Publications by authors named "Catherine M H Combelles"

32 Publications

Corrigendum: Bisphenol-A exposure and gene expression in human luteinized membrana granulosa cells in vitro.

Hum Reprod 2018 05;33(5):989

Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Tel Hashomer 52561, Israel.

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http://dx.doi.org/10.1093/humrep/dey068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527049PMC
May 2018

Spindle abnormalities and chromosome misalignment in bovine oocytes after exposure to low doses of bisphenol A or bisphenol S.

Hum Reprod 2018 05;33(5):895-904

Biology Department, Middlebury College, Middlebury, VT 05753, USA.

Study Question: What are the effects of exposure to bisphenol A (BPA) or bisphenol S (BPS) during IVM on bovine oocyte maturation, spindle morphology and chromosome alignment?

Summary Answer: Exposure to BPA or BPS during IVM resulted in increased spindle abnormalities and chromosome misalignment, even at very low concentrations.

What Is Known Already: BPA is an endocrine disrupting chemical that alters oocyte maturation, spindle morphology and chromosome alignment in a range of species. The use of BPA substitutes, such as BPS, is increasing and these substitutes often display different potencies and mechanisms of action compared with BPA.

Study Design, Size, Duration: Bovine cumulus-oocyte complexes (COCs) underwent IVM with BPA or BPS for 24 h, together with vehicle-only controls. Overall, 10 different concentrations of BPA or BPS were used ranging from 1 fM to 50 μM in order to detect low dose or non-monotonic effects. An incomplete block design was utilized for the study, with at least three replicates per block. A total of 939 oocytes (250 of which were controls) were used for the BPA experiments, and 432 (110 controls) for the BPS experiments. Following the IVM period, the oocytes were denuded and fixed for immunocytochemistry.

Participants/materials, Setting, Methods: Immunocytochemistry was used to label the chromatin, actin, and microtubules in the fixed oocytes. The meiotic stage was assessed using immunofluorescence, and the metaphase-II (MII) oocytes were further assessed for spindle morphology and chromosome alignment (in all MII oocytes regardless of spindle morphology) using immunofluorescence and confocal microscopy. Significant differences between the treatment and control groups were determined using chi-square and Fisher's exact tests.

Main Results And The Role Of Chance: There was no effect of BPA or BPS on the proportion of bovine oocytes that reached MII (P > 0.05). BPA and BPS increased spindle abnormalities in MII oocytes at almost all concentrations tested, including those as low as 1 fM (P = 0.013) or 10 fM (P < 0.0001), respectively, compared to control. Oocytes with flattened spindles with broad poles were observed at a higher frequency at some concentrations of BPA (P = 0.0002 and P = 0.002 for 10 nM and 50 μM, respectively) or BPS (P = 0.01 for 100 nM BPS), while this spindle phenotype was absent in the controls. BPA increased chromosome misalignment at concentrations of 10 fM, 10 nM and 50 μM (P < 0.0001 to P = 0.043 depending on the dose). BPS increased chromosome misalignment at concentrations of 10 fM, 100 pM, 10 nM, 100 nM and 50 μM (P < 0.0001 to P = 0.013 depending on the dose).

Limitations Reasons For Caution: Exposures to BPA or BPS were performed during the IVM of COCs to allow for determination of direct effects of these chemicals on oocyte maturation. Whole follicle culture or in vivo studies will confirm whether follicular cell interactions modify the effects of BPA or BPS on oocyte meiotic maturation. Investigation into the effects of BPA or BPS on other oocyte functions will determine whether these chemicals alter oocyte quality via mechanisms independent of the meiotic endpoints characterized here.

Wider Implications Of The Findings: The findings of this study show that both BPA and BPS induce spindle abnormalities and chromosome misalignment in bovine in a non-monotonic manner, and at concentrations that are orders of magnitude below those measured in humans. Taken in context with previous studies on the effects of BPA in a range of species, our data support the literature that BPA may reduce oocyte quality and lead to subsequent infertility. Additionally, these results contribute to the burgeoning field of research on BPS and suggest that BPS may indeed be a 'regrettable substitution' for BPA.

Study Funding/competing Interest(s): This study was supported by funding from the National Institutes of Health (NIH) (Grant 1R15ES024520-01). The authors declare no conflict of interest.
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http://dx.doi.org/10.1093/humrep/dey050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925783PMC
May 2018

In Vitro Exposure of Human Luteinized Mural Granulosa Cells to Dibutyl Phthalate Affects Global Gene Expression.

Toxicol Sci 2017 Nov;160(1):180-188

Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel-Aviv University, Israel.

Exposure to dibutyl phthalate (DBP) is ubiquitous among women of reproductive age. Previous studies in animal models and in human cells in vitro have shown that exposure to DBP disrupts ovarian function. Here, we examined the effect of DBP on global gene expression in mural granulosa cells (MGCs) in vitro. Primary cultures of MGC obtained from 48 patients undergoing IVF were treated with increasing concentrations of DBP (0, 0.01, 0.1, 1, 10, or 100 µg/ml) for 48 h. Microarray analysis was used to identify genes exhibiting expression changes following DBP exposure. When compared with untreated cells, exposure to 100 µg/ml DBP resulted in significant differences in expression of 346 annotated genes (> 2-fold; q value < .05). Of them, 151 were upregulated and 195 downregulated. The main functional annotations affected by DBP were associated with cell cycle, mitosis, Rho GTPases, PLK1, Aurora B signaling pathways, and E2F-mediated regulation of DNA replication. No significant differences in gene expression were observed for the lower concentrations of DBP (0.01, 0.1, 1, and 10 µg/ml) compared with controls for both the microarray analysis and genes validated by quantitative real-time (qRT)-PCR. This study provides important molecular inputs on the effect of short-term DBP exposure on human MGCs in vitro. Our results indicate that acute treatment with high concentrations of DBP alters gene expression pathways mainly associated with the cell cycle.
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http://dx.doi.org/10.1093/toxsci/kfx170DOI Listing
November 2017

Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro.

