Immature Human Oocytes Research Articles

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

This study was conducted to report the clinical-pregnancy outcome after vitrification of the blastocysts produced from in vitro maturation cycles. The survival rate after thawing was 92.0% (92/100). The clinical-pregnancy and implantation rates were 43.8% and 23.6%, respectively. These results suggest that the blastocyst-stage embryos produced from in vitro maturation cycles can be safely cryopreserved through vitrification.

Repeated pregnancies and live births after in vitro maturation treatment

To discuss and describe the successful treatment of women with primary infertility using in vitro maturation (IVM) of immature human oocytes resulting in repeated pregnancies and live births in the same individuals. Case report. McGill Reproductive Center, Royal Victoria Hospital, McGill University. Six patients who underwent IVM treatment. In vitro maturation and fertilization of immature human oocytes. Clinical pregnancies and live births. We report 11 singleton and 2 twin live births as a result of IVM treatment in six women with polycystic ovary syndrome. In vitro maturation of human oocytes is a novel treatment in the management of infertility. It is an effective method of conception, especially in women who have polycyctic ovaries.

Effect of rescuing donated immature human oocytes derived after FSH/hCG stimulation following in vitro culture with or without Follicular Fluid Meiosis Activating Sterol (FF-MAS)—an embryo chromosomal and morphological analysis

Studies in mice and humans have shown that Follicular Fluid - Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS. Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 microM or 20 microM. Seventy +/-2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis. Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups. Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results.

Principal findings from a multicenter trial investigating the safety of follicular-fluid meiosis-activating sterol for in vitro maturation of human cumulus-enclosed oocytes

To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes. Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial. Third-level referral academic centers, including reproductive biology and genetics laboratories. Endocrinologically normal women with a medical indication for IVF or intracytoplasmic sperm injection, or healthy volunteers. Subjects were randomized at a ratio 1 to 6 into either conventional GnRH-agonist and recombinant FSH stimulation (IVO) for oocyte retrieval, or minimally stimulated in vitro maturation (IVM) with the use of recombinant FSH. Retrieved immature oocyte cumulus complexes were cultured for 30 or 36 hours in one of six IVM culture conditions containing FF-MAS (range, 0.1-20 microM). Polar body-extruded oocytes from the IVO and IVM groups were processed for chromosomal analysis. The primary endpoint was the incidence of metaphase II stage oocytes with numeric chromosomal abnormalities, using full (spectral karyotyping) or partial (fluorescent in situ hybridization with seven probes) karyotyping or Giemsa count. A secondary objective was to document the frequency of metaphase II oocytes after IVM with FF-MAS supplements. Oocyte cumulus complexes obtained from the IVO (mean, 8.9) and IVM (mean, 6.2) groups had equal maturation rates. Compared to IVO, exposure of germinal-vesicle oocytes for a maturation period of 30 hours did not increase aneuploidy. An exposure period of 36 hours doubled the aneuploidy rate, but this was significant only for the 20-muM dose of FF-MAS. Inclusion of 1-10 microM FF-MAS in a 30-hour IVM protocol is safe.

Effect of aspiration vacuum on the developmental competence of immature human oocytes retrieved using a 20-gauge needle

In-vitro maturation (IVM) of immature oocytes has been proposed as a potential alternative to conventional IVF treatment following ovarian stimulation. However, the effects of the oocyte retrieval conditions on subsequent development have not been well understood. This study assessed the effects of different aspiration vacuums during oocyte retrieval on the developmental competence of immature oocytes following IVM, IVF and embryo transfer, retrospectively. Immature oocytes were aspirated with 20-gauge needles with a vacuum of 180 or 300 mmHg. Immature oocytes were cultured in IVM medium for 26 h. All mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was carried out 2 or 3 days after ICSI. The percentage of cumulus-cell enclosed oocytes and of transferable embryos per retrieved oocytes in 180 mmHg (69.7% and 23.8%, respectively) were significantly higher ( P < 0.01) than those in 300 mmHg (46.2% and 12.8%, respectively). The ongoing pregnancy rate per retrieval cycle in 180 mmHg (30%) was higher ( P < 0.01) than that in 300 mmHg (4.3%). The data indicate that lower pressure of vacuum aspiration with a 20-gauge needle improves the developmental competence of immature oocytes following IVM, IVF and embryo transfer.

