Gougeoun and Testart, 1986
). Similarly, fertilization rates by ICSI were similar. We believe that this observation confirms the power of ICSI, and suggests that there may be various factors related to the acquisition of fertilization and cleavage within the cytoplasm of the oocytes.
The high rate of blastocyst formation observed in group 1 (56.5%) is an interesting point of discussion. Cytoplasmic maturation is closely linked to all those processes that prepare the oocytes for fertilization, activation and embryo development. Evidence from goat, murine and bovine studies suggests that the capacity to reach blastocyst stage is higher for oocytes obtained from larger antral follicles than for oocytes from smaller follicles (Pavlok et al., 1992; Eppig and Wigglesworth, 1994
; Crozet et al., 1995
). Hence, nuclear and cytoplasmic maturation may occur in a coordinated fashion in larger follicles. In group 1 of the present study, all selected follicles were healthy and belonged to the class of larger non-dominant follicles where differentiation of GV stage oocytes could be related to acquisition of competence to undergo pre-implantational development.
We employed a simplified method for ovum retrieval using routine IVF tools, and obtained an acceptable retrieval rate of 5.9 oocytes per patient (61.1%). However, this was lower than the rate obtained using special needles for ovum retrieval (Trounson et al., 1994b; Russell et al., 1996
).
Table III summarizes the results of several reports concerning the outcome of 1207 in-vitro-matured oocytes obtained from unstimulated cycles in terms of maturation rate to MII stage, fertilization and cleavage rates, and pregnancies. Metaphase II stage oocytes obtained in different studies are similar, but there is more variability in fertilization and cleavage rates. This might be the consequence of differences in the developmental capability of the oocytes, which have not been obtained at the same stage of the cycle. Also, different culture conditions may influence the acquisition of cytoplasmic factors affecting oocyte competence to undergo fertilization and successful embryogenesis. Our fertilization and cleavage rates are higher than other reported figures, and the high rate of blastocyst formation (48.6%) suggests that collecting oocytes at the right time, and placing them in an optimal environment, might improve embryo quality.
|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnes, F., Kausche, A., Tiglias, J. et al. (1996) Production of embryos from in vitro matured human oocytes. Fertil. Steril., 65, 11511156.[ISI][Medline]
Carson, R. (1997) Maturation of human oocytes in vitro. J. Assist. Reprod. Genet., 14 (abstract book), p. 431.
Carson, R., Stangel, J., Crumm, K. et al. (1995) Maturation of oocytes in vitro. Fertil. Steril., Annual Meeting Program Supplement, O-075, S37.
Cha, K. and Chiang, R.C. (1998) Maturation in vitro of immature human oocytes for clinical use. Hum. Reprod. Update, 4, 103120.
Cha, K., Koo, J., Ko, J. et al. (1991) Pregnancy after in vitro fertilization of human follicular oocytes collected from non stimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil. Steril., 55, 109113.[ISI][Medline]
Crozet, N., Ahmed-Ali, M. and Dubos, M.P. (1995) Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilisation and culture in vivo. J. Reprod. Fertil., 103, 293298.[Abstract]
Edwards, R. (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 102, 493497.
Eppig, J.J. and Wigglesworth, K. (1994) Atypical maturation in oocytes in strain I/LnJ mice. Hum. Reprod., 9, 11361142.[Abstract]
Fauser, B. and van Heusden, A. (1997) Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr. Rev., 1, 71103.
Gil-Salom, M., Minguez, Y., Rubio, C. et al. (1995) Intracytoplasmic testicular sperm injection: an effective treatment for otherwise intractable obstructive azoospermia. J. Urol., 154, 20742077.[ISI][Medline]
Gómez, E., Tarín, J.J. and Pellicer, A. (1993) Oocyte maturation in humans: the role of gonadotropins and growth factors. Fertil. Steril., 60, 4046.[ISI][Medline]
Gougeoun, A. (1996) Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr. Rev., 2, 121155.
Gougeoun, A. and Testart, J. (1986) Germinal vesicle breakdown in oocytes of human atretic follicles during the menstrual cycle. J. Reprod. Fertil., 78, 389401.[Abstract]
Hillier, S.G., van der Boagaard, A., Reichert, L.E., Jr and van Hall, E.V. (1980) Intraovarian sex steroid hormone interactions and the regulation of follicular maturation: aromatization of androgens by human granulosa cells in vitro. J. Clin. Endocrinol. Metab., 50, 640647.[ISI][Medline]
Jaroudi, K., Hollanders, J.M.G., Sieck, U. et al. (1997) Pregnancy after transfer of embryos which were generated from in vitro matured oocytes. Hum. Reprod., 12, 857859.[Abstract]
Knezevich, K.M., Russell, B., Fabian, K.F. et al. (1996) Analysis of in vitro oocyte maturation and fertilization results. Fertil. Steril., Annual Meeting Program Supplement, O-019, S10.
