Effects of Cyclic Increase in Gonadotropins on the In Vitro Development of Primordial Follicles to Antral Stage

The aim of this study was to evaluate the effects of FSH and LH on follicle development during a long-term culture of cryopreserved human ovarian tissue, using morphological and ultrastructural examinations. Thawed ovarian tissue slices from a 4-year-old child with Wilms tumor were cultured for 32 weeks in two different culture conditions, without (medium A) and with (medium B) a monthly peaked increase in FSH and LH. At week 32, in the medium B cultured tissue, a cluster of preantral follicles associated with two oocytes prematurely ovulated was observed, suggesting that the cyclic increase of gonadotropins promoted thawed follicles to grow up to the antral stage. However, the integrity and coordinated follicle development were not maintained. Indeed, ultrastructural analysis showed a well-preserved “naked” oocyte with concomitant features of immaturity and maturity, as if this culture condition had led to an asynchronous maturation of oocyte cytoplasmic components.     

Effect of preantral follicle isolation technique on in-vitro follicular growth,oocyte maturation and embryo development in mice

BACKGROUND: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. METHODS: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. RESULTS: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. CONCLUSIONS: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.     

Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro

In two consecutive controlled experiments 160 early preantral follicles were cultured in order to evaluate effects of recombinant follicle stimulating hormone (r-FSH) on survival, differentiation, oestradiol and inhibin secretion, cumulus mucification and cumulus-corona-oocyte detachment by human chorionic gonadotrophin (HCG) stimulation. Nuclear maturation in oocytes was also assessed following addition of HCG. A histological analysis of cultured follicles was carried out on semi- thin sections at various culture stages. Addition of r-FSH was essential for follicle survival for 16 days: without r-FSH only 11% of the follicles survived for 12 days (with r-FSH: 79%) and none of these mucified after the HCG stimulus. r-FSH promoted granulosa cell proliferation and antral-like cavity formation. Without r-FSH, histology of the cultures demonstrated degeneration and reduced granulosa cell proliferation; oestradiol and inhibin production were reduced. This study illustrates the essential role of FSH in promoting the in-vitro growth of early preantral mouse ovarian follicles and in maintaining the oocyte under meiotic arrest.      

Ovary and ovulation: A morphological and functional study of the effect of slow freezing followed by complete in-vitro maturation of primary mouse ovarian follicles

Mechanically isolated intact early preantral follicles (100–130 µm diameter) from 14 day old mice were cryopreservedby a slow freezing protocol with dimethyl sulphoxide and thenmatured in vitro for 12 days after rapid thawing. Minor freezedamage observed after 1 day of in-vitro culture included ablationof the theca cell layer and granu-losa cell dehydration, resultingin disruption of intercellular contacts with the oocyte andbetween granulosa cells. Of the follicles, 24% were irreversiblydamaged and had a collapsed oocyte. The remaining majority ofthe follicles had an intact oocyte as evaluated by ultrastructuralanalysis. Follicles with an intact oocyte were cultured in vitroand, after an initial retarded development, the final numberof fully grown oocytes ovulated in vitro was not different fromthat of unfrozen controls. Cryopreserved early preantral folliclesmatured in vitro responded to stimulus with human chorionicgonadotrophin in a similar way to controls, with mucificationof the oocyte-cumulus complex, germinal vesicle breakdown andextrusion of the first polar body of the oocyte. These cryopreserved,in-vitro matured oocytes had the potential to fertilize anddevelop to hatched blastocysts.     

Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.     

Effects of varying gonadotrophin dose and timing on antrum formation and ovulation efficiency of mouse follicles in vitro