Reprod Toxicol 2017 04 16;69:13-18. Epub 2017 Jan 16.

Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Israel. Electronic address:

Exposure to di-butyl phthalate (DBP) exerts negative effects on female fertility in animal models, but human studies remain limited. Here, the effects of DBP exposure on mural granulosa cell function were investigated in primary cultures from women undergoing in vitro fertilization. Cultured cells treated with various doses of DBP (0, 0.01μg/mL, 0.1μg/mL, 1μg/mL, 10μg/mL, or 100μg/mL) for 48h were assessed using enzyme-linked immunosorbent assay and qRT-PCR. Treatment with 100μg/mL DBP resulted in significantly lower 17β-estradiol and progesterone production (p<0.01). It also resulted in altered mRNA expression of steroidogenic, angiogenic, and epidermal growth factor-like growth factor genes: CYP11A1 (p<0.001), CYP19A1 (aromatase) (p<0.001), VEGF-A (p<0.02), BTC (p=0.009), and EREG (p=0.04). StAR expression was impaired after exposure to both 10 and 100μg/mL (p<0.03 and p<0.001, respectively). Our results indicate that in vitro exposure of granulosa cells to high doses of DBP alters cell functions.
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http://dx.doi.org/10.1016/j.reprotox.2016.12.007DOI Listing
April 2017

Bisphenol-A exposure and gene expression in human luteinized membrana granulosa cells in vitro.

Hum Reprod 2017 02 15;32(2):409-417. Epub 2016 Dec 15.

Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Tel Hashomer 52561, Israel

Study Question: Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)?

Summary Answer: In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression.

What Is Known Already: Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in granulosa cells.

Study Design, Size, Duration: Primary cultures of MGC obtained from 24 patients undergoing IVF (for PGD, male factor infertility or unexplained infertility) were exposed to various concentrations of BPA (0, 0.02, 0.2, 2 or 20 µg/ml) for 48 h.

Participants/materials, Setting, Methods: The study was conducted in a university-affiliated hospital. Microarray analysis was used to identify genes exhibiting expression changes following BPA exposure. Genes significantly altered were identified based on changes greater than 2-fold relative to the control group (not treated by BPA) and a Student's t-test P-value <0.05. Statistical significance was adjusted for multiple comparisons using the Benjamini-Hochberg method. Alterations in the expression of genes that are involved in the enriched functional annotations altered by BPA at the concentration of 20 µg/ml were confirmed by real-time PCR.

Main Results And The Role Of Chance: A distinct pattern of gene expression was observed in primary cultures of MGC exposed to the highest BPA concentration compared with untreated cells. We identified 652 genes that exhibited at least 2-fold differences in expression after BPA exposure (all P < 0.05 versus untreated). These genes were significantly enriched for annotations related to cell cycle progression, segregation of chromosomes, steroid metabolism, apoptosis, lipid synthesis, oocyte maturation and chromosomal alignment. No significant changes in gene expression were found at the lower doses of BPA most relevant to human exposure.

Large Scale Data: N/A.

Limitations, Reasons For Caution: Human exposure to BPA in vivo occurs over long periods of time. In this in vitro model, cells were exposed to the chemical for 48 h only. Thus, the effects of BPA on the human follicle might be underestimated.

Wider Implications Of The Findings: As BPA exposure is ubiquitous, understanding the effects of the chemical on the ovary, specifically in women of reproductive age, has public health significance. The clinical evidence to date points to an association between BPA exposure and impaired IVF outcome, although not all studies have shown negative effects. Our study adds valuable mechanistic information showing that exposure to BPA alters granulosa cell gene expression at high and supra-physiological doses.

Study Funding/competing Interests: This study was supported by grant number 1936/12 from the ISF. The authors have nothing to disclose.
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http://dx.doi.org/10.1093/humrep/dew316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260419PMC
February 2017

Bisphenol-A and human oocyte maturation in vitro.

Hum Reprod 2013 Oct 30;28(10):2735-45. Epub 2013 Jul 30.

Department of Obstetrics, Gynecology and Reproductive Biology, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA.

Study Question: Does exposure to bisphenol-A (BPA) affect the maturation of human oocytes in vitro?

Summary Answer: There was a dose-response association of BPA exposure with altered human oocyte maturation in vitro.

What Is Known Already: There is widespread exposure of the general population to BPA. BPA has been detected in the human follicular fluid. Animal studies have shown that BPA exposure is associated with maturation arrest and spindle abnormalities in maturing oocytes.

Study Design, Size, Duration: A randomized trial, using 352 clinically discarded oocytes from 121 patients.

