Maturation Of Human Oocytes Research Articles

Vitrification of human mature oocytes in a straw to prevent the risk of liquid nitrogen contamination during storage

Vitrification of human mature oocytes in a straw to prevent the risk of liquid nitrogen contamination during storage

Freezing of human immature oocytes using cryoloops with Taxol in the vitrification solution

Objective: Oocytes freezing is very useful for Assisted Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including our own have been reported after the freezing of human mature oocytes. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer.Design: A prospective study.Materials and Methods: The immature oocytes allocated to this study were recovered from patients undergoing ICSI. Cumulus cells were removed by the hyaluronidase treatment and those having a visible germinal vesicle oocytes were used for the study (C(-)). Some oocytes that had a remaining two or three layers of cumulus cells, and had visible germinal vesicle oocytes were defined as C(+) immature oocytes. Vitrification using cryoloop was performed using ethylene glycol and dimethyl sulfoxide (DMSO) with or without Taxol (T(+) or T(-)). We divided four freezing group; C(+)T(+), C(+)T(-), C(-)T(+) and C(-)T(-). After thawing, immature oocytes were treated in a maturation culture, and metaphase a oocytes were treated using ICSI and cultured.Results: There were no significant differences in survival rate, maturation rate and fertilization rate. In the group of C(+)T(+), there was a significant difference in cleavage rate with C(+)T(-) and C(-)T(+) (87.5%, 42.9% and 44.4%, respectively). In the group of C(+)T(+), one embryo was developed to blastocyst at day 5 after ICSI.Conclusions: Our results showed that vitrification of immature oocytes is a good method of developing mature oocytes. Moreover, using vitrification solution with Taxol proved so useful that it developed one embryo to blastocyst stage. But most embryos were arrested until 8cell stages, and the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about immature oocytes on unstimulated cycles are needed. Objective: Oocytes freezing is very useful for Assisted Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including our own have been reported after the freezing of human mature oocytes. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. Design: A prospective study. Materials and Methods: The immature oocytes allocated to this study were recovered from patients undergoing ICSI. Cumulus cells were removed by the hyaluronidase treatment and those having a visible germinal vesicle oocytes were used for the study (C(-)). Some oocytes that had a remaining two or three layers of cumulus cells, and had visible germinal vesicle oocytes were defined as C(+) immature oocytes. Vitrification using cryoloop was performed using ethylene glycol and dimethyl sulfoxide (DMSO) with or without Taxol (T(+) or T(-)). We divided four freezing group; C(+)T(+), C(+)T(-), C(-)T(+) and C(-)T(-). After thawing, immature oocytes were treated in a maturation culture, and metaphase a oocytes were treated using ICSI and cultured. Results: There were no significant differences in survival rate, maturation rate and fertilization rate. In the group of C(+)T(+), there was a significant difference in cleavage rate with C(+)T(-) and C(-)T(+) (87.5%, 42.9% and 44.4%, respectively). In the group of C(+)T(+), one embryo was developed to blastocyst at day 5 after ICSI. Conclusions: Our results showed that vitrification of immature oocytes is a good method of developing mature oocytes. Moreover, using vitrification solution with Taxol proved so useful that it developed one embryo to blastocyst stage. But most embryos were arrested until 8cell stages, and the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about immature oocytes on unstimulated cycles are needed.

Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes

The purpose of this study was to determine if there is any additional benefit from FSH priming in addition to hCG priming on in-vitro maturation (IVM) programmes. Sixty women with polycystic ovary syndrome (PCOS) who underwent 68 IVM cycles were randomized by computer-generated numbers to receive FSH stimulation or not. Thirty-five cycles were pretreated with 75 IU rFSH for 6 days, and 33 cycles were not. Every cycle was given hCG 10,000 IU 36 h before oocyte retrieval. Immature oocytes were matured in vitro and fertilized by ICSI, and the resulting embryos were replaced on day 2 or 3. A total of 1528 immature oocytes were recovered. The overall maturation and fertilization rates were 74.2 and 72.8% respectively. After embryo transfer, 23 pregnancies resulted (33.8%). The oocyte numbers and endometrial thickness were similar between FSH-primed and non-FSH-primed groups. Serum estradiol level on the day of hCG injection was significantly higher in the FSH-primed group than in the non-FSH-primed group (377.2 pmol/l versus 143.8 pmol/l, P=0.001). The maturation rate, fertilization rate and pregnancy rate were 76.5, 75.8 and 31.4% respectively for FSH-primed group, and 71.9, 69.5 and 36.4% respectively for non-FSH-primed group (all not significant). IVM is a feasible treatment for women with PCOS. FSH priming has no additional beneficial effect on IVM.