BACKGROUND: This study tested factors affecting mouse follicle growth in vitro, to determine end-points marking follicle function in vitro. METHODS: Pre-antral follicles (mean 137 microm) from B6CBF1 mice were cultured in a substrate-adherent system for < or = 14 days. FSH (0-1000 mIU/ml) day of HCG (1.5 IU/ml days 9-14) protein supplement [fetal calf serum (FCS) (x2) mouse serum (x2) hypogonadal (hpg) mouse serum or human serum albumin (HSA)] were varied. Follicle survival timing of antrum formation incidence of ovulation within 16,24,40,48 h of HCG oocyte growth were assessed. RESULTS: FSH (100 mIU/ml) produced the best antral development (P < 0.001 versus 10 and 1000 mIU/ml). Antra were observed from day 5. Transient antra formed occasionally in the absence of FSH. By 14 days significant senescence had occurred (P < 0.001) but the proportion of follicles ovulating within 16 h of HCG declined from day 9 onwards indicating this to be a more sensitive marker of follicle responsiveness. Optimal growth occurred in 5% FCS (x2) or hpg mouse serum although fewer follicles ovulated in hpg serum (P < 0.05). No normal growth occurred in normal mouse serum (x2) or HSA. Oocytes grew to full size within 9 days with 100 mIU/ml FSH FCS. CONCLUSIONS: These data provide sensitive end-points for assessing follicle growth in vitro.     

Ultrastructure and viability of isolated bovine preantral follicles

Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes.     

Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary. In both the cases, oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of meiosis. The in vivo study revealed a significant decrease in the number of ovulated mouse oocytes and an increase in meiosis I-arrested and hyperploid metaphase II oocytes at a single i.p. dose of 70 mg/kg body weight of nocodazole. A significant increase was also observed in the number of meiosis I-arrested and hyperploid mouse oocytes from preantral follicle culture, when they were cultured in the presence of >or=30 nM nocodazole during the final stages of maturation. This concentration is slightly lower than that previously shown to induce nondisjunction in denuded mouse oocytes or in cultured human lymphocytes. The higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be related to a synergistic adverse influence of nocodazole on the oocyte, on somatic cell integrity and on cell-cell communication, which possibly also affects ovulation in vivo. When expressed in molarity relative to the mouse weight, the effective dose of the acute exposure in vivo is 3-4 orders of magnitude higher than the lowest effective concentration employed continuously in vitro. Reduced bioavailability of nocodazole to the target cells due to its poor water solubility may contribute to this difference. Preantral follicle culture can be helpful in analysing mechanisms in chemically induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.     

Effects of fibroblast growth factor-2 on the in vitro culture of caprine preantral follicles

The aims of the present study were to evaluate the effects of fibroblast growth factor-2 (FGF-2) on survival, activation and growth of caprine early-staged (preantral) follicles using histological and ultrastructural studies. Fragments of caprine ovarian cortex were cultured for 1 or 5 days in an enriched minimum essential medium, supplemented or not with different concentrations of FGF-2 (10, 50 or 100 ng/ml). Fragments from non-cultured ovarian tissue (control) and from tissues cultured for 1 or 5 days in a specific medium were processed for transmission electron microscopy (TEM) or classical histology to evaluate the morphological quality of caprine preantral follicles and to calculate the percentages of normal follicles. Additionally, effects of FGF-2 on oocyte and follicle diameter of cultured preantral follicles were investigated. Our results showed that, although the percentages of histologically normal follicles were lower in cultured than in non-cultured ovarian tissue fragments, there were no differences in this regard among treatments, neither on day 1 nor on day 5 of culture. After 1 and 5 days of culture, a significantly higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FGF-2. This FGF-2 treatment furthermore resulted in an increase in diameter of both oocytes and follicles that were cultured for 5 days. TEM showed that the ultrastructural integrity of caprine preantral follicles was maintained during their 5-day culture in the presence of 50 ng/ml FGF-2. In conclusion, this study demonstrated that at a concentration of 50 ng/ml FGF-2 not only maintains the morphological integrity of caprine preantral follicles cultured for 5 days, but also stimulates the activation of primordial follicles and the growth of activated follicles.     

Follicle culture after ovarian cryostorage

Objectives: Cryostorage of ovarian cortical tissue, before devastating chemo- and/or radiotherapy for cancer, permits survival of primordial and early preantral follicles. This work aims for a system allowing the long-term maturation in vitro (IVM) of small immature oocytes up to fertilisable metaphase II oocytes. Methods: A culture system allowing follicle attachment permitted the growth and maturation of isolated follicles (follicle diameter between 100 and 130 μm) from 14-day-old (prepuberal) mice. Follicle and oocyte development were observed under the inverted microscope and conditioned medium was used for biochemical analysis. Effects of recombinant gonadotrophins and oxygen tensions were studied for their specific effects on follicle development. Results: A 12-day culture period yielded full-grown oocytes which were able to complete meiosis (metaphase II). Live young were obtained after IVF and intra-uterine transfer of in vitro matured oocytes. Growth and maturation were only successful when recombinant gonadotropins were added and when the incubator had a 20% oxygen tension. This system enabled the growth of early preantral follicles after cryopreservation: 80% of frozen and thawed follicles survived up to culture-day 12 and yielded a comparable blastocyst formation rate as in controls. Conclusions: The mouse model suggests that IVM is a valuable option after oocyte storage. The development of a comparable system for long-term culture of human follicles will imply the acquisition of non-invasive techniques to appreciate oocyte's maturity and developmental capacity.     