Participants/materials, Setting, Methods: The study population was drawn from patients undergoing IVF/ICSI cycles in our program at Brigham and Women's Hospital from March 2011 to April 2012. Oocytes from only one cycle for each patient were included in the study. Cycles with at least two germinal vesicle stage oocytes were included with random allocation of one oocyte to culture for 30 h without BPA and remaining sibling oocytes to medium-containing BPA (20, 200 ng/ml or 20 µg/ml). Oocytes were fixed and labeled for tubulin, actin and chromatin and examined with immunofluorescence and confocal microscopy. Oocytes were assessed for meiotic stage (n = 292), and those at metaphase II (MII, n = 175) were further classified according to their spindle configurations and patterns of chromosome alignment. McNemar's test was used to compare dichotomized maturation status. Generalized estimating equations were used to account for the correlation between oocytes from the same woman and for the spindle analysis.

Main Results And The Role Of Chance: As the BPA dose increased, there was a decrease in the percentage of oocytes that progressed to MII (P = 0.002) and increases in the percentage of oocytes that were degenerated (P = 0.01) or that had undergone spontaneous activation (P = 0.007). Among MII oocytes, as the BPA dose increased, there was a significant trend (by test for trend) for a decreased incidence of bipolar spindles (P < 0.0001) and aligned chromosomes (P = 0.02).

Limitations, Reasons For Caution: Although we used sibling oocytes to overcome potential confounders, such as infertility diagnosis and maternal age, additional studies with a larger number of oocytes are required to confirm the present results. Having access only to clinically discarded oocytes, we were limited to evaluating only those oocytes that failed to mature in vivo despite having been exposed to gonadotrophin stimulation and the ovulatory trigger of HCG.

Wider Implications Of The Findings: To our knowledge, this is the first study investigating the effect of BPA on oocyte meiotic maturation, spindle morphology and chromosome alignment in human oocytes. Together with prior animal studies, the data support the negative influences of BPA on cell cycle progression, spindle architecture and chromosome organization during oocyte maturation. Furthermore, the increased rates of abnormal maturation in oocytes exposed to BPA may be relevant to our understanding of the decrease in fertility reported in the last decades.

Study Funding/competing Interest(s): This study was funded by the NIEHS Center Grant Pilot Project (P30-ES000002). R.M. was sponsored by a fellowship from the Environmental Health Fund, Israel and by the Frederick L. Hisaw Endowment, Harvard School of Public Health. There are no conflicts of interest.

Trial Registration Number: n/a.
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http://dx.doi.org/10.1093/humrep/det312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777571PMC
October 2013

The use of immature oocytes in the fertility preservation of cancer patients: current promises and challenges.

Int J Dev Biol 2012 ;56(10-12):919-29

Middlebury College, Biology Department, Middlebury, VT 05753, USA.

Improved oncological treatments permit increased survival rates, although cancer patients remain at risk of losing ovarian function. An attractive option for fertility preservation includes the use of immature oocytes, a strategy which can occur on a rapid timeline and without hormonal stimulation. As a result, cancer therapy can proceed promptly even in patients with hormone-sensitive tumors. Following retrieval, immature oocytes can be cryopreserved at either the immature germinal vesicle or the mature metaphase-II stage, i.e. either before or after in vitro maturation (IVM). We present a critical review of previous human studies on the cryopreservation of immature oocytes. Evaluations include in vitro developmental competence upon thawing/warming, or organization of the spindle and chromosomes. Reported successes vary, perhaps in relation to the source of the oocytes and protocols for cryopreservation and IVM. Weaknesses exist with the experimental designs implemented to date, so caution must be exercised before considering the use of immature oocytes to be a safe and reliable practice in the fertility preservation of cancer patients. To date, results indicate that with current protocols, it may be best to cryopreserve immature oocytes after IVM at the metaphase-II stage. Nonetheless, efficacy remains very low. Future efforts should tailor and optimize not only cryo-preservation, but also IVM protocols for use in either germinal vesicle or metaphase-II oocytes, together with a comprehensive assessment of oocyte function and developmental competence to term. Despite current challenges, the burgeoning field of immature oocyte cryopreservation constitutes a promising option for cancer patients with impaired ovarian function.
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http://dx.doi.org/10.1387/ijdb.120132ccDOI Listing
August 2013

The antral follicle: a microenvironment for oocyte differentiation.

Int J Dev Biol 2012 ;56(10-12):819-31

Middlebury College, Biology Department, Middlebury, VT 05753, USA.

Mammalian reproduction hinges upon the timely ovulation of a fully differentiated oocyte. This event is the culmination of a complex and dynamic developmental relationship between the oocyte and the antral follicle housing it; the antral follicle constitutes a specialized microenvironment or niche, uniquely suited to the needs of the oocyte as it approaches ovulation. During this time, the oocyte must complete its final growth, capacitation, and nuclear and cytoplasmic maturation. Its microenvironment--the antral follicle--is in turn responsible for the integrity of these processes and the production of a high quality oocyte. Components of the antral follicle, including three distinct somatic cell types (theca, granulosa and cumulus), the basal lamina, and follicular fluid, each have active and regulatory roles in oocyte differentiation. Several milestones in antral folliculogenesis also have an influence on oocyte development. This review will discuss the antral follicle microenvironment with specific attention to its importance in oocyte differentiation. As assisted reproductive technologies (ART) often require stages of oocyte differentiation to occur in vitro rather than in vivo, current knowledge of the antral follicle microenvironment will also be discussed with respect to its clinical applications.
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http://dx.doi.org/10.1387/ijdb.120133ccDOI Listing
August 2013

Over 40 years of mentoring, educating, and researching in the world of oocytes.