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

O-198: Leukemia inhibitory factor (LIF) induce cumulus expansion in immature human and mouse oocytes and improves mouse fertilization and delivery rates when it is present during mouse in vitro oocyte maturation (IVM)

Studies have demonstrated that LIF concentrations are higher in preovulatory follicles than in developing follicles and a positive correlation exists between follicular fluid LIF concentration and embryo quality. Therefore we decided to examine the role of LIF during human and mouse IVM on cumulus expansion and oocyte competence. Mouse and human oocyte cumulus expansion was evaluated during IVM. Mouse oocyte competence was determined by IVF, IVC and embryo transfer (ET). Adult B6CBAF1/J females were primed with 5 IU of PMSG. Oocytes were collected 48 hs later. Immature human oocytes (4 donors, 25 oocytes) were collected after COH with rhFSH at the time of anticipated hCG administration. IVM medium was Alfa-MEM (Gibco 32571-036) supplemented with 10 % HSS (human oocytes) or FCS (mouse oocytes) and 1.5 IU rhCG. Oocyte treatments during IVM were ± rFSH and ± LIF (mouse 18 h and human 48 h) and cumulus expansion was evaluated. Mouse IVM oocytes, but not human oocytes continued to IVF and IVC for 4 days. Experimental groups: IVM oocytes ± 0.2 IU rFSH; IVM oocytes ± rFSH + LIF (0.1, 1, 1000 ng/ml); and in vivo matured (ovulated) oocytes. Mouse embryo development in vitro was recorded to Day 4, and blastocysts from all groups (395 blastocysts), were transferred to the uterine horn of 2.5-Day pseudopregnant females. Delivery rates were recorded. Logarithmic transformed data were analyzed by ANOVA and Tukey’s test. Results are expressed as Mean ± SE. LIF alone in the IVM medium induced cumulus expansion in human and mouse oocytes. There were no significantly differences between the standard IVM medium supplemented with rFSH vs. supplemented with LIF (P>0.05). But, when 1000 ng/ml LIF was added in combination with rFSH, a significant increase in cleavage rate, embryo development rate (Blastocyst/Total oocytes) and birth rate was observed when compared to oocytes matured with FSH alone (P<0.05). The birth rate of the IVM + rFSH + 1000 ng/ml LIF group was comparable to the in vivo matured control (endogenously ovulated oocytes) (P>0.05). In summary, LIF induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and IVM with rFSH + LIF during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development and birth rate Our results demonstrate that LIF induces cumulus expansion in human and mouse oocytes and has a profound effect on mouse IVM oocyte competence improving live birth rates. These results strongly suggest that addition of LIF and FSH to IVM media may improves the efficiency of human IVM in ART.

P-694: Gene expression profile of human immature and mature oocytes: A microarray approach