Ovary and ovulation: In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

A simplified culture system was developed for the in-vitro maturationof early preantral mouse ovarian follicles. The follicles werecultured singly in 20 (µ droplets under oil in mediumsupplemented with recombinant follicle stimulating hormone (r-FSH)at 37°C and 5% CO² in air. The follicles grew and becameattached to the bottom of the dish, progressively lost theirspherical structure by outgrowth of the granulosa cells throughthe basal membrane and developed follicles with antral-likecavities. The normal three-dimensional follicular structurewas lost but all components, i.e. theca, granulosa and oocyte,remained functional, as was proven by the oestradiol, inhibinand progesterone secretion patterns. Follicle survival exceeded80% and histological analysis proved the absence of atresiaand cell death in granulosa cells up to day 16. Oocytes of 55(± 4) µm diameter on the day of isolation reached74 (± 3) µm by day 16 of culture. The optimal momentfor inducing the final meiotic maturation with human chorionicgonadotrophin was investigated: the highest absolute numbersof metaphase II oocytes were obtained on days 12 and 14 (39and 41%). The fertilizing potential of the in-vitro maturedoocytes was comparable to in-vivo matured controls. A 50% hatched-blastocystdevelopment rate was observed.     

Dynamic medium containing kit ligand and follicle-stimulating hormone promotes follicular survival, activation, and growth during long-term in vitro culture of caprine preantral follicles

The aim of this study was to evaluate the effects of a dynamic medium containing kit ligand (KL) and follicle-stimulating hormone (FSH) on the in vitro culture of caprine preantral follicles for 16 days. Ovarian fragments were cultured in α-MEM(+) containing or not containing KL (50 ng/ml) and/or FSH (50 ng/ml) added during the first (days 0-8) and/or second half (days 8-16) of the culture period. Noncultured (control) and cultured fragments were processed for histological and ultrastructural evaluation. After 1 day of culture, only the treatments performed with KL or FSH maintained a percentage of normal follicles similar to that of the control. After 16 days, all treatments using KL until day 8 (KL/KL, KL/FSH, and KL/FSH+KL) and only FSH during the entire culture period (FSH/FSH) showed higher rates of follicular survival compared to α-MEM(+) alone. After 1 and 8 days, the treatments initially cultured with KL increased the percentage of follicular activation in comparison to α-MEM(+) alone and other treatments. The highest follicular diameter after 16 days was observed in follicles cultured with KL until day 8 followed by FSH (KL/FSH). Furthermore, this treatment promoted, as early as after 1 day of culture, an increase in oocyte growth compared to α-MEM(+) alone. Ultrastructural analysis confirmed the integrity of follicles cultured in KL/FSH after 16 days. In conclusion, a dynamic medium containing KL and FSH maintained follicular integrity and promoted follicular activation and growth during the long-term in vitro culture of caprine preantral follicles.     

Follicle numbers are highly repeatable within individual animals but are inversely correlated with FSH concentrations and the proportion of good-quality embryos after ovarian stimulation in cattle