Int J Dev Biol 2012 ;56(10-12):765-70

Middlebury College, Biology Department, Middlebury, VT 05753, USA.

David Albertini has dedicated his life to illuminating our understanding of the most wondrous of cells--the oocyte. Beyond his powerful scientific contributions, he has mindfully and tirelessly mentored and educated scientists and clinicians in our field. In this essay which reports a dialogue, David Albertini shares some of the key experiences that have governed his career path. He has been a spokesperson to the public to ensure the accurate conveying of current findings in our field, and he has always strived to help others in communicating effectively. He also reflects (notably in light of where funding priorities may lie) on the imperative to use animal model systems that will be most suitable for addressing the pressing questions of reproductive biology today. Dr. Albertini pioneered the use of live cell imaging approaches over 30 years ago, and he has eagerly passed on his expertise to many others while these techniques were in their infancies. His career has been fueled by his passion for visualizing cellular events in live cells and tissues, as never undertaken or seen before. He took chances while always embracing opportunities as they arose. Dr. Albertini has also delineated the intersection between basic research on the oocyte and emerging trends in reproductive medicine--such as oocyte cryopreservation. Not only does he continue to advance the field of human oocyte biology, but he is also, and yet again, extending his role as educator and mentor by taking a lead in reproductive medicine as a journal editor and as a mentor to young and rising clinicians in the field.
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http://dx.doi.org/10.1387/ijdb.120176ccDOI Listing
August 2013

Follicular fluid hydrogen peroxide and lipid hydroperoxide in bovine antral follicles of various size, atresia, and dominance status.

J Assist Reprod Genet 2013 Mar 15;30(3):333-40. Epub 2013 Jan 15.

Biology Department, Middlebury College, Middlebury, VT 05753, USA.

Purpose: To avoid inducing a state of oxidative stress (OS), assisted reproductive technologies (ART) must maintain a balance of reactive oxygen species (ROS) and antioxidants during the in vitro culture of oocytes. However, oocyte requirements and tolerance thresholds for ROS during in vivo development are still unclear. Previous studies have examined ROS levels in follicular fluid (FF) using pooled samples or according to follicle size. This study sought to examine two OS markers, lipid hydroperoxides (LPO) and hydrogen peroxide (H2O2), in FF of individually sampled follicles from bovine ovary pairs according to follicle size, atresia, and dominance status.

Methods: TUNEL and cleaved Caspase-3 labeling were used to identify apoptotic granulosa cells and determine follicle atresia status. LPO were measured directly for the first time in FF.

Results: Non-atretic follicles and dominant follicles contained more FF H2O2 than atretic follicles and corresponding subordinate follicles, respectively. FF LPO did not vary in relation to atretic status, and no difference existed between dominant and subordinate follicles. However, FF LPO was significantly lower in first subordinate follicles than in the second subordinate follicles from each ovary pair. Neither H2O2 nor LPO levels correlated with follicle size.

Conclusions: These data provide clear evidence that the events of antral folliculogenesis are relevant to ROS dynamics in vivo. Furthermore, such studies will help to optimize in vitro conditions for oocyte culture protocols, particularly when combined with a comparison of oocyte quality with respect to source follicle characteristics.
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http://dx.doi.org/10.1007/s10815-012-9925-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607686PMC
March 2013

The association between severe obesity and characteristics of failed fertilized oocytes.

Hum Reprod 2012 Nov 11;27(11):3198-207. Epub 2012 Sep 11.

Department of Obstetrics, Gynecology and Reproductive Biology, Brigham & Women's Hospital and Harvard Medical School, 02115 Boston, MA, USA.

Study Question: Is the cytoskeletal and chromosomal organization of failed fertilized oocytes from severely obese patients (BMI ≥ 35 kg/m²) altered compared with that in patients with normal BMI (BMI 18.5-24.9 kg/m²)?

Summary Answer: Compared with normal BMI patients, severe obesity was associated with a greater prevalence of spindle anomalies and non-aligned chromosomes in failed fertilized oocytes.

What Is Known And What This Paper Adds: Obesity is associated with poor reproductive outcomes, but little is known regarding the underlying mechanisms. To address potential mechanisms, our study compared the cytoskeletal and chromosome organization in failed fertilized oocytes from severely obese and normal BMI patients.

Design: The study population was drawn from IVF patients treated in a hospital-based infertility clinic between February 2010 and July 2011. The prevalence of meiotic spindle and chromosome alignment anomalies in failed fertilized oocytes from patients with severe obesity (i.e. Class II and III; BMI 35.0-50.1 kg/m²) was compared with those from patients with normal BMI (BMI 18.5-24.9 kg/m²). Oocytes were fixed and then labeled for tubulin, actin and chromatin. Spindle number and integrity, as well as chromosome alignment, were assessed using immunofluorescence microscopy and, in some cases, confocal microscopy. Generalized estimating equations were applied, which account for the correlation among oocytes from the same patient to estimate odds ratio (OR), 95% confidence intervals (CIs) and two-sided Wald P-values. Models were adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and polycystic ovarian syndrome (PCOS) a priori.

Participants And Setting: University-affiliated infertility clinic. A total of 276 oocytes that failed to fertilize from 137 patients were evaluated: 105 oocytes from severely obese women (n = 47) and 171 oocytes from normal BMI patients (n = 90).

