Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P<0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

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237EFFECTS OF IN VITRO V. IN VIVO CULTURE ON EXPRESSION OF EMBRYO DERIVED GENE TRANSCRIPTS INVOLVED IN APOPTOSIS IN SINGLE BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab237 ◽

2004 ◽

Vol 16 (2) ◽

pp. 239

Author(s):

H.M. Knijn ◽

C. Wrenzycki ◽

P.L.A.M. Vos ◽

G.C. van der Weijden ◽

H. Niemann ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Relative Abundance ◽

Molecular Basis ◽

Culture Medium ◽

Specific Inhibitor ◽

Control Group ◽

Cell Stage ◽

Gene Transcripts

Earlier studies reported that the level of apoptosis in in vitro-produced bovine blastocysts is higher then in in vivo developed blastocysts (Gjorret et al., 2003: Biol. Reprod, in press). The molecular basis for this difference has not yet been studied. The regulation and execution of apoptosis is dependent on a cascade of events in which many proteins are involved. The aim of the present study was to analyze expression of BAX, a pro-apoptotic, and BCL-XL, a anti-apoptotic member of the BCL-2 family, and heat shock protein (HSP 70.1) transcripts in blastocysts cultured in vitro or in vivo. Furthermore, to verify if these transcripts detected in the blastocysts stages were newly expressed from the embryonic genome, a RNA polymerase II specific inhibitor, α-amanitin, was added to the culture medium from the zygote stage onwards, to block transcription. For the in vitro group, embryos were obtained from abattoir oocytes after IVM/IVF and IVC in SOF medium. For the in vivo group, embryos were collected from normally cyclic cows, superovulated with 3000 IU eCG (Intergonan;; Intervet, Tönisvorst, Germany) at day 7 po by non-surgical uterine flushing. The developmental stage of the embryos was determined with stereomicroscopy, and early blastocysts (eb), blastocysts (b), and expanded blastocysts (xb) were collected and frozen at −80°C. For transcription inhibition experiment, embryos were cultured in vitro as for the in vitro group until the 8- to 16-cell stage at 100h after the start of fertilization, one group with 10mM α-amanitin added to the culture medium (α-amanitin group) and the other group without (control group). A highly sensitive semi-quantitative RT-PCR assay (Wrenzycki et al., 1999) Mol. Reprod. Dev. 53, 8–18) was used to determine the relative levels of gene transcripts in single blastocysts and pooled 8- to 16-cell embryos. The relative abundance was calculated on a per cell basis per embryo. Assays were repeated on average eight times. Data on the relative expression of transcripts in blastocysts were analyzed by Anova followed by multiple pairwise comparisons using the Tukey test. No significant differences in relative abundance between in vitro and in vivo cultured embryos, for any of the developmental stages, were found for the three apoptosis related genes. The molecular basis for the difference in level of apoptosis between in vitro and in vivo cultured blastocysts is not related to the level of expression of BAX, BCL-XL and HSP transcripts but other genes involved in the apoptotic cascade may be responsible for the reported differences. The expression of BCL-XL and HSP 70.1 transcripts in the blastocysts was from embryonic origin as no expression of these transcripts was detected in the 8- to 16-cell stage embryos treated with α-amanitin. The expression of BAX gene transcripts was not affected by α-amanitin. Probably the maternally derived transcripts are very stable and not yet degraded at the 8- to 16-cell stage. Table 1 Relative abundance (±SEM) of BAX, BCL-XL, and HSP 70.1 transcripts in bovine single in vitro or in vivo-produced eb, b, and xb and in vitro produced pooled 8- to 16-cell stage embryos treated with or without α-amanitin

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Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality

Reproduction ◽

10.1530/rep.0.1260337 ◽

2003 ◽

pp. 337-346 ◽

Cited By ~ 106

Author(s):

P Lonergan ◽

D Rizos ◽

J Kanka ◽

L Nemcova ◽

AM Mbaye ◽

...

