The outcome of ICSI of immature MI oocytes and rescued in vitro matured MII oocytes

BACKGROUND: The use of immature oocytes is limited to cases where these are the only available oocytes, and they are usually only microinjected with sperm after having undergone maturation in vitro. This study compares the outcome of injection of sperm into metaphase I oocytes immediately after their denudation (MI) performed 2 h after their retrieval, with the outcome of injection of sperm into rescued in vitro matured metaphase II (IVM MII) oocytes after their short incubation in routine laboratory conditions. METHODS: ICSI was performed on MI oocytes, rescued IVM MII oocytes and on MI oocytes that were incubated but failed to extrude their first polar body (arrested IVM MI). Fertilization and cleavage rates were compared with those achieved in mature metaphase II oocytes (MII). RESULTS: ICSI of MI oocytes showed impaired performance compared with ICSI of rescued IVM MII oocytes and MII oocytes, in terms of oocyte degeneration rate (11 versus 6 versus 4%; P < 0.0001), fertilization rate (28 versus 44 versus 68%; P < 0.0001) and multipronucleated fertilization (10 versus 4 versus 4%; P < 0.01). The cleavage rate was lower in rescued IVM MII oocytes compared with MII oocytes (86 versus 95%; P < 0.01). Arrested IVM MI oocytes showed similar results to those of MI oocytes but had a lower cleavage rate (72 versus 96%; P < 0.01). CONCLUSIONS: The injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes.     

Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes

The objective of this study was to determine whether epidermal growth factor (EGF) promotes nuclear and cytoplasmic maturation of mouse oocytes grown in vivo or in vitro. In-vivo-grown oocytes were isolated at the germinal vesicle (GV) stage from gonadotrophin-primed (PR) or -unprimed (UPR) 22-day-old mice before in-vitro maturation (IVM). In-vitro-grown (IVG) oocytes were isolated from preantral follicles of 12-day-old mice and grown in vitro without gonadotrophins for 10 days before maturation (IVG/IVM oocytes). IVM and IVG/IVM oocytes were matured in medium supplemented with either EGF (10 ng/ml), follicle stimulating hormone (FSH) (100 ng/ml), EGF plus FSH, or with neither ligand (control). When oocyte-cumulus cell complexes were isolated from PR and UPR mice, IVM with EGF (10 ng/ml), alone or in combination with FSH (100 ng/ml), increased (P < 0.05) the incidence of nuclear maturation to metaphase II. Cytoplasmic maturation of oocytes from PR females, manifested as increased frequency of cleavage to the 2-cell stage and development to the blastocyst stage, was also enhanced with EGF (P < 0.05). Moreover, EGF increased the number of cells per blastocyst, but only in the absence of FSH (P < 0.01). In contrast, EGF, FSH, or EGF plus FSH did not affect the percentage of oocytes from UPR mice completing preimplantation development, but did increase the number of cells per blastocyst. These ligands also increased the proportion of IVG oocytes reaching metaphase II (53-57%) compared with controls (25%; P < 0.05). EGF alone or in combination with FSH increased (P < 0.05) the frequency of blastocyst formation (23% and 28%, respectively) compared with controls (13%). EGF treatment of maturing IVG oocytes produced blastocysts with more cells than other IVG groups (P < 0.05). It is concluded that gonadotrophins in vivo increase the sensitivity or responsiveness of cumulus cell-enclosed oocytes to EGF, thereby promoting both nuclear and cytoplasmic maturation. However, oocyte-granulosa cell complexes grown in vitro become responsive to EGF without gonadotrophin treatment. Thus, nuclear and cytoplasmic maturation of IVG oocytes is promoted by EGF treatment during meiotic maturation.     