Main Results And The Role Of Chance: (i) Significantly more oocytes from the severely obese group exhibited two spindles compared with those from the normal BMI group (58.9 versus 35.1%; OR = 2.68, CI = 1.39-5.15, P-value = 0.003). (ii) Among oocytes with a single spindle, those from severely obese patients showed a significantly higher prevalence of disarranged spindles with non-aligned chromosomes compared with those from normal BMI patients (28.6 versus 8.6%; OR = 4.58, CI = 1.05-19.86, P-value = 0.04).

Bias, Confounding And Other Reasons For Caution: Inclusion of only failed fertilized oocytes, small sample size, unknown factors such as non-PCOS comorbidity.

Generalizability To Other Populations: For this study, by design, it is unclear whether the findings are generalizable to successfully fertilized oocytes, and whether this oocyte-level influence of obesity is generalizable to infertile women who do not undergo stimulation or, more broadly, to spontaneous conceptions in fertile women.

Study Funding/competing Interest(s): none.

Trial Registration Number: n/a.
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http://dx.doi.org/10.1093/humrep/des308DOI Listing
November 2012

Media composition: antioxidants/chelators and cellular function.

Methods Mol Biol 2012 ;912:129-59

Biology Department, Middlebury College, McCardell Bicentennial Hall, Middlebury, VT, USA.

Protection of embryos against oxidative insults during culture is necessary to maintain viability. Generation of excessive levels of reactive oxygen species (ROS) is triggered by various components of the in vitro environment, most of which embryos do not normally encounter in vivo. To compensate for these deficiencies in the culture environment, antioxidants and chelators are often used to control or suppress ROS levels as embryos develop. However, there is no consensus regarding dosage, time of exposure, or appropriate combinations of antioxidants and chelators in embryo culture. In order to elucidate this aspect of an embryo's chemical surroundings in vitro, we present the current knowledge on the function and effect of each antioxidant or chelator that is often included in an embryo culture medium.
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http://dx.doi.org/10.1007/978-1-61779-971-6_9DOI Listing
November 2012

Is it best to cryopreserve human cumulus-free immature oocytes before or after in vitro maturation?

Cryobiology 2012 Oct 9;65(2):79-87. Epub 2012 Jun 9.

Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Freezing unfertilized oocytes is an option for females without a partner, either to preserve their fertility prior to sterilizing cancer treatment or for social reasons. Our study considered whether it is best to freeze immature human oocytes at the germinal vesicle (GV) stage, prior to in vitro maturation (IVM) or at metaphase-II (M-II), after IVM. Sibling GV-stage oocytes from stimulated ICSI cycles were allocated to freezing either prior to (n=109) or after (n=107) IVM. Cumulus-free oocytes were cryopreserved using a choline-substituted slow-freezing protocol and matured in a defined medium, with analysis of chromatin, microtubules, and microfilaments by three-dimensional imaging. Cryopreserved oocytes were compared with oocytes matured in vitro but never frozen (n=114). Survival was similar between oocytes frozen before or after IVM (69.7% vs. 70.5%). Polar body extrusion after IVM was lower in oocytes frozen at the GV stage versus those matured and then frozen (51.3% vs. 75.7%) or not frozen (75.4%). Stratification by patient age (<36 and ⩾36year) showed no difference in oocyte survival or maturation. Oocytes frozen as GVs showed elevated proportions of spontaneous activation (with or without polar body), an effect augmented by patient age. Spindle and chromosome configurations were disrupted to similar extents in both groups of frozen oocytes, with no further detrimental effect of patient age. The length, width, and volume of bipolar M-II spindles were comparable in all three groups. When frozen as GVs, oocytes exhibited decreased maturation and increased spontaneous activation, suggesting that it is best to freeze oocytes at M-II.
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http://dx.doi.org/10.1016/j.cryobiol.2012.06.001DOI Listing
October 2012

Release of superoxide dismutase-1 by day 3 embryos of varying quality and implantation potential.

J Assist Reprod Genet 2012 Apr 25;29(4):305-11. Epub 2012 Jan 25.

Biology Department, Middlebury College, Middlebury, VT 05753, USA.

Purpose: To determine if the antioxidant superoxide dismutase-1 (SOD1 or Cu,Zn-SOD) is released by cultured human cleavage-stage embryos and to assess any link between SOD1 and implantation potential.

Methods: Women (n = 91; ≤40 years old) undergoing IVF treatment with transfer of one or two 8-cell embryos that resulted in 0 or 100% implantation were included. Following individual embryo culture, spent medium samples (n = 122) were collected and levels of SOD1 protein were measured by an enzyme-linked immunosorbent assay. SOD1 detection and concentration in embryo spent medium were analyzed in relation to embryo fragmentation and symmetry scores, and implantation (viable fetus at >12 weeks).

Results: Cleavage-stage embryos release SOD1 protein into the spent culture medium. Neither detection nor concentration of SOD1 was related to implantation. There was a positive relationship between increased embryo fragmentation scores and SOD1 release, with no apparent association with symmetry. In non-pregnant cycles, the release of SOD1 decreased with increasing maternal age.

Conclusions: While SOD1 does not predict implantation potential of select good-quality embryos, our data support the need to evaluate the biological significance of released SOD1 by embryos of varying quality and from patients of varying age.
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http://dx.doi.org/10.1007/s10815-012-9711-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309978PMC
April 2012

Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing.

J Assist Reprod Genet 2011 Dec 17;28(12):1183-92. Epub 2011 Nov 17.

Biology Department, Middlebury College, Middlebury, VT 05753, USA.

