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1 RBMOnline - Vol 17 No Reproductive BioMedicine Online; on web 19 June 2008 Symposium: Innovative techniques in human embryo viability assessment Human oocyte respiration-rate measurement potential to improve oocyte and embryo selection? Lynette Scott has been working in the field of human IVF for 18 years, having started at King s College Hospital in London. Prior to moving into the human field she worked on the early development of mouse embryos, getting her training and love of embryology from David Whittingham and Anne McLaren at the Mammalian Development Unit in London. She is native of Zimbabwe, Africa and obtained her BSc from the University of Cape Town and MSc and PhD from the University of London, University College. Her interest lies in the development of the oocyte and early embryo. Dr Lynette Scott Lynette Scott 1,3, Jørgen Berntsen 2, Darlene Davies 1, Jens Gundersen 2, Joseph Hill 1, Niels Ramsing 2 1 Fertility Centres of New England, Reading, MA, USA; 2 Unisense FertiliTech, Science Centre Skejby, Aarhus, Denmark 3 Correspondence: Tel: , ext. 583; Fax: ; scottl@fertilitycenter.com Abstract Oocyte and embryo selection are not highly successful, with fewer than 10% of oocytes in assisted reproduction resulting in a delivery. Techniques for oocyte and embryo selection rely on highly subjective morphology assessment, with few true quantitative techniques available. One aspect of oocyte health that could be considered is the ability to produce ATP through respiration. Using a non-invasive technology, the respiration rates of individual human oocytes were recorded in an attempt to correlate respiration and oocyte health with probable subsequent development. Oocytes used were either immature or mature, non-fertilized oocytes from a clinical assisted reproduction programme. Differences in respiration rates between oocytes within a cohort and between cohorts of oocytes were recorded. The differences between cohorts reflected many of the currently known differences in oocyte health, related to age and FSH concentrations. However, within a cohort, differences between oocytes were observed, with some having high rates and others low. Oocytes with respiration rates of between 0.48 and 0.55 nl O 2 /h were viable, with lower rates consistent with lack of continued in-vitro maturation or atresia. This technology may have a future in the clinical laboratory as a predictor of oocyte health and ability to develop into an embryo with greater potential of delivery. Keywords: non-invasive technology, oocyte selection, oocytes, respiration Introduction Successful embryo formation and subsequent delivery of an offspring requires that both gametes, oocyte and spermatozoon, must be fundamentally normal. Delivery of an infant should be the only measure of success after assisted reproduction treatment. Sperm (gamete) and embryo morphology are the most common and standard techniques of selection in assisted reproduction, and these are subjective and visual. Neither is exact; the bell curve for success in these selection techniques can be rather wide, suggesting that there is an element of chance; approximately 10% of retrieved oocytes and fewer than 20% of transferred embryos result in a baby, worldwide (contributions from the Bertarelli Foundation, 2006). This is due most likely to patient profile and infertility aetiology, which can be reflected in the quality of the gametes. In a technical setting, it can also be due to operator variance, lack of standardization of what constitutes normal, as well as other laboratory factors. What is clear is that, although the field of assisted reproduction has become successful at a patient level, with more couples having deliveries; at the oocyte and embryo level, there is still substantial wastage Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 462 Even with cumulative delivery rates based on fresh and frozen cycles, there remains a large discrepancy between the numbers of oocytes retrieved and embryos created, and the number of babies delivered. Oocyte morphology has not been widely used in the selection of viable embryos, as most reported techniques have not shown strong correlations with outcome. Oocyte morphological criteria that have been reported to correlate with outcome include polar body morphology (Ebner et al., 2000); cytoplasm appearance (Ebner et al., 2006); and more recently zona pellucida thickness, appearance and birefringence (Shen et al., 2005; Montag et al., 2007; Pascale et al., 2007) and the position or shape of the spindle (Madaschi et al., 2007). Using any form of oocyte marker, morphological or metabolic, will require very carefully controlled prospective studies in which the contribution of the male gamete will not interfere with data analysis, otherwise vast numbers of oocytes will be required for true validation. Oocytes need to acquire genetic, cellular and metabolic competence in order to proceed through maturation, fertilization and embryo development. These factors include nuclear and cytoplasmic maturation, meiotic competence, spindle formation, activated mitochondria, the ability to mobilize and release calcium for internal stores and the ability to react to the incoming/penetrating spermatozoon (Dumollard et al., 2007). None