A successful model to assess embryo development after transplantation of prophase nuclei

BACKGROUND: Germinal vesicle transplantation (GVT) provides a means of investigating interactions between karyoplasts and cytoplasts isolated from different cells. Technically, GVT can be accomplished with a high degree of efficiency without compromising the maturation of either the human or mouse oocyte nucleus. Although maturation, fertilization and preimplantation development have been established using GVT, full-term development has been reported only after supplementation with fresh mature ooplasm. In this study, we assess the ability of immature oocytes collected from gonadotrophin-primed ovaries to mature in vitro after GVT and develop to full-term. METHODS: GV oocytes were retrieved from either non-stimulated or pregnant mare's serum gonadotrophin (PMSG)-primed female mice. Microsurgically isolated GV karyoplasts were transplanted into previously enucleated oocytes. Oocytes successfully reconstituted by electrofusion were cultured for 14 h to allow nuclear maturation. Metaphase II oocytes were subjected to Piezo-ICSI, and those fertilized normally were cultured to the blastocyst stage. Some such embryos were transferred to pseudopregnant female mice to examine their potential for normal development. Cumulus-denuded non-manipulated oocytes that were matured in vitro served as controls. RESULTS: The reconstitution and maturation rates were comparable in oocytes isolated from PMSG-primed and from unstimulated ovaries. The rate of normal fertilization in oocytes from primed ovaries was significantly higher than that of their non-primed counterparts (63.5 versus 39.6%; P < 0.01). This difference was also confirmed in terms of blastocyst development (31.8 versus 7.9%; P < 0.01). Of a total of 70 embryos transferred to the oviduct of five recipient mice, 21.4% developed to normal live offspring. All developed as normal adults and proved to be fertile. The live birth rate was comparable to that obtained using non-manipulated control oocytes (22.3%). CONCLUSIONS: Higher rates of fertilization and blastocyst formation were obtained after GVT of mouse oocytes isolated from PMSG-primed ovaries compared with their non-primed counterparts. These represent the first mouse offspring derived from in vitro matured, cumulus-denuded oocytes treated by allo-GVT and fertilized by ICSI. Thus, GVT appears not to impair oocyte maturation, fertilization and pre- and post-implantation development and, after gonadotrophin priming, allows generation of healthy mouse offspring without mature ooplasm supplementation.     

应用玻璃化冷冻技术保存人未成熟卵母细胞的研究

目的利用玻璃化冷冻保存技术合理利用常规促排卵周期获得的未成熟卵母细胞。方法1．对注射hCG后38h、43h、60h获得GV期卵母细胞进行玻璃化冷冻保存,冻融后的未成熟卵母细胞行体外成熟培养（IVM）,用ICSI技术对获得的成熟卵母细胞完成授精,并体外胚胎培养至囊胚期。比较各组间的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。2．对GV卵分别行先玻璃化冻融后IVM和先IVM后玻璃化冻融,比较它们的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。结果1．38h与43h卵龄GV卵在各项指标比较上没有差异,60h卵与前2组有相似的冻融存活率,但在体外成熟率、受精率和卵裂率上较前2组出现明显下降趋势。3组均无囊胚形成。2．先玻璃化冻融后IVM和先IVM后玻璃化冻融在未成熟卵母细胞的利用率上没有差异。结论1．冷冻卵龄较短的GV期卵母细胞能获得更好的冷冻保存效果。2．玻璃化冻融和IVM先后不影响未成熟卵母细胞的利用。     

Effects of myo-inositol on the in-vitro maturation and subsequent development of mouse oocytes

BACKGROUND: The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro. METHODS AND RESULTS: We performed a controlled prospective study using female ICR strain mice superovulated with pregnant mare's serum gonadotrophins. Cumulus-enclosed germinal vesicle (GV) oocytes were randomly cultured in medium with or without MI supplementation. The kinetics of GV breakdown after 4 h of incubation was significantly higher in oocytes incubated with 30 mmol/l of MI than in controls (P < 0.001). Accordingly, this concentration of MI was used for subsequent experiments. The proportion of metaphase II oocytes achieved after 24 h of culture, their fertilization and cleavage rates were significantly higher in the MI-treated group (P < 0.01, P < 0.05, P < 0.05 respectively). This group also demonstrated significant improvement in postimplantation development after transferring the 2-cell embryos to pseudopregnant mice. Confocal microscopy revealed spontaneous intracellular Ca(2+) oscillations within competent GV oocytes and treatment with MI caused an earlier onset of these Ca(2+) signals. CONCLUSIONS: Our results suggest that MI may affect meiotic progression of mouse GV oocytes possibly by enhancing the intracellular Ca(2+) oscillations. Supplementation of MI in culture medium may be useful for human oocyte maturation.     