Purpose: The cryopreservation of immature oocytes permits oocyte banking for patients at risk of losing their fertility. However, the optimum protocol for such fertility preservation remains uncertain.

Methods: The present study investigated the survival, maturation, cytoskeletal and chromosome organization of sibling immature oocytes leftover from controlled ovarian stimulation cycles, that were either slow-frozen (with choline-substitution) or vitrified. A comparison group included oocytes that were never cryopreserved.

Results: Among the three groups, comparable rates were observed for both survival (67-70%) and polar body extrusion (59-79%). Significantly more oocytes underwent spontaneous activation after IVM following slow-freezing compared with either vitrification or no cryopreservation. Likewise, the incidence of spindle abnormalities was greatest in the slow-frozen group, with no differences in spindle morphometrics or chromosome organization.

Conclusions: While the overall incidence of mature oocytes with normal bipolar spindles from warmed immature oocytes was low, the yield using Cryoleaf vitrification was slightly superior to choline-based slow-freezing.
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http://dx.doi.org/10.1007/s10815-011-9674-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241844PMC
December 2011

Fluctuations in total antioxidant capacity, catalase activity and hydrogen peroxide levels of follicular fluid during bovine folliculogenesis.

Reprod Fertil Dev 2011 ;23(5):673-80

Center for Reproductive Medicine, Obstetrics & Gynecology and Women's Health Institute, Glickman Urological & Kidney Institute, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA.

Follicular fluid is an important environment for oocyte development, yet current knowledge regarding its in vivo oxidant and antioxidant levels remains limited. Examining follicular fluid oxidants and antioxidants will improve understanding of their changes in vivo and contribute to optimisation of in vitro maturation conditions. The aim of the present study was to consider selected markers, namely catalase (CAT) enzyme activity, total antioxidant capacity (TAC) and hydrogen peroxide (H(2)O(2)) in follicular fluid samples (n = 503) originating from bovine antral follicles. The dynamic changes in two relevant antioxidant measures and one reactive oxygen species (ROS) were measured through stages of bovine follicular development and the oestrous cycle. CAT activity and H(2)O(2) levels decreased significantly as follicle size increased, whereas TAC increased significantly as follicle size increased. Lower TAC and higher H(2)O(2) in small follicles suggest increased ROS in the initial stages of folliculogenesis. Because CAT levels are highest in the follicular fluid of small follicles in the setting of an overall low TAC, CAT may represent a dominant antioxidant defence in the initial stages of folliculogenesis. Future studies must focus on other reactive oxygen species and their various scavenger types during antral folliculogenesis.
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http://dx.doi.org/10.1071/RD10270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235527PMC
September 2011

Cellular and genetic analysis of oocytes and embryos in a human case of spontaneous oocyte activation.

Hum Reprod 2011 Mar 11;26(3):545-52. Epub 2011 Jan 11.

Biology Department, Middlebury College, Middlebury, VT 05753, USA.

Unusual and consistent defects in infertility patients merit attention as these may indicate an underlying genetic abnormality, in turn necessitating tailored management strategies. We describe a case of repeated early pregnancy loss from in vivo conceptions, followed by cancelled embryo transfers after one IVF and one ICSI/PGD cycle. Following the unexpected presence of cleaved embryos at the fertilization check in the first IVF attempt, oocytes and embryos were subsequently analyzed in an ICSI/PGD case. Part of the oocyte cohort was fixed at retrieval for a cellular evaluation of microtubules, microfilaments and chromatin. The remaining oocytes were injected with sperm, and resultant embryos were biopsied for genetic analysis by fluorescence in situ hybridization (FISH), single-nucleotide polymorphism (SNP) microarray for 23 chromosome pairs, as well as with PCR for sex chromosomes. The presence of interphase microtubule networks and pronuclear structures indicated that oocytes were spontaneously activated by the time of retrieval. FISH revealed aneuploidy in all seven blastomeres analyzed, with all but two lacking Y chromosomes. Microarray SNP analysis showed an exclusively maternal origin of all blastomeres analyzed, which was further confirmed by PCR. From our multi-faceted analyses, we conclude that spontaneous activation, or parthenogenesis, was probably the pathology underlying our patient's recurrent inability to maintain a normal pregnancy. Such analyses may prove beneficial not only in diagnosing case-specific aberrations for other patients with similar or related failures, but also for furthering our general understanding of oocyte activation.
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http://dx.doi.org/10.1093/humrep/deq363DOI Listing
March 2011

Diagnosing cellular defects in an unexplained case of total fertilization failure.

Hum Reprod 2010 Jul 15;25(7):1666-71. Epub 2010 May 15.

Biology Department, Middlebury College, Bicentennial Hall 346, Middlebury, VT 05753, USA.

Despite the advent of ICSI, cases of total fertilization failure (TFF) often lead to cycle cancellation with limited diagnostic and therapeutic strategies currently available. We report on the case of an infertile couple who failed to conceive after repeated IVF and ICSI. Sperm of the husband were morphologically normal and passed a functional test assessing their ability to activate mouse oocytes. Whether oocytes were activated artificially with calcium ionophore after injection of husband's or with donor sperm, all oocytes failed to fertilize. Multiple polar bodies and two disorganized spindle structures were predominantly observed, pointing towards a cytoplasmic defect in the oocytes as the primary cause of the couple's infertility. In fact, injection of husband's sperm into donor oocytes resulted in the delivery of healthy twins. This report describes a course of action that may be applied for couples with TFF after both IVF and ICSI.
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http://dx.doi.org/10.1093/humrep/deq064DOI Listing
July 2010

Profiling of superoxide dismutase isoenzymes in compartments of the developing bovine antral follicles.