The understanding of the mechanisms regulating human oocyte maturation are still rudimentary. The aim of this study is to determine gene expression profiles of human nuclear mature and immature oocytes (germinal vesicle and metaphase I). We have identified transcripts differentially expressed between imature and mature oocytes and cumulus cells. The patients referred for cIVF or ICSI were stimulated with a combination of gonadotropin-releasing hormone agonist (GnRH-a) and recombinants FSH or Menopur. Germinal vesicle (GV, n=20), metaphase I (MI, n=20) and unfertilized MII (n=16) oocytes were collected 21 hours or 44 hours post insemination and frozen at -80°C in RLT buffer from the RNeasy kit (Qiagen) before RNA extraction. Pools of oocytes were analyzed by DNA microarrays on Affymetrix® HG-U133 plus 2.0 GeneChip oligonucleotide array chips made of 54675 probesets and representing roughly 35000 unique human known or predicted genes. The Affymetrix® GeneChip Operating Software 1.2 (GCOS) was used to evaluate the signal intensity for each probeset (detection call “present” or “absent”) and also to perform pair wise comparison between single gene expression raw values from oocytes of different stages. We determined genes exclusively expressed during one stage and a list of genes which expression progressively increased during oocyte maturation by selecting the probesets with the following ratios constraints: MI/GV > 1.73, MII/MI > 1.73 and MII/GV > 3, with a significant change p-value (P < 0.01) and where GV, MI and MII stands for signals values in these samples. Immature and mature oocytes express in average 8728 genes, but there is a clear decline from the GV and MI stages (n = 10 892 and 9682 respectively) to MII oocytes (n = 5 633). Nevertheless, within this contingent expression varies little between the GV, MI and MII samples (tight scatter plots = 0.63 and high correlation coefficients = 0.92). The most specific genes only found in one category were HKR3, ZNF165 and MAK for the GV stage, PTPN12 for the MI stage. Despite showing the lowest diversity of gene expression, MII oocytes displayed the most specific pattern among all oocytes by up-regulation of 444 genes. Some are specific to MII oocytes (SOX2OT, DMBX1/OTX3, TGFB3, BCL2L11 and CAS2) whereas others were gradually induced during maturation (HIC2, CDC25A, CCNJ, CCNG2, SOCS7 and CUL5). Very few genes were specifically over- or under-expressed in GV and MI cells. Interestingly, a marker like Epithin (ST14) is very specific of both GV and MI oocytes whereas it is absent from mature MII oocytes. Such gene represents a potential marker of human immature oocytes. We identified human genes that are specific to three oocyte developmental stages as well as genes which expression increase gradually with the oocyte maturation process. Understanding these factors’ function will greatly improve our command of in vitro fertilization procedures. These genes already give insights about vital regulation pathways and will provide candidate cytoplasmic and nuclear markers of human oocyte maturation.

P-103: Addition of melatonin in in vitro maturation (IVM) medium increases maturation and fertilization of immature human oocytes

Melatonin (N-acetyl-5-methoxytryptamine), a major hormone of pineal gland in vertebrates, is known to be associated with regulation of the dynamic physiological functions in general. This hormone is present in human preovulatory follicular fluid and may relate reproduction. And its antioxidant properties as a scavenger are also reported. We have reported that the addition of melatonin to the in vitro maturation (IVM) medium improved nuclear maturation of the mouse GV oocytes and reduced apoptosis in cumulus cells.

P-697: The developmental competency of immature oocytes obtained from stimulated IVF cycles; comparison of two culture media

P-697: The developmental competency of immature oocytes obtained from stimulated IVF cycles; comparison of two culture media

Control of a particular micro-macro positioning system applied to cell micromanipulation

Biological research requires new tools for cell micromanipulations. Currently, biological cell sizes range from a few to hundreds of micrometers, their manipulation therefore belonging to the field of microrobotics. This paper presents a new wireless micromanipulation system which allows cells placed in a droplet of liquid to be pushed on a glass slide. The cell micropusher is a ferromagnetic object which follows the movement of a permanent magnet located under the glass slide. It has been proved in previous works that two kinds of micropusher movements can induce a movement of the pushed object: turning the micropusher around the contact point (rotation), or moving the micropusher in translation. Rotation allows an object to be placed with a precision below 1 /spl mu/m, but acts within a narrow range. Translation allows placement of an object with lower accuracy, but within a wide range. We propose a specific coarse-fine control strategy to push an object, with good precision, within a wide range. Furthermore, experimentation on polystyrene balls of 50 /spl mu/m in diameter, and immature human oocytes of 150 /spl mu/m in diameter are presented. Note to Practitioners-Biological research requires new tools for cell micromanipulations. Currently, biological cell sizes range from a few hundred micrometers; their manipulation, therefore, belongs to the field of microrobotics. This paper presents a new wireless micromanipulation system which allows cells placed in a droplet of liquid to be pushed on a glass slide. The cell micropusher is a ferromagnetic object which follows the movement of a permanent magnet located under the glass slide. It has been proven in previous works that two kinds of micropusher movements can induce a movement of the pushed object: turning the micropusher around the contact point (rotation), or moving the micropusher in translation. Rotation allows an object to be placed with a precision below 1 /spl mu/m, but acts within a narrow range. Translation allows placement of an object with lower accuracy, but within a wide range. We propose a specific coarse-fine control strategy to push an object, with good precision, within a wide range. Furthermore, experimentation on polystyrene balls of 50 /spl mu/m in diameter, and immature human oocytes of 150 /spl mu/m in diameter are presented.