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.     

Perivitelline space granularity: a sign of human menopausal gonadotrophin overdose in intracytoplasmic sperm injection

The significance of the presence of coarse dark granules in the perivitelline space of oocytes has not been studied before. The study included 2288 intact oocytes [2063 in metaphase II (MII), 136 in metaphase I (MI), and 89 in germinal vesicle (GV)] retrieved in 206 intracytoplasmic sperm injection cycles stimulated by a long agonist protocol. The incidence of granules varied with oocyte maturity. It was detected in 34.3% and 4% of the MII and MI oocytes respectively, while none of the GV oocytes contained granules. The woman's age, hormonal values (oestradiol and progesterone), human chorionic gonadotrophin/oocyte retrieval interval, number of oocytes retrieved, and oocyte retrieval/injection interval were not related to the percentage of granular oocytes. Moreover, there was no correlation between the percentage of granular oocytes and the fertilization and cleavage rates, pregnancy outcome, as well as the implantation rate. Patients were divided into three groups according to the total human menopausal gonadotrophin (HMG) dose they received. There was a statistically significant difference between the three groups in the percentage of granular oocytes [17.4 +/- 5.2% versus 26.7 +/- 3.2% versus 45.4 +/- 4.2% in the low-dose (< 30 ampoules), intermediate dose (31-45 ampoules), and high-dose (> 45 ampoules) groups respectively]. We conclude that granularity in the perivitelline space is probably a physiological phenomenon related to the maturational events in oocytes and enhanced by exposure to high dosages of HMG.      

体外成熟卵母细胞受精方式及发育能力的影响因素

目的 探讨体外成熟卵母细胞的受精方式及影响其胚胎发育的因素.方法 收集本院2008年7月至12月因男性因素需进行卵母细胞胞浆内单精子注射术(ICSI)助孕的135对夫妇未成熟的卵母细胞(DO)354枚,置入P1培养体系中观察培养20～24、26～30、36～40 h后的成熟情况.去除了退化的成熟卵母细胞,随机分为常规体外受精(IVF)和ICSI组,比较2组的正常受精率、卵裂率、第3天优质胚胎率及各组优质胚胎和非优质胚胎的患者年龄、不孕年限、促排时间及卵母细胞体外成熟时间.结果 卵母细胞体外成熟率为82.5%(292/354),IVF组正常受精率56.5%(70/124)低于ICSI组69.9%(107/153)(P<0.05).两组卵裂率和优质胚胎率差异无统计学意义(P>0.05).2组组内优质胚胎的患者年龄、体外成熟时间均小于非优质胚胎(P<0.05);不孕年限及促排时间差异无统计学意义(P>0.05).结论 ICSI能够提高体外成熟卵母细胞的受精率.体外成熟卵母细胞具有常规受精的能力,第3天胚胎质量与ICSI相似.患者年龄和卵母细胞体外成熟时间是体外成熟卵母细胞发育能力的主要影响因素.     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33- 40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in- vitro fertilization.      

Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in- vitro fertilization or intracytoplasmic sperm injection

The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using frozen-thawed spermatozoa. Follicular fluid did not increase the maturation of oocytes to metaphase II stage compared to control. After IVF, there was no difference in fertilization rates between FF- supplemented oocytes and controls (7/87, 8.4% of oocytes showing two pronuclei with FF versus 7/116, 6% with EMS; not significant). However, after ICSI, FF-supplemented oocytes showed significantly increased normal fertilization (32/85, 37.6% of two-pronuclear oocytes) and developmental potential (15/31, 48% cleavage) compared to the control oocytes (7/47, 14.9%, P < 0.01; and 2/48, 4%, P < 0.01, respectively). Overall, ICSI resulted in increased fertilization rates compared to IVF, regardless of the presence or absence of FF (39/132, 29.5% with ICSI versus 14/203, 6.9%). These results suggest that follicular fluid supplementation may improve the maturity of equine cumulus-enclosed oocytes sufficiently for the successful use of ICSI, but not sufficiently for normal sperm-egg interaction occurring during IVF.      

Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome

The present study examined whether the rates of oocyte maturation, fertilization and development, as well as pregnancy rate could be improved by human chorionic gonadotrophin (HCG) priming 36 h before immature oocyte retrieval in patients with polycystic ovarian syndrome (PCOS). Immature oocyte retrieval was performed on day 10-14 of the cycles and patients were randomly allocated either to be primed with 10 000 IU of HCG before the retrieval, or not primed. Immature oocytes were cultured for 24-48 h in TC-199 medium with 20% (v/v) inactivated fetal bovine serum (FBS) supplemented with 75 mIU/ml follicle stimulating hormone (FSH) and luteinizing hormone (LH). Intracytoplasmic sperm injection (ICSI) was performed in all mature oocytes and the resulting embryos were transferred on day 2 or 3 after ICSI. A total of 17 patients underwent 24 completed treatment cycles. Thirteen cycles were primed with HCG and 11 other cycles were not primed. The mean number of oocytes retrieved was comparable in the two groups (7.8 +/- 3.9 versus 7.4 +/- 5.2). The percentage of oocytes achieving maturation at 48 h was significantly higher (P < 0.05) in the HCG-primed group (84.3%, 86/102) than in the non-HCG-primed group (69.1%, 56/81). Oocyte maturation was hastened in the HCG-primed group. Following 24 h of culture, 78.2 +/- 7.1% of oocytes were matured in the HCG-primed group compared with 4.9 +/- 2.5% of oocytes in the non-HCG-primed group (P < 0.001). There were no significant differences in the rates of oocyte fertilization and cleavage in these two groups. There were five clinical pregnancies (38.5%) in the HCG-primed group, and three pregnancies (27.3%) in the non-HCG-primed group.     

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.     

Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor

BACKGROUND: The use of hormones for controlled ovarian stimulation results in follicular heterogeneity, with oocytes at diverse stages of nuclear and cytoplasmic development. This study evaluated the impact of temporary nuclear arrest by a specific phosphodiesterase 3-inhibitor (PDE3-I), cilostamide, on nuclear and cytoplasmic maturation of cumulus-free germinal vesicle (GV) human oocytes from controlled ovarian stimulated cycles. METHODS: GV oocytes (n = 234) were cultured in: (i) medium without the inhibitor (control); (ii) medium supplemented with 1 microM cilostamide and (iii) medium supplemented with 10 microM cilostamide. Oocytes in groups (ii) and (iii) were exposed to cilostamide for 24 h. The PDE3-I was subsequently removed by transfer of oocytes to fresh in vitro maturation (IVM) medium and the reversibility of GV arrest was assessed during IVM culture for maximum 48 h. RESULTS: Cilostamide (1 and 10 microM) could maintain >80% of the oocytes at the GV stage, without affecting subsequent maturation to metaphase II. Oocytes exposed to 1 microM cilostamide were more likely to have normal bipolar spindles with aligned chromosomes than control oocytes (P < 0.05). When GV chromatin configurations before and after arrest were compared, a significantly higher proportion of oocytes had acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (P < 0.05). CONCLUSIONS: Temporary nuclear arrest of human GV oocytes with PDE3-I proved to be beneficial for obtaining normal spindle and chromosome configurations after IVM. It resulted also in synchronization within the population of GV oocytes.     

Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice

BACKGROUND: Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as growth differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. METHODS: Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. RESULTS: Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. CONCLUSIONS: Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.     