Avaliação da aplicabilidade da técnica de maturação in vitro de oócitos humanos e posterior fertilização

PURPOSE: to evaluate the usefulness of the in vitro maturation technique of human oocyte and subsequent fertilization. METHODS: this is a prospective nonrandomized, descriptive study, carried out during the period of November 1999 to March 2001, with 20 cycles of in vitro fertilization of 15 patients with tubal infertility. All signed the written informed consent before the beginning of the study. The selected patients were at least 18 and at most 32 years of age, with only tubal infertility, and body mass index less than 25 kg/m². The patients received 300 UI of recombinant follicle stimulating hormone (FSH) by intramuscular injection at the second day of the cycle and additional doses of 150 IU at the fourth and sixth days of cycle. The oocyte retrieval was performed at the seventh day of the cycle. Those oocytes classified as immature were cultured in tissue culture medium 199 (TCM-199) with antibiotics, pyruvate, FSH, human chorionic gonadotropin (hCG) and serum (serum substitute supplement - Irvine Scientific®). After 48 h of culture, the oocytes that achieved metaphase II stage were inseminated, and the fertilized ones were transferred. RESULTS: one hundred and forty-four follicles were aspirated. There were 67 (46.5%) immature retrieved oocytes and 43 (64.2%) reached the metaphase II stage and were inseminated. Thirty fertilized oocytes and 25 embryos were transferred to 10 patients. There was one pregnancy with a baby born. CONCLUSION: we conclude that to mature human oocytes in vitro before in vitro fertilization is a procedure able to achieve pregnancy.

Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro.

This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 microM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.

Chromatin configuration and transcriptional control in human and mouse oocytes.

In vitro maturation of human oocytes at the germinal vesicle (GV) stage could offer an alternative in several cases of female infertility. It however rests on a better knowledge of the quality of human oocyte. Using fluorescence imaging of DNA and of the transcription sites, combined with electron microscopy, we show that human oocytes follow size-dependent changes in chromatin configuration, transcription sites distribution and nuclear ultrastructure that follow those observed in mouse GV oocytes. We thus analyzed in mouse GV oocytes the phosphorylation dependence of the transcriptional activity. We show by Western blot that, while active GV oocytes have approximately the same proportion of hypo- and hyperphosphorylated forms of the RNA polymerase II (RNAP II), the hyperphosphorylated form is almost absent from inactive oocytes. We also show that (1) RNAP II-dependent transcription is much less sensitive to various kinase inhibitors in mouse oocytes than in somatic cells or mouse one-cell embryos, although the phosphorylation equilibrium of RNAP II was largely shifted towards the hypo-phosphorylated form upon treatment with these inhibitors (2) RNAP I is completely insensitive to kinase inhibitors in GV oocytes.

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

The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro. 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. 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.

Oocyte Maturation in Humans: the Potential Relevance to Reproductive Medicine

Oocyte Maturation in Humans: the Potential Relevance to Reproductive Medicine

Multi-locus (ML)-FISH is a reliable tool for nondisjunction studies in human oocytes

In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes.

High xylosyltransferase activities in human follicular fluid and cultured granulosa-lutein cells.

Follicular fluid proteoglycans play an important role in human oocyte maturation, including the development of a fluid-filled compartment and maintenance of the hypocoagulative state of the follicular fluid. Human xylosyltransferase (EC 2.4.2.26, XT) is the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans and is secreted into body fluids together with large proteoglycans. We investigated the XT activities in human follicular fluid and granulosa-lutein cells from women undergoing IVF procedures. The mean XT activity was determined as 17.7 mU/l, which is 20-fold higher than in serum and the highest XT activity ever found in body fluids. Cultured human granulosa-lutein cells secreted large amounts of XT (14.52 micro U/10(6) cells), indicating that these cells are the main source of this enzyme in human follicular fluid. The XT from human follicular fluid was found to be associated with large chondroitin sulphate-containing proteoglycans. Furthermore, heparin was shown to bind strongly to the follicular fluid XT and to inhibit its enzyme activity. These findings indicate that XT may play a role in maintaining the haemostatic potential of the follicular fluid.

Culture environment modulates maturation and metabolism of human oocytes.

