The influence of amifostine administration prior to cyclophosphamide on in vitro maturation of mouse oocytes

Purpose

The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes.

Materials and methods

Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined.

Results

In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p < 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased.

Discussion

Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.     

Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles

The objective of our study was to compare to ability of collagen-treated membranes and bovine collagen gels to maintain murine preantral follicle growth and development in-vitro. To fulfill that objective, murine follicle and oocyte growth rates were followed for ten days in culture. Meiotic competence and the capacity to reach the two-cell stage after in-vitro maturation and fertilization, respectively, were then assessed. We used preantral follicles from 12 day-old CF-1 female mice that were isolated by enzymatic digestion from ovaries. Follicles were placed either on collagen-treated membranes or embedded in a bovine collagen matrix. The follicles were grown, changing the media and obtaining measurements every other day for ten days. Following culture, the granulosa-oocyte complexes were matured; the resultant metaphase II arrested oocytes were inseminated and cultured to the two-cell stage. The data was analyzed with significance considered for probability values of p < 0.05. We performed individual measurements on 650 follicles in seven separate experiments. Forty-eight hours after initial seeding and throughout the entire length of culture, both the follicles and oocytes grown in the collagen matrix were larger than follicles cultured on collagen-treated membranes (p < .0001). However, oocyte recovery rates were higher among follicles cultured on collagen-treated membranes (p < .01). Similar percentages of meiotically competent oocytes, fertilization and cleavage rates were observed in both groups. Our results show that mouse preantral follicles display a greater growth rate when grown embedded in a collagen gel matrix. This may be due to the maintenance of a normal three-dimensional organization of the follicle within the collagen matrix. However, this system does not enhance meiotic competency or fertilization rates in the mouse when compared to culture on collagen-treated membranes.     

Effect of preantral mouse follicle culture period on meiotic maturation and developmental competence of oocytes

Purpose  

The aim of this study is to determine the optimal culture period for meiotic maturation and developmental competence of in vitro-grown mouse oocytes.     

Expression of G protein estrogen receptor (GPER) on membrane of mouse oocytes during maturation

Purpose

To determine expression of G-protein estrogen receptor (GPER) in mouse oocyte membrane during maturation.

Methods

The expression of GPER from different maturation stages of oocytes, in vivo and in vitro matured oocytes as well as aging oocytes was examined by immune-fluorescence GPR30 antibody and the images were analyzed by laser scanning confocal microscope. Further confirmation was performed by Western blots for cell fractionation.

Results

Significant fluorescent signal was observed on the surface of mouse oocytes. The image expression was lower in germinal vesicle (GV) stage than mature metaphase-II (M-II) stage oocytes. There was high expression in in-vivo matured oocytes compared to in vitro matured oocytes. The highest expression was observed in aging oocytes compared with other oocytes.

Conclusions

The changes of expression of GPER on mouse oocytes plasma membrane confirm oocyte membrane maturation, suggesting that those changes of GPER may be related to the functional role of oocyte maturation.     

Developmental competence of antral follicles and their oocytes after gonadotrophin treatment of sows with gene polymorphisms for leptin and melanocortin receptors (Iberian pig)

Purpose

To evaluate possible differences in follicle and oocyte developmental competence after gonadotrophin treatment in sows of obese and lean genotypes.

Methods

Follicle dynamics, ovulation rate and oocyte developmental competence to embryo were compared between females, of obese (n = 7) and lean genotypes (n = 10), treated with 1,250 I.U. of eCG and 500 I.U. of hCG.

Results

The obese genotype showed lower numbers of follicles growing to preovulatory stages (12.4 ± 1.8 vs 18.6 ± 1.0, P < 0.05), of corpora lutea (16.0 ± 0.9 vs 23.5 ± 0.9, P < 0.05), and of recovered oocytes/embryos (8.0 ± 1.3 vs 12.9 ± 0.9, P < 0.05). Thereafter, embryo viability rates also decreased when compared to lean genotypes (62.5 vs 77.6%, P < 0.05).