Looney, C., Lindsay, B., Gonseth, C. and Johnson, D. (1994) Commercial aspects of oocyte retrieval and in vitro fertilization for embryo production problem in cow. Theriogenology, 41, 6772.[ISI]
Mason, H.D., Wills, D., Beard, R. et al. (1994) Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentration of gonadotropins and sex steroids in follicular fluids. J. Clin. Endocrinol. Metab., 79, 13551360.[Abstract]
McNatty, K.P., Hillier, S.G., van den Boogaard, A.M.J. et al. (1983) Follicular development during the luteal phase of the human menstrual cycle. J. Clin. Endocrinol. Metab., 56, 10221031.[Abstract]
Nagy, Z., Cecile, J., Liu, J. et al. (1996) Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal vesicle stage oocytes: case report. Fertil. Steril., 65, 10471050.[ISI][Medline]
Pavlok, A., Lucas-Hahn, A. and Niemann, H. (1992) Fertilisation and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol. Reprod. Dev., 31, 6367.[ISI][Medline]
Pellicer, A., Lightman, A., Parmer, T.G. et al. (1988) Morphological and functional studies of immature rat oocytecumulus complexes after cryopreservation. Fertil. Steril., 50, 805810.[ISI][Medline]
Pellicer, A., Parmer, T.G., Stoane, J.M. and Behrman, H.R. (1989) Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle. Mol. Cell. Endocrinol., 64, 179188.[ISI][Medline]
Pincus, G. and Enzmann, E. (1935) The comparative behaviour of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J. Exp. Med., 62, 655675.
Pincus, G. and Saunders, B. (1939) The comparative behaviour of mammalian eggs in vivo and in vitro. IV. The maturation of human ovarian ova. Anat. Rec., 75, 537545.
Russell, J.B., Knezevich, K.M., Fabian, K.F. et al. (1995) Unstimulated immature oocyte retrieval: early vs mid follicular endometrial priming. Fertil. Steril., Annual Meeting Program Supplement, O-1, A1.
Russell, J.B., Knezevich, K.M., Fabian, K.F. et al. (1996) Unstimulated immature oocyte retrieval: early vs midfollicular endometrial priming. Hum. Reprod., 11 (Abstract Book 1), 2.[ISI][Medline]
Sasano, H., Okamoto, M., Mason, J.I. et al. (1989) Immunolocalization of aromatase, 17-hydroxylase and side chain cleavage cytochrome P-450 in the human ovary. J. Reprod. Fertil., 85, 163169.[Abstract]
Shroder, A. and Eppig, J. (1984) The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev. Biol., 102, 493497.[ISI][Medline]
Simon, C., Gomeno, M.J., Mercader, A. et al. (1997) Embryonic regulation of integrins ß3, 3, and
1, in human endometrial epithelial cells in vitro. J. Clin. Endocrinol. Metab., 82, 26072616.
Simon, C., Mercader, A., García-Velasco, J. et al. (1999) Coculture of human embryos with autologous endometrial epithelial cells in patients with implantation failure. J. Clin. Endocrinol. Metab. (in press).
Tamura, T., Kitawaki, J., Yamamoto, T. et al. (1992) Immunohistochemical localization of 17-hydroxylase/C17-20 lyase and aromatase cytochrome P-450 in the human ovary during the menstrual cycle. J. Endocrinol., 135, 589595.[Abstract]
Tesarik, J. and Mendoza, C. (1995) Nongenomic effects of 17ß-estradiol on maturing human oocytes: relationship to oocyte developmental potential. J. Clin. Endocrinol. Metab., 80, 14381443.[Abstract]
Trounson, A., Pushet, D., MacLellan, L. et al. (1994a) Current status of IVM/IVF and embryo culture in humans and farm animals. Theriogenology, 41, 5766.[ISI]
Trounson, A., Wood, C. and Kausche, A. (1994b) In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil. Steril., 62, 353362.[ISI][Medline]
Veeck, L., Edward, J., Witmyer, J. et al. (1983) Maturation and fertilization of morphologically immature oocytes in a program of in vitro fertilization. Fertil. Steril., 39, 594602.[ISI][Medline]
Wu, Tch J., Wang, L. and Wan, Y.J.Y. (1993) Detection of estrogen receptor messenger ribonucleic acid in human oocytes and cumulusoocyte complexes using reverse transcriptasepolymerase chain reaction. Fertil. Steril., 59, 5459.[ISI][Medline]
Submitted on December 17, 1998; accepted on March 25, 1999.