The clinical use of oocytes matured in vitro for IVF is increasing, but little is known about the effect of culture conditions on oocyte maturation. Denuded immature oocytes identified following superovulation prior to ICSI were individually matured in one of two commercial media: tissue culture medium (TCM) 199 or modified Eagle medium with Earle's modified salts (MEME). During maturation, depletion of pyruvate and accumulation of lactate in the culture medium were non-invasively measured. Maturing oocytes took up pyruvate (20-30 pmol/oocyte/h) and produced lactate (2-10 pmol/oocyte/h). Oocytes matured faster in MEME, with significantly more oocytes reaching metaphase II by 24 h after oocyte retrieval compared with TCM 199 (P = 0.03). The oocytes that matured more quickly in MEME had significantly lower lactate production than oocytes that matured more slowly (P = 0.02). In TCM 199, pyruvate uptake by rapidly maturing oocytes was lower than by slowly maturing oocytes (P = 0.05). During the second incubation, from 24 to 48 h post-oocyte retrieval, pyruvate uptake in MEME was 30% lower than in TCM 199 (P = 0.007). Pyruvate uptake and lactate production differed depending on the stage of nuclear maturation: pyruvate uptake and lactate production were greater during germinal vesicle breakdown than during polar body extrusion in MEME (P < 0.05). We have shown that: (i). pyruvate is a major energy source during oocyte maturation; (ii). the composition of the culture medium can affect the rate of maturation; and (iii). the culture medium and stage of nuclear maturation can affect pyruvate uptake and lactate production.

Enhancement of developmental capacity of meiotically inhibited bovine oocytes by retinoic acid.

Although high vitamin A may be teratogenic to the embryo, retinol has been shown to support oocyte developmental potential in vivo. Similarly, addition of retinol metabolite 9-cis-retinoic acid to in-vitro cultured oocytes could promote cytoplasmic maturation and subsequent early embryonic development. The objective of this study was to evaluate the effects of 5 nmol/l retinoic acid during in-vitro pre-maturation and maturation of bovine oocyte-cumulus complexes. Oocytes were aspirated from cows and either kept under meiotic arrest with 25 micro mol/l roscovitine and matured, or allowed to mature in permissive conditions (control). Cortical granule migration was analysed both after pre-maturation and maturation by fluorescent labelling of oocytes and subsequent laser confocal and fluorescence microscopy. Variables studied after in-vitro fertilization and culture in modified synthetic oviduct fluid were: (i) in-vitro embryonic development; (ii) ability of blastocysts to survive vitrification and warming; and (iii) differential cell counts measured by fluorescence microscopy. Although the presence of 5 nmol/l retinoic acid throughout in-vitro maturation was harmful, its presence during pre-maturation alone improved cytoplasmic granular migration, embryonic development, cryopreservation tolerance, total cell numbers and, as a consequence, embryonic quality. Pre-maturation in the presence of retinoic acid improves cytoplasmic competence of in-vitro matured bovine oocytes. Until more is known of the molecular mechanisms it would be irresponsible to use retinoic acid in maturation of human oocytes, especially in view of the narrow time window and possible species-specific differences in susceptibility and protection of the oocyte from epigenetic influences of retinol.

Higher cortisol:cortisone ratios in the preovulatory follicle of completely unstimulated IVF cycles indicate oocytes with increased pregnancy potential.

Conception following gonadotrophin-stimulated IVF and embryo transfer has been associated with a higher intrafollicular cortisol:cortisone ratio and decreased metabolism of cortisol to cortisone. The role of glucocorticoids in human oocyte maturation is not fully understood, but active glucocorticoid (cortisol) may be important. This study relates intrafollicular cortisol and cortisone concentrations to oocyte fertilization and embryo implantation in unstimulated cycles. Patients aged <40 years with favourable sperm underwent unstimulated IVF-embryo transfer. Study 1 related intrafollicular cortisol levels to oocyte and IVF outcome: (i) fertilized, pregnant (n = 9); (ii) fertilized, not pregnant (n = 21); and (iii) unfertilized (n = 12). Study 2 was a case-control study of 27 patients (same outcome groups of equal size) which measured intrafollicular cortisol, cortisone and the cortisol:cortisone ratio. Conception cycles demonstrated higher cortisol concentrations compared with the fertilized group (study 1) [median (95% confidence interval): 299 (249-330) versus 227 nmol/l (185-261); P < 0.05] and higher cortisol:cortisone ratios when compared with the unfertilized group (study 2) [7.38 (5.23-9.19) versus 3.56 (1.75-7.46) respectively; P = 0.02]. Of the women with cortisol:cortisone ratios greater than the outcome independent mean of 5.90, 58% conceived compared with only 13% with ratios <5.90 (P < 0.02). Higher cortisol:cortisone ratios in conception cycles suggest that active glucocorticoid may be important for final oocyte maturation and embryo implantation in unstimulated cycles.