of these parameters can be scored visually and measuring any of these factors may necessitate destruction of the oocyte, which is not acceptable in a clinical setting. Oocytes experience a very active growth phase during maturation, but arrest and become quiet once fully mature, at ovulation. During the process of oocytes maturation, mitochondria function actively. Mature oocytes contain a large number of genes coding for mitochondrial DNA (mtdna), but these originate from a few (approximately 10) that are present in primordial cells; these are then amplified during oocyte growth. The small size of the original pool ensures the integrity of the mtdna from generation to generation (Cummins, 2002). As the primordial germ cells begin to grow and develop into oocytes, which arrest at the prophase stage of meiosis I, the mtdna has been amplified to approximately 6000 copies in an average oocyte (Jansen and de Boer 1998). During its final growth and development, following release from arrest at meiosis I, the oocyte increases in size, and there is also a large increase in the number of mitochondria, up to copies of mtdna. A number of studies using immature or non-fertilized mature human oocytes have quantified the mtdna load, and the data show differences both in cohorts and in individual oocytes, which indicates that not all oocytes are equivalent at the mitochondrial or mtdna level (Reynier et al., 2001; May- Panloup et al., 2005, 2007; Pascale et al., 2007). Mitochondrial function is the key to fertilization and development, as they are the main generators of energy, and also contribute to aspects of calcium homeostasis, which is essential to development and the supply of intermediate metabolites during energy generation (Van Blerkom, 2004). Very few of the total mitochondria are fully active in the oocyte, and the majority of potentiated (active) organelles lie at the periphery of the oocyte, under the oolemma. Activation of these mitochondria at fertilization is key to all subsequent development, and probably also contributes to embryo quality and all subsequent scoring parameters, characteristics that are carried through to the blastocyst stage (Wilding et al., 2001; Dumollard et al., 2004, 2007; Van Blerkom 2004). Considering the vital role of mitochondria in oocyte competence and subsequent development, it was postulated that measuring one aspect of mitochondrial function, respiration (oxidative phosphorylation), in single oocytes may indicate both mitochondrial load and activation; this could be a means of selecting oocytes with subsequent enhanced potential for embryo development. Oocyte selection has the advantage of minimizing the number of embryos created, a source of moral dilemma in many parts of the world. It could aid oocyte cryopreservation programmes, and could be used in countries with tight restrictions on the number of oocytes allowed for fertilization, culture or transfer. It may also become a means of screening the developmental potential of in-vitro matured (IVM) oocytes, and help in the development of refined IVM systems. Respiration has been previously measured in human oocytes, but in very few oocytes from unstimulated cycles and in a system that did not allow recovery of the oocytes (Magnusson et al., 1986). An EmbryoScope has been developed that allows automated, accurate, oxygen consumption measurements on small numbers of cells, or single oocytes and embryos. The assessment is conducted within a controlled incubator environment, is non-invasive and could be compatible with clinical use. The EmbryoScope has been used non-invasively with live offspring in bovine (Lopes et al., 2005) and murine (Ottosen et al., 2007) models. The current study was designed to utilize oocytes unsuitable for clinical use from a human IVF programme, in order initially to validate the concept and technique of using the EmbryoScope to measure realtime respiration in human oocytes. The aim was to elucidate any measurable differences between oocytes of differing developmental stages, from different sources (infertility aetiologies, age, etc.), and which had different fates, and document the feasibility of using oocyte respiration as a clinical selection tool. As this is an experimental procedure and the EmbryoScope has not been verified or validated for clinical use, only material unsuitable for treatment could be used. This included immature and unfertilized mature oocytes in a clinical assisted reproduction programme. Although this material might be considered as abnormal, we felt it necessary to use human material for initial validation of the EmbryoScope. Human oocytes and embryos from stimulated cycles do not fit the normal pattern of development seen in laboratory animal models, and it should become a requirement in new technology development that human material should be used to validate any system aimed for use in a human assisted reproduction programme. The data showed reproducible differences in respiration rates between individual oocytes, both within and between cohorts, and also between sources of oocytes. From these data, it is suggested that oocyte respiration measurements could be a viable oocyte selection technique that deserves further research, development and prospective validation in a human assisted reproduction setting.