The in vitro maturation (IVM) of human oocytes for in vitro fertilization (IVF): is it time yet to switch to IVM-IVF?

The recent study by Li et al. observed that human oocytes from patients with polycystic ovary syndrome (PCOS) matured in vitro exhibited a higher proportion of abnormal spindle structures and disturbed chromosomal configurations compared with in vivo-matured oocytes from a control group of PCOS patients. This article discusses the obstacles that must be overcome and factors that must be monitored when attempting to optimize conditions for the in vitro maturation of human oocytes, with particular attention to the strengths and weaknesses of the study by Li et al.

Mammalian Oocyte in Vitro Maturation: The Endomembrane System Dynamics

The process of oocyte maturation encompasses a serie of molecular and structural events, culminating in the arrest of the oocyte chromosomes on the metaphase II plate (MII). Contrary to bovines, maturation in vitro can be accomplished in humans, but it is associated with a loss of developmental and functional competence. This loss can be associated with a poorly understood absence or abnormal distribution of specific proteins and organelles during attempts of germinal vesicles (GV) culture to MII in vitro. Cyclin B-Cdc2 kinase phosphorylates Golgi apparatus (GM130), while PP2A phosphatase dephosphorylates the residue during telophase. Since mammalian oocytes contain large stockpiles of cyclin B1, cyclin B2 and cdc2, it is likely that these molecules act on Golgi matrix proteins during meiosis to partition the organelle. In this report we focus on the dynamics of the Golgi matrix and Endoplasmic Reticulum (ER) in bovine and human oocytes during meiosis. Differences between these two animal models and between in vivo and in vitro human oocyte maturation were considered for Golgi and ER distribution. Prospective and descriptive basic study performed in an IVF private clinic affiliated to University of Buenos Aires. Confocal Scanning visualization of bovine and human immature and mature oocytes was performed after in vivo and in vitro maturation (220 bovine and 16 human oocytes). Human in vitro oocyte maturation was performed in human tubal fluid supplemented with 15% of synthetic serum in 5% CO2 ,100% humidity at 37°C for 24hs. Bovine in vitro oocyte maturation was performed in M199 media; 5% fetal calf serum and FSH in 5% CO2 , 100% humidity at 39°C for 24hs. Fixation and permeabilization were done with formaldehyde and Triton X-100 respectively. Golgi and ER were identified using GM-130 and Calreticulin antibodies respectively. DNA was labeled using TOTO-3 and samples were imaged using a confocal microscope. Golgi and ER dynamics was also studied after microtubule stabilization and depolymerization (taxol and nocodazole respectively). Our preliminary results show bovine and human immature oocytes (GVs,) with independent Golgi structures that vesiculate following Germinal Vesicle Breakdown (GVBD). The matrix fraction of the Golgi fragments after GVBD and disperses in bovine and human MII oocytes. In preliminary observations, human oocyte fragmentation of the Golgi matrix during in vitro maturation redistributes punctate to clusters of ER in a similar way to bovines. No apparent differences were found in terms of the endomembranes distribution between in vivo and in vitro human oocyte maturation. In vitro disruption of microtubules during oocyte maturation affects Golgi and ER distribution and the maturation rate, suggesting that a microtubule-dependent movement of these organelles is essential during in vitro oocyte maturation. Although differences in terms of bovine and human oocyte competence during in vitro maturation have been described, these two models are comparable in terms of Golgi and ER distribution until MII arrest. Enlargement of the sample and more research is needed to better describe the presence of checkpoints and the role of translation and protein uptake on completing oocyte maturation in vitro and in vivo. These investigations will enhance our understanding of mammalian oocyte maturation with special emphasis on clinically valuable applications in humans.