Discussion

To our knowledge, this is the first study analyzing the effect of obese genotypes on the ovarian response to exogenous gonadotrophins in a non-rodent animal model, the pig. A lower efficiency of gonadotrophin treatments for stimulation of follicle development and induction of ovulation was observed.     

人窦前卵泡的体外发育及其卵母细胞成熟过程的观察

目的探讨在体外培养条件下,人窦前卵泡发育及其卵母细胞成熟的可能性。方法建立适宜的体外培养环境,应用正交设计将不同剂量的人绝经期促性腺激素（ｈＭＧ）、卵泡液、表皮生长因子（ＥＧＦ）组合,作用于来自体外受精－胚胎移植（ＩＶＦ－ＥＴ）抽吸的卵泡液及冲洗液中获得的窦前卵泡。结果窦前卵泡于培养第６～１２天形成窦卵泡,培养２１～２８天时直径≥３００μｍ的卵泡数显著增多（Ｐ＜０．００５）,当以ｈＭＧ１５０ＩＵ／Ｌ、卵泡液４０％、ＥＧＦ６μｇ／Ｌ作用于卵泡时,卵母细胞排出率与再经培养后成熟率均达最高,分别为８３％和３８％（Ｐ＜０．００５）。结论在适宜的培养条件下,来自ＩＶＦ－ＥＴ病人的抽吸卵泡液的窦前卵泡在体外能够发育,且其卵母细胞可达到成熟,是一新的人卵来源。窦前卵泡的体外发育和体内不同     

The effect of alpha lipoic acid on the developmental competence of mouse isolated preantral follicles

Purpose  

This study was designed to investigate the effect of alpha-lipoic acid (ALA) on reactive oxygen species (ROS) production, total antioxidant capacity (TAC) and developmental competence of cultured pre-antral follicles derived from mouse ovarian tissue.     

Correlation of somatic cell steroid secretion and quality of generated oocytes after in-vitro stimulation of mouse follicles

Purpose: To test the possibility of follicular somatic cell steroidogenesis as a marker for quality of their embraced oocytes. Methods: Mechanically isolated mouse preantral follicles were cultured and matured in-vitro (IVC/IVM) for study. Results: During IVC/IVM, oogenesis occurred concomitantly with folliculogenesis in a coordinated manner and simultaneously with progressive increments of somatic cell steroidogenesis. Follicular E₂ production of matured oocytes were significantly higher than that of immature ones. The majority of MII oocytes (32/36) and all developed blastocysts(12/12) were associated with active E₂ production prior to ovulation. In this study, 18 MII oocytes met both requirements for active and optimal E₂ production. 13 of them were fertilized and 10 developed into blastocysts. Conclusion: Active somatic cell steroidogenesis prior to ovulation and an optimal steroid milieu at ovulation are prerequisites for generation of competent oocytes after follicular maturation in-vitro.Predictability of somatic cell steroidogenesis for oocyte competency after follicular IVC/IVM.     

Association between the proportion of dominant follicles and oocyte developmental competence

Purpose

To explore the optimal timing for hCG triggering by investigating the impact of different proportion of dominant follicles on the oocyte developmental competence.

Methods

One hundred ninety-eight infertile women were divided into three groups according to the proportion of dominant follicles on hCG day: (1) low: <15 % (n = 66); (2) middle: 15–27 % (n = 66); (3) high: >27 % (n = 66). The grouping criteria were the bottom and top tertiles of the proportion of dominant follicles.

Results

The gonadotropin dosage, duration and maximum follicle diameter in the low proportion group were lower than those in the middle and high proportion groups. Oocyte maturation and the abnormal fertilization rate in the low proportion group were lower than those in the middle and high proportion groups. The normal fertilization rate did not differ among the three groups. The cleavage rate and number of transferable embryos in the low proportion group were significantly higher than those in the high proportion group. The high-quality embryo rate, implantation rate, and pregnancy rate in the low proportion group were significantly higher than those in the middle and high proportion groups.