3 Materials and methods The EmbryoScope Oxygen respiration rate measurements were made with an EmbryoScope TM (Unisense FertiliTech, Aarhus, Denmark). The instrument is housed within a standard IVF CO 2 incubator (a commercial version of the EmbryoScope with built-in trigas incubator is currently under development). The EmbryoScope uses amperometric Clark-type oxygen microelectrodes to measure the steady-state diffusive oxygen flux to samples placed on the bottom of narrow wells in custom-made culture trays. In other words, oxygen consumption by the sample generates a slight local oxygen depletion, which is steadily replenished by molecular diffusion of oxygen from above. The ensuing oxygen concentration gradient is proportional to the respiration rate of the sample and is directly measurable with the oxygen microelectrode (Figure 1). The trays are constructed with a depression and a straight-sided columnar well, into which the sample is placed (Figure 1). The tray construction makes the entire system impermeable to gaseous exchange, which enables accurate measurements of gases (oxygen in this case) directly around the sample (oocytes in these experiments), and any changes in O 2 concentration can be recorded. Each tray contains wells for individual samples/blanks, covered with a common oil-layer. The respiration rate is calculated from the measured oxygen concentration gradient within the well containing the sample, according to Fick s first law of diffusion, i.e. by multiplying the gradient with the molecular diffusion co-efficient for oxygen ( cm 2 s 1 at 37ºC and 9% salinity) and the cross-sectional area of the well (0.49 mm 2 ). The calculated oxygen respiration rate is expressed in nanolitres of oxygen at standard temperature and pressure (STP) consumed per hour by the sample, using oxygen solubility in the media of µmol/l at 37ºC and 9% salinity, and a molar gas volume for oxygen at STP of l. fibre-optic oxygen meter) made by PreSens (Regensburg, Germany), to measure oxygen depletion in a custom-made glass vial with a volume of approximately 20 µl containing 20 mouse embryos. The closed system (traditional) is not as sensitive as the EmbryoScope, thus a large number of embryos were required in these validations. The electrode was inserted through a long narrow opening in the vial that was not closed during the experiment, but the dimensions (length 8 mm, diameter mm) were such that molecular diffusion through the opening can safely be ignored The glass trays used for embryo culture required validation for embryo development capacity, as did the entire incubator system. For this, a mouse embryo model was again adopted, with embryos being grown in current standard Nunc dishes or the glass trays, in both the EmbryoScope and a laboratory culture incubator. An F1-hybrid mouse embryo was utilized, and these were required to develop from the 1-cell stage to a hatching blastocyst at a rate >60% in order for the system to be validated as sound. The quality control tests were performed three times with 10 embryos per treatment per test (30 in total). Human oocyte respiration measurements Material source All oocytes were obtained through the IVF programme at the Fertility Centres of New England. The oocytes were fresh (non-frozen) and all were deemed discarded or nonclinical samples, but scored as normal or viable. Patients sign an informed consent for the use of discarded/non-clinical gametes for use in both research and teaching. No Institutional Review Board (IRB) authorization was required, as none of the material used was suitable for clinical use, and there was no attempt to fertilize any of the oocytes. The oxygen microelectrode is recalibrated automatically at the onset of each new measurement cycle by taking measurements in oxygen-saturated sterile media and an anoxic solution of alkaline sodium ascorbate (ph 13). These calibration solutions are kept in oil-covered wells of a separate calibration tray, which also contains 0.1 mol/l HCl (ph 1) and sterile PBS and culture media for automatic rinsing of the electrode tip between cycles. The culture trays were loaded with media the day prior to use, with an overlay of mineral oil to prevent media evaporation. The media and oil used was that in current use in the IVF laboratory, Sage Complete Culture medium (Sage Pharmaceuticals). All trays were equilibrated for at least 18 h prior to use. Validation of the system In order to validate the system s methodology, a traditional method of respiration measurement was employed alongside the current system. The method used a mouse model system, with closed respirometry using optical oxygen microsensors (NTH micro-optodes and a Microx TX3 single channel Figure 1. Graphic depiction of the oxygen microelectrode entering the well containing the oocyte/embryo and calculation of oxygen concentration or flux measured as the probe moves down the well. 463