Successful healthy birth in vitro maturation of human immature oocytes · Attempt of the unstimulated menstrual cycles and Frozen-Thawed embryo cycles

Objective: The IVM of human oocytes retrieved during unstimulated cycles is a reproductive technology of increasing interest. This study was examined about IVM in the unstimulated menstrual cycles (Fresh cycle) and frozen-thawed embryo transfer in hormone replacement cycles (Frozen cycle).Design: A retorospective randomized studyMaterials/Methods: Immature oocytes were retrieved transvaginally between days 8–23 of unstimulated menstrual cycles from women. Oocytes were cultured in TCM199 medium containing 20% heat-inactivated Follicular fluid, 10IU/ml FSH, 20IU/ml hCG, 10ng/ml Estradiol for 24 hours at 37°C and under 5% CO2, 5% O2, 90% N2 humidified atmosphere. All oocytes matured in metaphase2 were injected with husband sperm, and then cultured in fertilization medium for the first 16–18 hours. Fertilized embryos were changed medium to cleavage medium for 3 days until embryo transfer in Fresh cycle. In Frozen cycle, Zygotes on day 1 after ICSI were frozen, and then the thawed embryos were transferred at 2–8 cells stage with hormone replacement.Results: 19 cycles in Fresh cycle and 9 cycles in Frozen cycle from our program were treatment this study. The mean of oocytes aspirated per patients was 8.9 ± 5.9. After 24 hours of maturation 51.8% (88/170) of oocytes had a visible first polar body. After insemination, 78.4% (69/88) of the oocytes were fertilized and cleavage rate were 97.4% (37/38). The embryos were cultured for 3 days after insemination. Frozen-thawed embryos, 78.6% (22/28) were recovered and cleavage rate were 100% (22/22). The average of embryo transfer was 2.8 ± 1.5 in Fresh cycle, 2.4 ± 1.0 in Frozen cycle. The clinical pregnancy rate of Fresh cycle and Frozen cycle was 23.1% (3/13) and 22.2% (2/9). The implantation rate was 8.1% (3/37) and 13.6% (3/22). One patient delivered a healthy singleton in Fresh cycle and healthy twins in Frozen cycle.Conclusions: This study demonstrates that human immature oocytes retrieved from women with unstimulated menstrual cycles can undergo maturation, fertilization, pregnancy, and that the resulting embryo can establish healthy birth. The hormone replacement cycle is a good method. This method can solve synchronization to embryo and endometrium. Pregnancy rate is never high, but it is big progress for the PCO patients. We confirm that further studies are good oocyte retrieval timing, IVM medium of high maturation rate and synchronization method.Supported by: None. Objective: The IVM of human oocytes retrieved during unstimulated cycles is a reproductive technology of increasing interest. This study was examined about IVM in the unstimulated menstrual cycles (Fresh cycle) and frozen-thawed embryo transfer in hormone replacement cycles (Frozen cycle). Design: A retorospective randomized study Materials/Methods: Immature oocytes were retrieved transvaginally between days 8–23 of unstimulated menstrual cycles from women. Oocytes were cultured in TCM199 medium containing 20% heat-inactivated Follicular fluid, 10IU/ml FSH, 20IU/ml hCG, 10ng/ml Estradiol for 24 hours at 37°C and under 5% CO2, 5% O2, 90% N2 humidified atmosphere. All oocytes matured in metaphase2 were injected with husband sperm, and then cultured in fertilization medium for the first 16–18 hours. Fertilized embryos were changed medium to cleavage medium for 3 days until embryo transfer in Fresh cycle. In Frozen cycle, Zygotes on day 1 after ICSI were frozen, and then the thawed embryos were transferred at 2–8 cells stage with hormone replacement. Results: 19 cycles in Fresh cycle and 9 cycles in Frozen cycle from our program were treatment this study. The mean of oocytes aspirated per patients was 8.9 ± 5.9. After 24 hours of maturation 51.8% (88/170) of oocytes had a visible first polar body. After insemination, 78.4% (69/88) of the oocytes were fertilized and cleavage rate were 97.4% (37/38). The embryos were cultured for 3 days after insemination. Frozen-thawed embryos, 78.6% (22/28) were recovered and cleavage rate were 100% (22/22). The average of embryo transfer was 2.8 ± 1.5 in Fresh cycle, 2.4 ± 1.0 in Frozen cycle. The clinical pregnancy rate of Fresh cycle and Frozen cycle was 23.1% (3/13) and 22.2% (2/9). The implantation rate was 8.1% (3/37) and 13.6% (3/22). One patient delivered a healthy singleton in Fresh cycle and healthy twins in Frozen cycle. Conclusions: This study demonstrates that human immature oocytes retrieved from women with unstimulated menstrual cycles can undergo maturation, fertilization, pregnancy, and that the resulting embryo can establish healthy birth. The hormone replacement cycle is a good method. This method can solve synchronization to embryo and endometrium. Pregnancy rate is never high, but it is big progress for the PCO patients. We confirm that further studies are good oocyte retrieval timing, IVM medium of high maturation rate and synchronization method. Supported by: None.

