Catherine M.H. Combelles

Day 3 and day 5 morphological predictors of embryo viability

Controlling multiple pregnancies in patients undergoing artificial reproductive procedures requires consideration of single embryo transfers. Therefore, refinements for embryo evaluation are needed that select for the most developmentally competent embryo. The present study was designed to identify day 3 and day 5 morphological predictors of viability following transfers in which the morphology and fate of each embryo was precisely determined. Assessments on day 3 included cell number, and the extent of fragmentation and asymmetry, and on day 5, the developmental stage. Embryos resulting in a viable fetus at 11 weeks gestation were considered developmentally competent. The relationships among individual and collective embryo morphological characteristics were evaluated. Analysis of the interactions among morphological characteristics of embryos transferred on day 3 enabled identification of a multivariable selection order. Assessment of day 5 embryos revealed that expanding and expanded blastocysts exhibited comparable developmental potential that was superior to that of either morulae or early blastocysts. However, expanding or expanded blastocysts derived from 7-cell or 8-cell embryos were developmentally superior to those derived from other cleavage stages, regardless of fragmentation or asymmetry. Collectively, these findings further understanding of morphological predictors of viability, thereby improving the ability to select the most viable embryo for transfer.

COULD OXIDATIVE STRESS INFLUENCE THE IN-VITRO MATURATION OF OOCYTES?

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.

Sorting and reorganization of centrosomes during oocyte maturation in the mouse

In animal oocytes, the centrosome exists as an acentriolar aggregate of centrosomal material that is regulated in a dynamic manner throughout the process of meiotic maturation. Recently, it has been demonstrated that in female meiotic systems spindle assembly is likely regulated by chromosomal and microtubule/microtubule‐associated influences. The purpose of this study was to analyze the distribution of the integral centrosomal protein, pericentrin, during the course of meiotic maturation. The function of the centrosome during meiotic progression was evaluated by exposing oocytes to pharmacological agents that perturb cytoplasmic homeostasis (cycloheximide, nocodazole, cytochalasin D, taxol, and vanadate). Pericentrin was localized to the spindle poles during metaphase of meiosis‐I as O‐ and C‐shaped structures. At anaphase, these structures fragment, become displaced from the spindle poles, and associate with the lateral spindle margin. The metaphase spindle at meiosis‐II had incomplete pericentrin rings at both spindle poles. Vanadate treatment, a known inhibitor of dynein‐ATPase, resulted in meiotic arrest, constriction of the spindle pole, and an aggregation of pericentrin at the spindle poles. After taxol exposure, pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased. Treatment of oocytes with cycloheximide, nocodazole, and cytochalasin D, influenced early events associated with chromosome capture and spindle assembly and altered the number and distribution of cytoplasmic centrosomes. Thus, although pericentrin incorporation is not required for meiotic spindle formation, the dynamic reorganization of pericentrin and changes in centrosome microtubule nucleating capacity are involved in critical cell cycle transitions during meiotic maturation. Microsc. Res. Tech. 49:435–444, 2000.

Assessment of Oocyte Quality Following Repeated Gonadotropin Stimulation in the Mouse

Abstract The present study assessed the effects of repeated ovarian stimulation on oocyte quality. Female mice were stimulated with eCG and hCG at 1-wk intervals for 4 wk. Germinal vesicle (GV)-stage oocytes were evaluated in relation to size, somatic cell association, and chromatin organization after each week of stimulation. In addition, ATP content and expression of meiotic competence were monitored in GV and in vivo (IVO) or in vitro (IVM)-matured oocytes. The developmental competence of ovulated oocytes was determined after in vitro fertilization and embryo culture, and reproductive outcome was evaluated after mating following repeated cycles of stimulation. In GV oocytes, the degree of somatic cell association, size, and timing of transcriptional repression were altered when comparing repeated with single cycle(s) of stimulation. Meiotic competence expression was unaffected for IVO oocytes while IVM oocytes exhibited a progressive decrease in meiotic competence with repeated stimulation. The ATP content of immature and IVO oocytes decreased with repeated stimulation. Although after one cycle of stimulation ATP content was lower in IVM than IVO oocytes, IVM oocytes exhibited stable levels of ATP across cycles of stimulation. Last, the in vitro developmental competence of IVO oocytes retrieved after repeated stimulation was not significantly different, and in vivo, similar implantation and resorption rates were observed following mating of animals subjected to repeated stimulation. Therefore, despite measurable consequences of repeated stimulation on specific parameters of follicular oocyte quality, compensatory mechanisms may exist in vivo to optimize the developmental competence of ovulated oocytes in the mouse.

A novel system for in vitro maturation of human oocytes

OBJECTIVE To compare in vitro maturation of cumulus-free oocytes in glucose-free medium (P1) and standard medium (TC199). DESIGN Prospective, cohort study. SETTING Assisted reproductive technology program. PATIENT(S) One hundred eight patients undergoing ICSI. INTERVENTION(S) Germinal vesicle-stage or metaphase I--stage oocytes were allocated to culture with P1 or TC199. Metaphase II oocytes were fixed for immunofluorescence analysis or fluorescence in situ hybridization at 24 or 48 hours (or both). Media were compared by performing conditional logistic regression analysis that controlled for egg-specific factors. MAIN OUTCOME MEASURE(S) Proportion of mature oocytes and appearance of normal spindle-chromosome cytoarchitecture. RESULT(S) At 24 hours, more P1 oocytes than TC199 oocytes reached metaphase II (59.7% vs. 44.9%). At 48 hours, 71.7% of P1 oocytes and 61.0% of TC199 oocytes reached metaphase II, but this difference was not significant. Metaphase II oocytes in P1 were 34.3% more likely than those in TC199 to have a bipolar spindle with aligned chromosomes. Compared with oocytes at the germinal vesicle stage at 0 hour, those at metaphase I at 0 hour were more likely to progress to metaphase II (72.6% vs. 46.1% at 24 hours; 84.1% vs. 60.6% at 48 hours). CONCLUSION(S) P1 is superior to TC199 for in vitro maturation of granulosa-free human oocytes.

The antral follicle: a microenvironment for oocyte differentiation.

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.

The association between severe obesity and characteristics of failed fertilized oocytes

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.

Bisphenol-A and human oocyte maturation in vitro

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 Womens 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. McNemars 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.

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

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.

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

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.