4 464 Oocytes were obtained at two different time points: on the day of oocyte retrieval, D0, and the day following retrieval and insemination, D1. D0 oocytes were from ICSI procedures and were immature, either germinal vesicle (GV) or immature metaphase I (MI) oocytes at h post-hcg, the time at which the ICSI procedure was performed. Day 1 oocytes were immature (GV and MI) and mature metaphase II (MII) oocytes that had failed to fertilize after standard insemination. These were h post-insemination or h post-hcg. These times were kept constant for all experiments, since the timing of HCG, oocyte retrieval, insemination/icsi, and fertilization assessment are also kept constant in the clinical IVF laboratory. No pre-selection of patients or oocytes was performed; all available material was used whenever possible. This method of oocyte use and measurement allowed for a large variety of material to be sampled, maximum use of the system and no built-in bias as the system was being validated in the human system. Clearly, carefully controlled groups with strict inclusion/exclusion criteria will be required for true clinical validation. The results were collected over an 8-month time period and the data analysed retrospectively. Oocytes were washed free of all granulosa and cumulus cells and single oocytes were loaded into the wells in the glass trays. Each oocyte had a unique designated number for tracking. This unique number was also linked to the patient of origin, so that the infertility history, medication and outcome of treatment from the cycle for each oocyte were known. The media were kept constant for washing and culture and were the same as used in the IVF laboratory at the time, Sage Complete Embryo Culture medium and Sage oil (Sage Invitro Fertilization, Inc., Trumbull, CT, USA). The oocytes were placed in the glass trays, the trays loaded into the EmbryoScope and respiration measurements obtained over an initial 1 5 h phase (baseline respiration rate). This measurement reflected the respiration rate of the oocyte at the time of scoring and its initial developmental stage. Oocytes were then monitored over a further h for respiration and fate of the oocyte; the oocytes either became atretic, arrested in development or continued to mature (e.g. MI to MII development). Measurements were taken at 15 min to 1 h intervals. In each experiment the first three wells (of 15 total) were blank, containing culture media only, and at least one, and usually two, other wells were left empty to act as zero points for calibration. During the experiments the temperature was also closely monitored with continual read-out on the screen, since temperature can affect respiration. The data were analysed by day of oocyte source (D0 or D1) and stage of maturation. The data were further analysed by the fate of the oocyte, whether the oocyte continued to mature or not; by the age of the patient, infertility diagnosis, type of medication; the rate of fertilization of the cohort from which it originated; whether the oocyte originated from a cohort of oocytes that resulted in a positive outcome or not. Results Results of validation In the mouse embryo quality control experiments for the trays and system, 86.6% of embryos reached the blastocyst stage (26/30) and 60.0% hatched (18/30) in the glass trays in the culture incubator. In the EmbryoScope incubator, 83.3% reached blastocyst stage (25/30) and 63.3% hatched (19/30). Control Nunc dishes gave 66.6% blastocyst formation (20/30) and 53.3% hatching (16/30) in the standard incubator (NS, passed QC test). Using the EmbryoScope, single mouse embryos and groups of 1, 2, 3 and 5 embryos resulted in average respiration rates of 0.26 nl O 2 /h (SD 0.03). When 20 embryos were used together in the closed system (1 cell), the average respiration rate was 0.15 nl O 2 /h over a 4-h measuring period. Eight of these embryos were subsequently transferred to the EmbryoScope, where the respiration rate recorded over a 4-h period gave average measurements of 0.1 nl O 2 /h. The similarity of the average respiration rates for mouse embryos in the EmbryoScope and those measured by closed respirometry in glass vials indicate that the respiration rate calculations and results from the EmbryoScope are consistent with