Conclusions

A high proportion of dominant follicles are closely associated with impaired oocyte developmental competence and low pregnancy rate. These findings suggest that follicular overgrowth induced by delayed hCG triggering may undermine oocyte developmental competence and the proportion of dominant follicles may be a potential parameters for hCG triggering.     

Comparison of maturation, meiotic competence, and chromosome aneuploidy of oocytes derived from two protocols for in vitro culture of mouse secondary follicles

PURPOSE: To compare maturation rates of mouse preantral follicles cultured using two previously reported follicle in vitro follicle culture protocols, and to compare the aneuploidy of oocytes derived from the two protocols with in vivo-matured control oocytes. METHODS: Mouse preantral follicles were either mechanically isolated then cultured in individual microdroplets, or enzymatically isolated and cultured in groups in a modified culture medium. Maturation of the follicles/oocytes and resulting oocyte aneuploidy rates were evaluated and compared. RESULTS: The mechanical isolation and individual culture protocol (M/I) resulted in higher follicle survival than the enzymatic isolation and group culture protocol (E/G) (89.1% versus 79.1%, p < 0.01), and better maturation to the metaphase 2 stage (61.5% versus 39.5%, p = 0.01) The rate of aneuploidy of oocytes not significantly higher in oocytes from the E/G group than the M/I group (15.4% versus 9.9%), but hypoploidy was significantly increased (4.7% versus 0.9%, p < 0.05). Both groups had a higher rate of aneuploidy than the control oocytes (2.9%, p < 0.02). CONCLUSIONS: These results indicate an increased survival and competency of oocytes derived from the M/I protocol, compared to the E/G protocol. The data highlight an increased susceptibility to meiotic errors in early stage follicles undergoing in vitro culture, compared to in vivo-matured oocytes.     

Effect of <Emphasis Type="Italic">Papaver rhoeas</Emphasis> extract on in vitro maturation and developmental competence of immature mouse oocytes

Purpose  

This experiment examined the effect of Papaver rhoeas L. extract on in vitro maturation, in vitro fertilization (IVF) and subsequent developmental competence of mouse oocytes.     

In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence

Purpose  

The in-vitro environment influences oocyte competence and gene expression in cumulus cells and oocytes. Effects of culturing under non-attachment conditions and varying follicle exposure to FSH were investigated at the mRNA level and on oocyte developmental capacity.     

Comparison of fertilization and embryonic development in sibling in vivo matured oocytes retrieved from different sizes follicles from in vitro maturation cycles

Purpose

To examine the fertilization and developmental potential of sibling mature oocytes collected from different follicle sizes on day of retrieval in in vitro maturation (IVM) cycles.

Methods

Two hundred thirty eight hCG-primed IVM cycles were performed in 213 patients with polycystic ovaries. If sibling mature oocytes were retrieved on day of collection, they were divided into two groups, Group 1 (n = 78): M-II oocytes obtained from follicles size 10–14 mm; Group 2 (n = 192): M-II oocytes obtained from follicles size <10 mm.

Results

Of the 238 cycles, 63 cycles had more than one M-II oocytes retrieved (total M-II oocytes = 270) both from Groups 1 and 2. There were no significant differences between the two groups for oocyte diameter (117.2 mm vs. 116.9 mm), fertilization (79.5% vs. 72.4%) and good quality embryo on day 3 (66.1% vs. 56.8%).

Conclusions

The M-II oocytes retrieved from the cohort of follicles (<10 mm diameter) can produce the same quality of embryos as that from large follicles, likely contributing to improve the clinical outcome.     

Evaluation of maturation competence of metaphase II oocytes in mice based on the distance between pericentriolar materials of meiotic spindle

Purpose  

To ascertain whether metaphase II (MII) spindle shape influences oocyte competence, we examined the meiotic spindle organization in in vivo ovulated (IVO) oocytes and in spontaneously matured or follicle stimulating hormone (FSH)-induced oocytes.     