Keyword(s):

Relative Abundance ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Blastocyst Development ◽

Blastocyst Quality ◽

Gene Transcripts ◽

Culture Environment ◽

Bax Gene

The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.

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Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

Veterinary Sciences ◽

10.3390/vetsci8060110 ◽

2021 ◽

Vol 8 (6) ◽

pp. 110

Author(s):

Nathalie Meijerink ◽

Jean E. de Oliveira ◽

Daphne A. van Haarlem ◽

Guilherme Hosotani ◽

David M. Lamot ◽

...

Keyword(s):

Immune System ◽

Nk Cells ◽

Relative Abundance ◽

Nk Cell ◽

Feed Additives ◽

Microbiota Composition ◽

In Ovo ◽

Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

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Investigating the Intrafollicular Factors Affecting Oocyte Developmental Competency

10.26686/wgtn.17068070.v1 ◽

2021 ◽

Author(s):

◽

Zaramasina Clark

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Granulosa Cells ◽

Embryo Quality ◽

Cumulus Cells ◽

Significant Finding ◽

High Quality ◽

Final Maturation

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos. The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes. The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation. The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>

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P–199 A case report to suggest that there must be other mutations than PATL2 or TUBB8 to cause oocyte maturation arrest

Human Reproduction ◽

10.1093/humrep/deab130.198 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

E Molinari ◽

M Yang ◽

J Hu ◽

L Zhang ◽

D F Albertini ◽

...

Keyword(s):

Case Report ◽

Oocyte Maturation ◽

Fertilization Rate ◽

Functional Study ◽

Embryo Morphology ◽

Loss Of Function ◽

Maturation Arrest ◽

Polar Bodies

Abstract Study question What causes our patient’s repeated almost complete oocyte maturation arrest (OMA)? Summary answer Since we did not detect PATL2 and TUBB8 mutations, both known to cause OMA, this case was likely caused by mutations in HUS1 and ITGB3 What is known already OMA has been associated with loss-of-function in key genes, such as PATL2 and TUBB8. Such patients have, however, uniformly have been unable to conceive with IVF Study design, size, duration We here report the case of repeatedly presenting patient between 2009 until 2020 (age 30 at 1st and 41 at last visit). Participants/materials, setting, methods The couple underwent 7 IVF treatments under several ovarian stimulation protocols at different gonadotropin dosages and in different preparations to try to recruit mature eggs. She conceived in her 2nd IVF cycle in 2009 and delivered uneventfully in 2010. She then conceived spontaneously and delivered a healthy boy in 2014. The couple since then has been attempting another pregnancy. Remarkably, in all IVF cycles all eggs but one arrested at prophase. Main results and the role of chance The female demonstrates abnormally high ovarian reserve for age (AMH=5.9 ng/mL in 2019) (mean, 10.6 oocytes). In all cycles, all but one retrieved were immature. In vitro maturation rate for the GV oocytes was 28%. Resultant M2s, however, demonstrated morphological abnormalities, such as giant polar bodies. In vivo M2s, in contrast, were always morphologically unremarkable, and their fertilization rate was 85%. Embryo morphology deteriorated appreciatively with advancing age. Sanger sequencing for TUBB8 and PATL2 genes were unremarkable. Whole genome sequencing of her and her sister (who had no fertility problems) revealed mutations of genes belonging to the integrin family (ITGB3) and DNA repair checkpoint (HUS1), both of which could be determinants in the observed maturation arrest. Limitations, reasons for caution A functional study, coupled with imaging of the discarded material, will likely offer further information regarding the mechanisms leading to OMA in this female. Wider implications of the findings: This case report represents a new phenotype of female infertility, characterized by almost complete maturation arrest which, however, still offers opportunity for pregnancy. Further isolation of underlying mutation(s) may offer additional insights about checkpoints required for the transition of prophase to metaphase in human oocytes. Trial registration number NA

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P–442 Cancer does not adversely affect oocyte maturation in vitro with the exception of breast cancer