rates obtained in other verified systems. Human oocyte respiration measurements All results in which the control wells were of quality 3 (low), or when they were high, variable and not reliable, were removed from the data. Results from wells in which bubbles were noted, which can also affect the readings, and in which the oocyte adhered to the side of the well, were also excluded from the data. Where MII oocytes were used, data were excluded when the oocyte spontaneously cleaved or fragmented on continued culture; this occurs only in activated oocytes, and the possibility that these were silently fertilized cannot be excluded (Van Blerkom et al., 1987). All other readings were included in the data analysis. The data are presented as the initial or baseline respiration rate (BRR), the first five or six readings, i.e. the average rate, for a defined group of oocytes, including the standard deviation, for this time period. The oocytes were generally cultured further (18 h in total) but the measurements for this time frame are not presented as they are not relevant to this initial analysis. D0 and D1 oocytes Of the 502 oocytes used in the EmbryoScope, data were assessed as reliable for 446 oocytes (88%). Rejection of the data on 56 oocytes was due to the criteria outlined above. Of the 446 oocytes with readings, seven were large or giant oocytes (see below) and 15 were grossly abnormal, with vacuoles, abnormal zona, spontaneous fragmentation and cleavage. These 22 oocytes were also removed from the data. Abnormal oocytes had very high BRR, and oocytes that fragmented had rates that were lower than 0.45 nl O 2 /h.

5 Of the remaining 424 oocytes, 161 were D0 (100 GV and 61 MI) and 263 D1 (29 GV, 83 MI and 151 MI1). The data were analysed as BRR for each oocyte according to initial stage, then by the fate or development of that oocyte in culture over 18 h. The data are presented by oocyte stage and fate, regardless of source, age or infertility aetiology of the patient. Overall, on both D0 and D1, regardless of the stage of maturity, oocytes that became atretic in culture had a BRR that was significantly lower than their counterparts (P < 0.01). When this was plotted for all included oocytes (Figure 2), oocytes that became atretic in culture were on the lower phase of the graph. The data for the rest of the D0 and D1 oocytes are summarized in Table 1. Healthy GV and MI oocytes, those that continued maturation in vitro, had higher respiration rates than those that arrested in vitro. Oocytes that arrested/had no development in vitro, but did not become atretic, had intermediate respiration rates (Table 1 and Figure 2). Both GV and MI oocytes had higher BRR on D0 than on D1. MII oocytes (D1) had the lowest BRR rates, which could indicate that they were resting. However, D1 versus D0 GV and MI oocytes had lower rates, thus lowered D1 rates for MII oocytes could also be an artefact of ageing and invitro culture. The data were considered as real, as placing one, two and three oocytes together in wells produced BRR approximately one, two and three times greater than for a single oocyte. These data represent the first consistent large cohort study of individual human oocyte respiration rates in vitro. Large/giant oocytes Giant/large oocytes are occasionally identified at retrieval. When screened by fluorescence in-situ hybridization analysis (Munné et al., 1994) these oocytes are generally polyploid or haploid. The average initial respiration rates of the giant/large oocytes in this study (n = 7) ranged from to nl O 2 /h, the higher value representing the highest rates recorded for any oocyte used in the system (Table 1). As these are abnormal oocytes, their rates are also excluded from the data in Table 1 and Figure 2. Some giant oocytes (n = 4) had very high respiration rates, which could reflect their increased volume and possibly increased numbers of mitochondria. Oocyte cohort differences Fertilization results It has previously been shown that, in a cohort of mature oocytes, different oocytes have differing mtdna loads, according to the fate of the oocytes in the whole cohort (Reynier et al. 2001; May-Panloup et al. 2005, 2007). One of the differences noted occurred when the oocytes originated from cohorts that had poor or failed fertilization in the absence of a male factor problem, perhaps indicating an oocyte origin for the failed fertilization (Reynier et al., 2001). The data from the 442 oocytes with reliable readings were again analysed according to D1 MII non-fertilized oocytes where there was no male factor and where there was a varying