BACKGROUND: The significance of the high variation in numbers of follicles produced during reproductive cycles in humans and cattle is unknown. METHODS: We selected beef heifers with high (> or =25) or low (< or =15) numbers of ovarian follicles and determined the association with alterations in FSH and estradiol concentrations, as well as responsiveness to superstimulation and embryo quality. The variation in follicle numbers was also compared with oocyte quality in natural cycles using IVF and abattoir-sourced bovine ovaries. RESULTS: Results show that: (i) FSH was lower (P < 0.03) in animals with high compared with low follicle numbers per follicle wave; (ii) after superovulation, in the high versus low follicle number group, the number of oocytes/embryos recovered after insemination (10.6 +/- 2.7 versus 4.7 +/- 0.7) and the number of transferable embryos (5.4 +/- 1.3 versus 3.8 +/- 0.8) per animal were greater (P < 0.05), whereas the proportion of transferable embryos (50.7% versus 79.8%) was lower (P < 0.05); (iii) in unstimulated animals, the numbers of high-quality oocytes harvested and in-vitro fertilized oocytes developing into blastocysts were up to 4-fold greater (P < 0.05) for ovaries with high versus low numbers of follicles, but the proportions of oocytes developing into blastocysts were similar in the two groups. CONCLUSION: Phenotypic classification based on numbers of follicles may be useful to improve superovulation procedures. The lower proportion of transferable embryos following superovulation of ovaries with high numbers of follicles is probably not the result of differences in the quality of oocytes before superovulation.     

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established. It permits the identification of direct and also indirect effects of environmental chemicals on the somatic compartment, the follicle and theca cells, that may lead to disturbances of oocyte growth, maturation and chromosome segregation. Early preantral follicles from prepubertal female mice are cultured in microdroplets for 12 days under strictly controlled conditions. The follicle-enclosed oocytes resume maturation, develop to metaphase II and become in vitro ovulated within 16 h after a physiological ovulatory stimulus with recombinant human gonadotrophins and epidermal growth factor. These oocytes grown and matured in vitro possess normal barrel-shaped spindles with well-aligned chromosomes. Their chromosomes segregate with high fidelity during anaphase I. The model aneugen colchicine induced a meiotic arrest and aneuploidy in these in vitro grown, follicle-enclosed oocytes in a dose-dependent manner, comparable to in vivo tests. Therefore, preantral follicle culture appears to provide an effective and reliable method to assess the influences of environmental mutagens, pharmaceutical agents and potentially endocrine disrupting chemicals on the fidelity of female meiosis.     

Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile

A model culture system has been developed whereby individual,primary ovarian mouse follicles can be grown in vitro to theGraafian stage in the normal physiological time course, andthen ovulated in response to luteinizing hormone. We reporthere on the successful fertilization and subsequent embryo developmentof the oocytes from such follicles. This is the first time thatoocytes from in-vitro matured whole follicles have been fertilizedand shown to produce viable offspring in host animals. The studydemonstrates that the culture system mimics physiological conditionsfor normal follicle development.     

Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination.

BACKGROUND: Birefringent spindles imaged with the Polscope can predict fertilization rates after intracytoplasmic sperm injection (ICSI). The present study examined the development of human oocytes with or without birefringent spindles, imaged with the Polscope before ICSI. METHODS: Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. Spindles were imaged with the Polscope combined with a computerized image analysis system. After imaging and ICSI, oocytes with or without spindles were cultured separately for examination of fertilization and embryo development. A total of 1544 oocytes from 136 cycles were examined with the Polscope and inseminated by ICSI. RESULTS: Spindles were imaged in 82% of oocytes. After ICSI, more oocytes (P < 0.05) with spindles (69.4%) fertilized normally, forming 2 pronuclei, than oocytes without spindles (62.9%). At day 3, more oocytes (P < 0.01) with spindles (66.3%) developed to 4-11 cell stages than oocytes without spindles (55.4%). Significantly more (P < 0.001) oocytes with spindles developed to morula and blastocyst by day 5 (51.1 versus 30.3%) and day 6 (53.2 versus 29.3%) compared with oocytes without spindles. CONCLUSIONS: The results indicate that the presence of a birefringent spindle in human oocytes can predict not only higher fertilization rate, but also higher embryo developmental competence.     

Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes

In this prospective study we investigated whether the maturation and fertilization of immature oocytes can be improved by administration of recombinant follicle stimulating hormone (rFSH) starting in the late luteal phase in two groups of women: group 1 (n = 6) women with regular menstrual cycles; and group 2 (n = 6) women with irregular cycles and polycystic ovaries (PCO) on ultrasound examination. Low-dose (37.5 IU) rFSH was commenced 11 days after LH surge during a spontaneous menstrual cycle and on the ninth day of progesterone administration in an irregular cycle. Recombinant FSH was continued until the leading follicle was approximately 10 mm in diameter. The oocytes were retrieved after withdrawing rFSH for 2-5 days. In total, 136 oocytes were recovered (group 1, 67 oocytes; group 2, 69 oocytes). Nine of the oocytes from PCO women were atretic at retrieval. Oocytes complete with cumulus cells were cultured for 44 h in complex tissue culture medium supplemented with gonadotrophins and fetal calf serum. After maturation, the cumulus cells were removed and metaphase II oocytes were injected with spermatozoa. Respectively, the oocyte maturation and fertilization rates were 64 and 72% in group 1, and 78 and 57% in group 2 (not significant). After fertilization, the zygotes (group 1, n = 22; group 2, n = 11) and cleavage stage embryos (group 1, n = 9; group 2, n = 15) were frozen in propanediol. All women except one (11/12) had approximately five zygotes or cleaved embryos frozen. The viability of in-vitro matured frozen-thawed embryos was generally poorer than that (81%) seen after conventional intracytoplasmic sperm injection, with 61% survival in group 1 and 23% in group 2. Fifteen embryo transfers resulted in one miscarriage at 6 weeks gestation. The late luteal start of low-dose rFSH yielded a good number of immature oocytes in women with both regular and irregular cycles. Two out of three of these oocytes matured and fertilized. However, cryosurvival of the zygotes and cleaved embryos was unsatisfactory and thus cryopreservation of in-vitro matured embryos may not be an optimal procedure.     

Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size.

The potential use of immature oocytes for in-vitro fertilization (IVF) requires the conditions for successful maturation to be defined. This study focused on the day of oocyte retrieval. The selection of a dominant follicle may induce endocrine changes in the remaining cohort that may be detrimental to their subsequent fertilization and embryonic development. Natural cycles in volunteer donors were followed by measurement of serum oestradiol and by vaginal ultrasound, starting on day 3 of the cycle. Cycles were randomly allocated to one of two groups: group 1 (n = 10), in which follicles were aspirated before the leading follicle was 10 mm in diameter; and group 2 (n = 9), in which follicles were aspirated when a dominant follicle was clearly visible with diameter >10 mm. Oocytes were cultured in vitro to metaphase II (MII) stage, donated, and inseminated by intracytoplasmic sperm injection (ICSI) with husband's spermatozoa. Those that became fertilized within 24 h were further co-cultured in autologous endometrial epithelial cells up to the blastocyst stage, and cryopreserved. There was a significantly (P < 0.05) increased rate of oocyte retrieval in group 1 (70.8% of aspirated follicles) compared with group 2 (50.5%). Maturation to MII and fertilization were similar between the groups. However, development to blastocyst stage was significantly (P < 0.05) higher in group 1 embryos (56.5%) compared with group 2 (35.7%). There was a positive correlation (r2 = 0.1978) between the appearance of the cumulus cells and the ability to develop to blastocyst stage when both parameters were analysed in group 1, whereas no such correlation was found in group 2. In conclusion, our data suggest the importance of retrieving immature oocytes before follicular selection, and define the conditions for the first stage in the use of immature oocytes. Further stages must be defined before this technique can be used clinically.     

Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation

The objective of this study was to investigate the effect of follicle stimulating hormone (FSH) priming on the in-vitro maturation (IVM) of human oocytes from healthy ovaries using a chemically defined culture system. Seventeen patients donating oocytes for research received a truncated course of 600 IU FSH over 5 days and a further control group of nine patients received no FSH treatment. Mid-follicular phase cumulus-enclosed oocytes (n = 160) were aspirated from follicles < or =4 mm diameter under transvaginal ultrasound guidance and were cultured for 48 h in microdrops of medium containing 10 mIU/ml FSH and 100 mIU/ ml human chorionic gonadotrophin (HCG). The results demonstrated that human oocytes will efficiently undergo IVM under serum-free conditions. After mild FSH stimulation, a greater number of cumulus-enclosed oocytes was collected, and following culture, a lower rate of degeneration was observed. Significantly more oocytes completed nuclear maturation to metaphase II following FSH stimulation (71.1 versus 43.5%). In conclusion, a truncated course of FSH stimulation in vivo improved the oocyte maturation rate in vitro, giving a mean of 4.8+/- 0.7 metaphase II oocytes per patient compared with only 2.1+/-0.7 from control patients, thus yielding more mature oocytes for future IVF treatment.