Comparison of two freezing methods for human immature oocytes with cumulus or cumulus free: Slow freezing versus vitrification (including the effect of Taxol)

Objective: Oocyte freezing is very useful for Assisited Reproductive Technology (ART), especially for malignant diseases, premature ovarian failure, and oocyte donation. But there has only been one successful delivery using frozen human immature oocytes (Tucker et al 1998), whereas many successful cases including ours were reported after the freezing of human mature oocytes in both slow-freezing (SF) and vitrification (V). We examined the feasibility using either enclosed cumulus cells or cumulus free cells using both SF and V methods (including the effect of Taxol) to find the best results of IVF-IVC. Design: A prospective study. Materials/Methods: At first, mature oocytes were collected from more than 10 mm diameter follicles of the patients stimulated by HMG-HCG with GnRH agonist. After that, cumulus-enclosed oocytes were collected from less than 10mm diameter follicles for this study. SF using straw was performed as cryoprotectants 1,2-propanediol(PROH) and sucrose(S). V using cryoloop was performed using ethylene glycol (EG) + dimethyl sulfoxide (DMSO)+S. We divided human immature oocytes into 5 groups: (Group A;SF, with cumulus, Group B;SF, cumulus free, Group C; V, with cumulus, Group D; V, cumulus free, Group E; V, with cumulus, Taxol 1μM). Results: Survival rate, maturation rate, fertilization rate, cleavage rate (Group A, B, C, D,E) were 100%(2/2), 100%(2/2), 100%(2/2), 100%(2/2): (GroupA), 84.0%(21/25),71.4%(15/21), 60.0%(9/15) 66.7%(6/9), (GroupB) 69.2%(9/13), 88.9%(8/9), 62.5%(5/8), 40%(2/5): (GroupC), 79.3%(23/29), 82.6%(19/23) 57.9%(11/19), 63.6%(7/11): (GroupD), 88.2%(15/17), 93.3%(14/15), 71.4%(10/14), 90.0%(9/10) (GroupE) respectively. There was a statistically significant difference between GroupC and GroupE in cleavage rate (p <0.05). Almost embryos arrested until 8 cell stages. Only one embryo of Group E developed to morula stage. Conclusions: Both slow freezing and vitrification methods using either enclosed cumulus or cumulus free immature oocytes were similar good results. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of human immature oocytes.But it is said that the arrested embryos derived from immature oocytes on stimulated cycles showed a high incidence of multinuclear blastomeres and aneuploidy. Further studies about human immature oocytes on unstimulated cycles are needed. Supported by: None.

Aging and infertility in women: a committee opinion

Aging and infertility in women: a committee opinion

Oocyte maturation in vitro.