Human Reproduction ◽

10.1093/humrep/deab130.441 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

I Viran. . Klun ◽

J Bedenk ◽

N Jancar

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Hormonal Stimulation ◽

Infertile Women ◽

Maturation In Vitro ◽

Infertile Patients

Abstract Study question Do different types of cancer affect the success of oocyte maturation in vitro compared to infertile women included in the in vitro fertilization (IVF) program? Summary answer Cancer does not adversely affect oocyte maturation in vitro, with the exception of breast cancer, compared to infertile women in the in vitro fertilization program. What is known already Vitrification and storage of oocytes in liquid nitrogen is one of the real options for maintaining reproductive function in cancer patients. Despite careful hormonal stimulation of the ovaries, however, the proportion of oocytes is immature and lost to the patient. In vitro maturation of oocytes can play an important role in resolving immature oocytes and increasing the chances of conception in cancer patients. Moreover, it can mean a safe way to store oocytes when ovarian hormonal stimulation could worsen the disease. Therefore, the aim of this study was to determine whether different types of cancer affect oocyte in vitro maturation. Study design, size, duration After ovarian stimulation in 18 cancer patients, the number and maturity of oocytes were compared to 21 infertile patients in the IVF program over a three-year period. In both groups, 119 germinal vesicle-GV oocytes were matured in vitro to compare the maturation rate. After IVF in a subset of 17 infertile patients, the fertilization of in vitro and in vivo matured oocytes was compared in the same cycles. The procedure was considered in cancer patients. Participants/materials, setting, methods In this prospective study, forty-five GV oocytes in cancer patients and 74 GV oocytes in infertile patients underwent in vitro maturation procedure. Each oocyte was matured in vitro in the MediCult IVM System by conditioning in LAG medium and maturation for up to 28 hours in IVM medium with added hormones FSH and hCG, in coculture with cumulus cells from mature oocytes in the same patients. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Main results and the role of chance After controlled ovarian hormonal stimulation, 198 oocytes were retrieved in cancer patients and 259 oocytes in infertile women and there were no significant differences in the number of retrieved oocytes, proportion of degenerated oocytes and proportion of GV oocytes. In cancer patients, the proportion of oocytes that matured in vitro was lower than in infertile patients (66.0 vs. 80.0%), but the difference was not significant. Among cancer patients, the oocyte maturation rate tended to be lower in patients with breast cancer than in patients with other cancers (54.5% vs. 81.2%; difference not significant). However, in patients with breast cancer, significantly fewer oocytes matured in vitro than in infertile patients (54.5% vs. 80.0%; P < 0.05, Chi-Square test) even though they tended to be younger (29.3 ± 7.4 vs. 33.4 ± 5.0 years; non-significant difference). After in vitro maturation, there was a 13% increase in mature oocyte yield in cancer patients and a 20.1% increase in infertile women with no significant difference observed. After ICSI in a subset of infertile women, there was approximately the same fertilization rate between oocytes matured in vitro and in vivo (55.1% vs. 57.0%) in the same cycles. Limitations, reasons for caution For ICSI in oocytes matured in vitro, we had to use semen collected the day before, while oocytes matured in vivo were fertilized with fresh semen in the same cycle. Therefore, we could not compare the development of embryos in both groups. Wider implications of the findings: In vitro maturation of oocytes in connection with their vitrification or vitrification of embryos after their fertilization appears to be a valuable way to maintain the fertility of young cancer patients, but a worse outcome is expected in breast cancer patients. Trial registration number National Medical Ethical Committee Approval, No. 0120–222/2016–2; KME 115/04/16.

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The Usefulness of Retinoic Acid Supplementation during in Vitro Oocyte Maturation for the in Vitro Embryo Production of Livestock: A Review

Animals ◽

10.3390/ani9080561 ◽

2019 ◽

Vol 9 (8) ◽

pp. 561 ◽

Cited By ~ 4

Author(s):

Abdelnour ◽

El-Hack ◽

Swelum ◽

Saadeldin ◽

Noreldin ◽

...