Maturation arrest of human oocytes as a cause of infertility: case report

Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case series of infertile couples for whom all oocytes repeatedly failed to mature during IVF/ICSI. Eight couples, all presenting with unexplained infertility, underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped of cumulus cells prior to the ICSI procedure and their maturity status was defined. In each couple, oocyte maturation was repeatedly arrested at the germinal vesicle (GV) (n = 1), metaphase I (MI) (n = 4) and metaphase II (MII) (n = 3) stage. Oocyte maturation arrest may be the cause of infertility in some couples previously classified as having unexplained infertility. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the yet poorly defined entity currently known as 'oocyte factor'. The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject of further investigation.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

卵丘细胞分泌的趋化因子5与辅助生育结局的相关性

目的探讨卵丘细胞分泌的趋化因子5能否预测卵母细胞质量和辅助生育妊娠结局。方法收集75个体外受精周期中获得的562枚卵母细胞的卵泡液、受精液和其后的胚胎培养液,根据卵母细胞的发育命运进行分组：A组为未受精卵母细胞;B组受精后发育为非优质胚胎;C组受精后发育为优质胚胎;D组受精后发育为妊娠胚胎。ELISA方法测定4组卵泡液、受精液和胚胎培养液中CCL5的含量,并将结果进行比较。并分析卵母细胞成熟与CCL5水平的关系。结果A组卵泡液和受精液中CCL5的含量最低。C组与B组卵泡液和受精液中CCL5的含量比较有显著差异,但与D组比较无明显差别。B、C、D3组胚胎培养液中CCL5含量比较无明显差异。MII期卵子卵泡液中CCL5含量明显高于MI期和GV期卵子。结论卵丘细胞分泌的CCL5水平与卵母细胞的成熟和妊娠结局相关,可为选择移植胚胎和预测妊娠结局提供新的参考标准。     

Birth of a healthy infant after in vitro oocyte maturation and ICSI in a woman with diminished ovarian response: case report

In vitro maturation of oocytes (IVM) has been developed as a treatment option for subjects with good prognosis in assisted reproduction. We present successful IVM treatment in connection with a woman from whom low numbers of embryos were obtained after repeated failed conventional IVF cycles. A 35 year old woman, after 5 years infertility and two intrauterine insemination and three conventional IVF cycles, underwent first an IVM cycle with low dose FSH stimulation, and after failure, another natural IVM cycle. Three oocytes were obtained. After 36 h of IVM the oocytes had reached metaphase II stage, and fertilization using ICSI resulted in one 4-cell stage embryo, which was transferred 2 days later. The result was an uneventful pregnancy and birth of a healthy female infant weighing 4150 g. IVM may be an option for women from whom only low numbers of oocytes are obtained after gonadotrophin stimulation.     

Technical approaches to correction of oocyte aneuploidy

BACKGROUND: This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes. METHODS: GV stage mouse oocytes were collected from unstimulated ovaries, and human GV oocytes were donated from consenting patients undergoing ICSI. Stromal (somatic) cells were isolated from uterine biopsies of consenting patients. Mouse cumulus cells were obtained after ovarian stimulation. GV ooplasts prepared by removing nuclei were transplanted either with GV nuclei or with somatic cells by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were inseminated or analysed cytogenetically. Ooplasmic dysfunction was induced by photosensitization with a mitochondria-specific fluorescent dye. RESULTS: GV transplantation had an overall efficiency of 87 and 73% in the mouse and humans respectively. Maturation rates of 95 (mouse) and 64% (human) following reconstitution were comparable with those in control oocytes, as was the incidence of aneuploidy for five chromosome-specific probes after aneuploidy among the reconstituted oocytes. Photosensitization of oocytes significantly reduced the maturation rate to 4.2%, whereas 61.9% of oocytes matured after transfer of photosensitized GV karyoplasts into healthy ooplasts, with 52% of these mature oocytes being successfully fertilized by ICSI. Enucleated immature oocytes receiving mouse cumulus or human endometrial cell nuclei extruded a polar body in >40% of cases. Five out of seven successfully transferred aged human nuclei exhibited the expected number of signals with five chromosome-specific probes suggesting an appropriate chromosome separation in young ooplasm. CONCLUSIONS: Nuclear transplantation itself does not appear to interfere with chromosome segregation and can possibly rescue oocytes with damaged mitochondria. Finally, immature mouse ooplasm supported separation of somatic chromosomes to expected numbers, implying that haploidization may be occurring. The roles of genetic imprinting and fidelity of chromosome segregation are unknown.