In vitro culture and in vitro maturation of mouse preantral follicles with recombinant gonadotropins

OBJECTIVE: To develop an effective method for in vitro maturation of preantral follicles isolated from mice ovarian tissue. DESIGN: Isolated preantral follicles were randomly allocated to designed experimental groups for study. SETTING: University-based research lab. PATIENT(S): Healthy, normal mice. INTERVENTION(S): Superovulation with pregnant mare serum gonadotropin and hCG. MAIN OUTCOME MEASURE(S): Morphological changes and E(2) production were assessed. RESULT(S): To obtain competent oocytes, preantral follicles must be cultured with medium containing insulin and recombinant gonadotropins (i.e., recombinant FSH and recombinant LH), with a change of medium daily. A high initial recombinant LH or recombinant FSH facilitates E(2) secretion, enhances granulosa cell outgrowth, and has earlier antral formation. However, prolonged culture in high-recombinant LH or recombinant FSH triggers early differentiation and luteinization of granulosa cells, which results in low metaphase II oocyte and blastocyst formation. CONCLUSION(S): We have developed a culture system that allows the successful maturation of preantral follicles in vitro. The matured follicles are a physiologically functional unit that not only secrete E(2) but also generate competent oocytes. In a special condition, 90% of the cultured follicles survived, 53.5% of them produced MII oocytes, and 50% of the derived MII oocytes were fertilized and reached the blastocyst stage after culture in vitro.     

Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice

Purpose

To investigate whether single-culture systems influence the quality of in vitro-matured oocytes, we examined the maturation and developmental competence of oocytes obtained by grouped in vitro maturation (IVM) or single IVM.

Methods

In vitro-matured oocytes were obtained using the culture drop (CD) method for the grouped IVM experiments, and the CD and hanging drop (HD) method for the single IVM experiments. To evaluate oocyte developmental competence, we performed in vitro fertilization and culture, and counted the number of blastocysts. To evaluate the oocyte cytoplasmic maturation, we measured the maturation promoting factor (MPF) expression levels.

Results

Oocytes cultured singly had lower maturity and developmental competence than the grouped IVM oocytes. However, enhanced oocyte fertility and blastocyst quality was achieved by the HD single IVM method. Additionally, the MPF activity level increased in all culture methods, compared to the control; however, it lagged behind nuclear maturation.

Conclusions

These results suggest that the HD method is efficient for single IVM.     

Clinical outcomes from mature oocytes derived from preovulatory and antral follicles: reflections on follicle physiology and oocyte competence

Purpose

The aim of this retrospective study was to compare the competence of oocytes obtained from preovulatory and antral follicles.

Methods

Mature oocytes from preovulatory follicles were retrieved from women selected for standard IVF treatment (Group A). Mature oocytes from antral follicles were recovered from women undergoing hCG-primed in vitro maturation (IVM) treatment (Group B). Patients groups were matched for age, BMI, FSH, AMH and antral follicle count (AFC) values. In vivo matured oocytes from both groups were microinjected and resulting embryos were culture and selected on day 3 for embryo transfer.

Results

Oocyte pick-ups (OPU) were 315 and 204 in Groups A and B, respectively. Fertilization rates were comparable (72.8 and 75.9 %, respectively; P = 0.137). In Group A, in which the average number of embryos transferred was higher, clinical pregnancy rates per OPU (37.5 %) and embryo transfer (38.4 %) were superior in comparison to Group B (27.0 %, P = 0.013; 29.4 %, P = 0.041; respectively). On the other hand, implantation rates (Group A, 23.7 %; Group B, 20.8 %) and proportions of babies born per transferred embryo (Group A, 19.5 %; Group B, 16.9 %) were similar (P = 0.528 and 0.332, respectively).

Conclusions

Overall, this suggests that oocyte competence is already achieved at the antral stage of follicle development.