Mammalian Oocyte Maturation and Microtubule-Associated Proteins Dynamics

Mammalian Oocyte Maturation and Microtubule-Associated Proteins Dynamics

Comparison of Basic Medium for the In Vitro Maturation for Human Immature Oocytes

Although in vitro maturation-in vitro fertilization (IVM-IVF) program has been successfully applied into human ART program for PCO patients, the pregnancy rate of this procedure is still low. This disadvantage is caused by poor maturation of human immature oocytes and no synchronization with implantation windows. So, one of the key points for successful IVM procedure is the establishment of optimal maturation medium for proper cytoplasmic maturation, fertilization and development. This study was performed to find a proper basic medium for better quality of oocytes and embryos by comparing two types of commercial IVF media.

Preimplantation Genetic Diagnosis of Embryos Developing From Intracytoplasmic Sperm Injection of In Vitro Matured Metaphase II Human Oocytes

The recovery of immature human oocytes is a common occurrence in routine IVF. While in vitro maturation (IVM) of germinal vesicle (GV) or Metaphase I (MI) stage oocytes can be accomplished in conventional culture medium, the viability of these oocytes is questionable. Cell cycle regulatory mechanisms could be altered, and although fertilization can be achieved, the downstream affects of IVM are largely unknown. Here, we assessed chromosomal status of the embryos that developed from in vitro matured MI oocytes which had reached the Metaphase II stage at the time of ICSI which was performed within four hours post egg retrieval.

RNA interference in meiosis I human oocytes: towards an understanding of human aneuploidy

Although female meiosis I errors account for the majority of human aneuploidy, their molecular basis is largely unknown. By elucidating gene function, gene knockdown using RNA interference (RNAi) could shed light on this enigmatic process. In practice, however, the extreme paucity of immature human oocytes makes the evaluation of gene-targeting tools difficult. Here, we undertake RNAi in human oocytes and describe an approach employing mouse oocytes which could overcome the problem of limited biological material. We designed a short interfering RNA (siRNA) designated si539 to target the human mitotic arrest deficient 2 (hMad2) spindle checkpoint component. In human oocytes microinjected with si539, the hMad2 signal detected by Western blotting was 85-92% less intense than in oocytes injected with control siRNA indicating efficient silencing. Further examination of si539's targeting efficiency was undertaken using a green fluorescent protein (GFP)-tagged hMad2 mRNA construct in mouse oocytes. Consistent with Western blot analysis, si539 reduced hMad2-GFP expression in mouse oocytes by approximately 94% and relieved the meiosis I arrest otherwise induced by hMad2-GFP in mouse oocytes. By facilitating the investigation of candidate genes involved in regulating human female meiosis I, this approach can bring us closer to understanding the origins of aneuploidies such as Down's syndrome.

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa

In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems. Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA. Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients. This result demonstrates that the successful pregnancies and live births can be established from embryos produced from in vitro matured oocytes that fertilized with testicular sperm.

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

In human frozen immature oocytes, there has been little successful delivery. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. We set four experimental groups that immature oocytes has cumulus cells or not, or including Taxol or not in the vitrification solution. Frozen-thawed oocytes have been performed IVM, ICSI, and IVC. There were no significant differences in survival, maturation, and fertilization rate, respectively. However, in the group enveloped by cumulus cells and including Taxol in the vitrification solution, one embryo was developed to blastocyst. Our results showed that using vitrification solution with Taxol proved so effective.

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes.

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.