Reproduction 2010 May 2;139(5):871-81. Epub 2010 Mar 2.

Biology Department, Middlebury College, McCardell Bicentennial Hall 346, Middlebury, Vermont 05753, USA.

The antral follicle constitutes a complex and regulated ovarian microenvironment that influences oocyte quality. Oxidative stress is a cellular state that may play a role during folliculogenesis and oogenesis, although direct supporting evidence is currently lacking. We thus evaluated the expression of the three isoforms (SOD1, SOD2, and SOD3) of the enzymatic antioxidant superoxide dismutase in all the cellular (granulosa cells, cumulus cells, and oocytes) and extracellular (follicular fluid) compartments of the follicle. Comparisons were made in bovine ovaries across progressive stages of antral follicular development. Follicular fluid possessed increased amounts of SOD1, SOD2, and SOD3 in small antral follicles when compared with large antral follicles; concomitantly, total SOD activity was highest in follicular fluids from smaller diameter follicles. SOD1, SOD2, and SOD3 proteins were expressed in granulosa cells without any fluctuations in follicle sizes. All three SOD isoforms were present, but were distributed differently in oocytes from small, medium, or large antral follicles. Cumulus cells expressed high levels of SOD3, some SOD2, but no detectable SOD1. Our studies provide a temporal and spatial expression profile of the three SOD isoforms in the different compartments of the developing bovine antral follicles. These results lay the ground for future investigations into the potential regulation and roles of antioxidants during folliculogenesis and oogenesis.
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http://dx.doi.org/10.1530/REP-09-0390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244472PMC
May 2010

Could oxidative stress influence the in-vitro maturation of oocytes?

Reprod Biomed Online 2009 Jun;18(6):864-80

Biology Department, MBH 346, Middlebury College, Middlebury, VT 05753, USA.

In the efforts aimed at improving the quality of in-vitro-matured human oocytes, the dynamic balance and roles of pro-/antioxidants merit further consideration. In-vitro maturation (IVM) is emerging as a popular technology at the forefront of fertility treatment and preservation. However, standard in-vitro culture conditions exert oxidative stress or an imbalance between oxidants and antioxidants. Reactive oxygen species (ROS) are oxygen-derived molecules formed as intermediary products of cellular metabolism. By acting as powerful oxidants, ROS can oxidatively modify any molecule, resulting in structural and functional alterations. ROS are neutralized by an elaborate defence system consisting of enzymatic and nonenzymatic antioxidants. This review captures the inherent and external factors that may modulate the oxidative stress status of oocytes. It discusses the suspected impacts of oxidative stress on the gamut of events associated with IVM, including prematuration arrest, meiotic progression, chromosomal segregation, cytoskeletal architecture and gene expression. In-vivo and in-vitro strategies that may overcome the potential influences of oxidative stress on oocyte IVM are presented. Future studies profiling the oxidative stress status of the oocyte may permit not only the formulation of a superior IVM medium that maintains an adequate pro-/antioxidant balance, but also the identification of predictors of oocyte quality.
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http://dx.doi.org/10.1016/s1472-6483(10)60038-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235363PMC
June 2009

What are the trade-offs between one-cell and two-cell biopsies of preimplantation embryos?

Hum Reprod 2008 Mar 24;23(3):493-8. Epub 2007 Dec 24.

Biology Department, Middlebury College, McCardell Bicentennial Hall 346, Middlebury, VT 05753, USA.

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http://dx.doi.org/10.1093/humrep/dem396DOI Listing
March 2008

Optimum number of embryos to transfer in women more than 40 years of age undergoing treatment with assisted reproductive technologies.

Fertil Steril 2005 Dec;84(6):1637-42

Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Objective: To determine whether increasing the number of embryos transferred beyond five increases pregnancy rates in women aged > 40 years.

Design: Retrospective analysis of cycles performed between January 1998 and July 2003.

Setting: University-affiliated teaching hospital.

Patient(s): Women aged > 40 years undergoing a fresh cycle with a day-3 ET (n = 863).

Intervention(s): None.

Main Outcome Measure(s): Pregnancy, chemical pregnancy, miscarriage rates, number of viable fetuses at 12 weeks' gestation, live birth rates, and number of babies delivered.

Result(s): Compared with patients with fewer than five embryos transferred, those having five or more embryos transferred had significantly increased pregnancy rates and live birth rates, more viable fetuses at 12 weeks, and significantly decreased miscarriage rates. None of these outcome variables differed between the five-embryo and more-than-five-embryo groups. There were no differences in outcome when only five embryos were transferred, regardless of whether five or more than five embryos were available. The number of embryos transferred did not significantly influence multiple birth rates.

Conclusion(s): The present study demonstrates that in women aged > 40 years, five embryos is the optimum number to transfer, and transferring more than five does not confer any additional benefit to clinical outcome.
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http://dx.doi.org/10.1016/j.fertnstert.2005.04.070DOI Listing
December 2005

Genetic strain variations in the metaphase-II phenotype of mouse oocytes matured in vivo or in vitro.

Reproduction 2005 Dec;130(6):845-55

Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA.