Keyword(s):

Retinoic Acid ◽

Embryo Development ◽

Oocyte Maturation ◽

Mammalian Species ◽

Antioxidant Properties ◽

Reproductive Function ◽

Embryonic Growth ◽

Oxidative Damages

Retinoic acid (RA) is an indigenous metabolite and descriptive physiologically functioning constituent of vitamin A. Retinoids were documented as vital regulators for cell development and distinction, embryonic growth, and reproductive function in both male and female livestock. Previously, RA has been shown to have several positive impacts in vivo and in vitro and critically control many reproductive events, such as oocyte development, follicular growth, and early embryonic growth. In addition, RA manages apoptotic signaling and oxidative damages in cells. Recently, RA has been used widely in assisted reproductive technology fields, especially during in vitro embryo development in various mammalian species, including buffaloes, bovine, goats, sheep, pigs, and rabbits. However, the optimum concentration of RA greatly differs based on the condition of maturation media and species. Based on the obtained findings, it was generally accepted that RA enhances nuclear oocyte maturation, cleavage and maturation rates, blastocyst formation, and embryo development. As such, it possesses antioxidant properties against reactive oxygen species (ROS) and an anti-apoptotic effect through enhancing the transcription of some related genes such as superoxide dismutase, prostaglandin synthase, glutathione peroxidase, peroxiredoxins, and heme oxygenase. Therefore, the current review concludes that an addition of RA (up to 50 nM) has the potential to improve the oocyte maturation media of various species of livestock due to its antioxidant activity.

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In vitro and in vivo culture effects on mRNA expression of genes involved in metabolism and apoptosis in bovine embryos

Reproduction Fertility and Development ◽

10.1071/rd05038 ◽

2005 ◽

Vol 17 (8) ◽

pp. 775 ◽

Cited By ~ 43

Author(s):

Hiemke M. Knijn ◽

Christine Wrenzycki ◽

Peter J. M. Hendriksen ◽

Peter L. A. M. Vos ◽

Elly C. Zeinstra ◽

...

Keyword(s):

Glucose Metabolism ◽

Mrna Expression ◽

Critical Period ◽

Glucose Transporter ◽

Expression Patterns ◽

Culture Conditions ◽

Glut 4 ◽

Gene Transcripts

Bovine blastocysts produced in vitro differ substantially from their in vivo-derived counterparts with regard to glucose metabolism, level of apoptosis and mRNA expression patterns. Maternal embryonic genomic transition is a critical period in which these changes could be induced. The goals of the present study were twofold: (1) to identify the critical period of culture during which the differences in expression of gene transcripts involved in glucose metabolism are induced; and (2) to identify gene transcripts involved in apoptosis that are differentially expressed in in vitro- and in vivo-produced blastocysts. Relative abundances of transcripts for the glucose transporters Glut-1, Glut-3, Glut-4 and Glut-8, and transcripts involved in the apoptotic cascade, including BAX, BCL-XL, XIAP and HSP 70.1, were analysed by a semiquantitative reverse transcription–polymerase chain reaction assay in single blastocysts produced in vitro or in vivo for specific time intervals, that is, before or after maternal embryonic transition. Whether the culture environment was in vitro or in vivo affected the expression of glucose transporter transcripts Glut-3, Glut-4 and Glut-8. However, the critical period during culture responsible for these changes, before or after maternal embryonic transition, could not be determined. With the exception of XIAP, no effects of culture system on the mRNA expression patterns of BAX, BCL-XL and HSP 70.1 could be observed. These data show that expression of XIAP transcripts in expanded blastocysts is affected by in vitro culture. These findings add to the list of bovine genes aberrantly expressed in culture conditions, but do not support the hypothesis that maternal embryonic transition is critical in inducing the aberrations in gene expression patterns studied here.

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Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽

10.1530/rep.0.1210051 ◽

2001 ◽

pp. 51-75 ◽

Cited By ~ 221

Author(s):

A Trounson ◽

C Anderiesz ◽

G Jones

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Growth Phase ◽

Developmental Competence ◽

Minimum Size ◽

Success Rates ◽

Human Oocytes ◽

Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

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