In contrast to conventional assisted reproduction techniques in which metaphase II oocytes are retrieved for fertilization in vitro, during the in-vitro maturation of oocytes, immature germinal vesicle stage oocytes are retrieved and matured in the laboratory before fertilization and embryo transfer. Whereas in-vitro maturation is technically more demanding than in-vitro fertilization it carries many potential advantages in terms of lower treatment costs and greater safety, as a result of reducing both the number of clinical consultations and the amount of pharmacological intervention. Although the technology is still experimental, in-vitro maturation has been successfully used for the treatment of patients with polycystic ovarian syndrome, and a number of pregnancies and live births have been reported. Despite these successes and much continuing research effort, the overall efficiency of in-vitro maturation remains low, and neither clinical nor laboratory procedures can be considered to be as robust and routinely feasible as conventional in-vitro fertilization techniques. The immediate goals for in-vitro maturation programmes are therefore to increase germinal vesicle oocyte recovery rates, optimize culture conditions, improve oocyte maturation potential, and after fertilization to produce pregnancy rates that are at least equivalent to those obtained using conventional in-vitro fertilization. This review focuses on the physiology and application of human oocyte maturation in vitro, and will detail the recent advances reported in this rapidly advancing field.

Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development.

During the early preimplantationes of human embryos, pyruvate and lactate, but not glucose, are the preferred energy substrates. Transport of these monocarboxylates is mediated, in mammalian cells, by a family of transporters, designated as monocarboxylate transporters (MCTs). Human and mouse genetic expression of MCT members 1, 2, 3, 4 and basigin, a chaperone protein of MCT1 and MCT4, was qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in immature oocytes (germinal vesicle stage; GV), in non-fertilised metaphase II (MII) oocytes and in embryos from 2-cell stage to blastocysts. Transcripts encoding for MCT1 and MCT2 were present, under a polyadenylated form, in the majority of the human and mouse oocytes and early embryos. MCT3 transcripts were not detected in either human or mouse. MCT4 mRNA was not detected in human oocytes and embryos, but was present in mouse oocytes and embryos. This fact could imply differences in lactate transport and regulation of intracellular pH between human and murine early embryos. Basigin transcripts were present in mouse and human MII oocytes and preimplantation embryos, but were not detected at GV stage. However, using 3' end-specific primers in the RT reaction instead of Oligo(dT)12-18 primers, transcripts encoding for this protein were then detected at GV stage in both species. This result suggests that a regulated polyadenylation process occurs during oocyte maturation for these transcripts. Thus, basigin mRNA can be considered as a marker of oocyte cytoplasmic maturation in human and mouse species.

Identification and characterisation of known and novel transcripts expressed during the final stages of human oocyte maturation.

The final stages of oocyte maturation, from the germinal vesicle (GV) stage to metaphase II (MII) oocytes, are characterised by a series of dynamic events. These include germinal vesicle break down (GVBD), resumption of meiosis, and nuclear and cytoplasmic maturation to produce MII oocytes ready for fertilisation. To investigate the specific genes transcribed during these stages of oogenesis, we have prepared and analysed amplified cDNA representing the transcribed genes in a series of GV and MII oocytes. Differential display analysis disclosed that the overall gene expression profiles between different samples of GV oocytes are very similar, regardless of their source, while those between the MII oocytes are markedly variable. A comparison of expression profiles in oocytes and somatic (cumulus) cells identified several known genes preferentially-expressed in oocytes (e.g., a zona pellucida gene), as well as five novel sequences. Two of the five novel sequences are homologous to retrotransposon sequences, long terminal repeat (LTR) and long interspersed nuclear element (LINE) 1, and two other sequences show partial homology to known ESTs and genomic sequences. The remaining sequence, which is identical to shorter ESTs isolated from germ cell tumor cDNA libraries, was extended towards its 5' end by PCR, using the original cDNA preparation from which it was isolated as a template. Expression of the resultant 1.1-kb transcript is restricted to the testis and ovary, and its expression correlates with cell pluripotency in that it is expressed in embryonal carcinoma cells, but not in their differentiated derivative cells.

Cryopreservation of immature and mature human oocytes.

Cryopreservation of oocytes facilitates the long-term storage of oocytes for patients in danger of losing ovarian function. It also alleviates many of the ethical concerns associated with embryo cryopreservation. Problems associated with metaphase II oocyte cryopreservation include zona pellucida hardening and spindle damage. The cryopreservation of germinal vesicle-stage oocytes has been undertaken as a means of circumventing the problem of spindle damage in mature oocytes. One of the main disadvantages of immature oocyte cryopreservation is the fact that in vitro maturation is required post-thaw. The majority of live births from oocyte cryopreservation have involved the use of 1,2-propanediol and slow freezing protocols. Various methods have been used in an attempt to improve survival rates. These include vitrification and use of novel cryopreservatives. Future areas of concentration should include in vitro maturation, vitrification, and alternate cryopreservatives.