The interplay between genetic and epigenetic factors plays a central role in mammalian embryo production strategies that superimpose ex vivo or in vivo manipulations upon strain background characteristics. In this study, we examined the relationship between genetic background and the phenotypic properties of mouse metaphase-II (M-II) oocytes that were matured under in vivo (IVO) or in vitro conditions, either in a basal (IVM) or a supplemented (IVM + ) medium. Differences existed amongst inbred (C57BL/6), outbred (CF-1, Black Swiss, NU/NU) and hybrid lines (B6D2F1) induced to superovulate with regard to cytoplasmic microtubule organizing center (MTOC) number but not spindle size or shape, except for larger and asymmetrical spindles in Black Swiss oocytes. When oocytes were matured in culture, meiotic spindle and cytoplasmic phenotypic properties of M-II oocytes were affected relative to in vivo conditions and between strains. Specifically, measures of meiotic spindle size, shape, polar pericentrin distribution and cytoplasmic MTOC number all revealed characteristic variations. Interestingly, the overall reduction in cytoplasmic MTOC number noted upon IVM was concomitant with an overall increase in spindle and polar body size. Maturation under IVM + conditions resulted in a further decrease in cytoplasmic MTOC number, but spindle and polar body characteristics were intermediate between IVO and IVM. How these oocyte phenotypic properties of maternal origin may be linked to predictive assessments of fecundity remains to be established.
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http://dx.doi.org/10.1530/rep.1.00558DOI Listing
December 2005

Assessment and optimization of oocyte quality during assisted reproductive technology treatment.

Semin Reprod Med 2005 Aug;23(3):277-84

Biology Department, Middlebury College, Middlebury, Vermont 05753, USA.

The health of fetuses, neonates, and adults depends on normal development of the oocytes and embryos from which they arise. In addition, the ability of an embryo to implant, sustain a pregnancy, and give rise to a viable offspring is unquestionably rooted in oogenesis. Therefore, defining markers that can reliably predict the best quality oocytes will prove invaluable in the care and management of infertility patients. Furthermore, identification of the best quality oocyte will also permit the transfer of a single embryo, thereby increasing overall pregnancy rates and reducing multiple rates. Although a few potentially important predictors of oocyte quality have been identified, their application for routine use in the assisted reproductive technology (ART) laboratory remains to be established. With the critical need for markers to be noninvasive, reliable, and assayable with a rapid turnaround, key areas that will take advantage of recent technological advances are identified in this article. Oocyte quality also needs to be improved in the management of human infertility by ART programs. Given that mechanisms determining oocyte quality are multifactorial and complex, several possible areas that continue to necessitate optimization are discussed.
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http://dx.doi.org/10.1055/s-2005-872456DOI Listing
August 2005

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system.

Hum Reprod 2005 May 3;20(5):1349-58. Epub 2005 Feb 3.

Brigham and Women's Hospital, Harvard Medical School, Department of Obstetrics and Gynecology, Boston, MA 02115, USA.

Background: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes.

Methods And Results: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels.

Conclusions: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.
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http://dx.doi.org/10.1093/humrep/deh750DOI Listing
May 2005

Hormonal control of somatic cell oocyte interactions during ovarian follicle development.

Mol Reprod Dev 2004 Nov;69(3):347-55

Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, USA.

In the mammalian ovarian follicle, paracrine signaling between the oocyte and somatic granulosa cells is bidirectional but the structural basis and physiological regulations of communication between gametic and somatic compartments remain unknown. The present experiments were designed to test the hypothesis that follicle stimulating hormone (FSH) regulates the ability of granulosa cells to make connections with the oocyte. We show that in prepubertal unprimed mice and mice carrying a targeted deletion of the FSHbeta subunit gene, granulosa cells exhibit orientation towards the oocyte manifest by the elaboration of transzonal projections (TZPs) and "apical" centrosome positioning at sites of granulosa-zona contact. In vivo FSH treatment results in a retraction of TZPs. Coincident with TZP retraction induced by FSH are changes in oocyte transcriptional activity and meiotic competence, which suggests one means by which the oocyte-granulosa cell dialogue may be modulated during development of ovarian follicles.
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http://dx.doi.org/10.1002/mrd.20128DOI Listing
November 2004

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation.

Hum Reprod 2003 Oct;18(10):2124-30

Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.

Background: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human.

Methods: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I.

Results: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control.

Conclusions: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.
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http://dx.doi.org/10.1093/humrep/deg419DOI Listing
October 2003

Origins and manifestations of oocyte maturation competencies.

Reprod Biomed Online 2003 Jun;6(4):410-5

Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.

Mammalian oocytes acquire a series of competencies during follicular development that play critical roles at fertilization and subsequent stages of preimplantation embryonic development. These competencies involve remodelling of chromatin and the cytoskeleton in the oocyte at critical stages of folliculogenesis when gametes and somatic cells communicate by paracrine and junctional mechanisms. While the detailed steps involved in bi-directional signalling between oocytes and granulosa cells remain unknown, studies from mice bearing targeted deletions in essential 'communication' genes reveal selective disturbances in oocyte maturation competencies that compromise the oocyte's developmental potential. Recent data are reviewed that illustrate the general principle that competencies acquired at sequential stages of oogenesis are manifest during oocyte growth, maturation, or following fertilization. The recognition that oocyte-specific genes are called into play at key developmental transitions in mammalian embryogenesis emphasizes the importance of monitoring genetic and epigenetic determinants when using current assisted reproductive technologies manipulations.
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http://dx.doi.org/10.1016/s1472-6483(10)62159-1DOI Listing
June 2003
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