degree of fertilization. A total of 134 MII oocytes failed to fertilize in couples with no severe male factor diagnosis (all had > /ml spermatozoa post-processing, >4% normal forms by Kruger strict criteria, no testicular aspiration/ biopsy). These were further categorized by the cohort results, 0% fertilization; 1 40; 41 59; 60 79; 80 90%. These cut-off values were chosen to coincide with the published data on mtdna load (Reynier et al., 2001). The comparative group of oocytes for these experiments was severe male factor with female age being less than 37 years with no known pathological (not structural) female infertility. These may be considered abnormal in that they had undergone ICSI with no fertilization; however, only oocytes in which there was no suspected female contribution to the infertility were used. The results for oocyte respiration rates by cohort fertilization rates are shown in Table 2 and Figure 3. There was a significant correlation between fertilization rate and respiration rates, with lowered fertilization being consistent with lowered respiration. The cut-off value for fertilization was approximately 40%, after which there appeared to be no correlation with respiration. The BRR of failed-fertilized MII in severe male factor ICSI cases were equivalent to the upper levels of fertilization. Although the numbers are small in some of the groups, the data support the concept that not all fertilization failure is sperm based. Day 2 FSH It is well known that oocyte quality and therefore treatment outcome can be closely linked to baseline/day 2 FSH concentrations in stimulated cycles. The BRR of all oocytes that originated from oocyte cohorts where the FSH was low, increasing to high levels was compared, to analyse the potential effect of follicle/ovarian ageing on oocyte respiration. The results are shown in Table 3 and Figure 4. The cut-off values for FSH were 5, , and miu/ml (the highest allowed value in the programme). All oocytes were used in this data. For comparative purposes, rates in young fertile donors, all of whom had low FSH concentrations, were taken as indicating a normal range, using an average BRR of nl O 2 /h. The results (Table 3) show a decreasing BRR of oocytes with increasing FSH concentration, which reached significance at FSH 11 miu/ml. It is clear from these data that the respiration rates of oocytes originating from women with elevated FSH are depressed compared with those with lower FSH. These data are consistent with clinical findings, and the linear curve of BRR mimics that of declining fecundity as FSH rises. Age As there is abundant literature on declining fecundity with age, the BRR was analysed by age, using the Society for Assisted Reproductive Technology (SART) age categories. When the data were analysed by age for all oocytes on D0 and D1, the data had a wide spread and was variable. When analysed by D0 and stage of maturity there was a significant effect of age on BRR, with decreasing respiration as the patient age increased (Table 4). This was more pronounced 465

6 Table 1. Baseline respiration rates of day 0 (D0) and day 1 (D1) oocytes over 1 3 h, according to their in-vitro fate after 18 h of culture. Oocyte type n Baseline rate and fate respiration (nl O 2 /)h a Figure 2. Scatter plot of baseline respiration rates (BRR) of oocytes included in the study. The plot was grouped by the bracketed mean, minimum and maximum BRR for any oocyte that became atretic in vitro; or remained unchanged; or matured in vitro. D0 oocytes All GV (0.020) GV atretic (0.031) GV GV (0.010) GV M (0.022) GV M (0.014) All M (0.015) M1 atretic (0.027) M1 M (0.020) M1 M (0.014) D1 oocytes All GV (0.050) GV atretic (0.030) GV GV (0.017) GV M (0.015) GV M (0.004) All M (0.015) M1 atretic (0.012) M1 M (0.011) M1 M (0.015) All M (0.017) M2 atretic (0.031) M2 M (0.012) a Values are mean (SE). GV = germinal vesicle stage; MI = immature metaphase I; MII = mature metaphase II. Table 2. Baseline respiration rates in failed fertilized MII oocytes according to cohort fertilization rates. Fertilization MII Baseline P-value rate (%) oocytes respiration (n) rate (nl O 2 /h) a (0.015) (0.035) % (0.010) NS (0.020) NS (0.013) NS Control (0.014) NS a Values are mean (SE). Figure 3. The mean baseline respiration rates (BRR) for oocytes in non-male factor patients was plotted by cohort fertilization rates of MII oocytes, which varied from 0 to 90%. The comparison group was oocytes from male factor cases without a female contribution where ICSI was used and there was no fertilization. 466

7 Table 3. Baseline respiration rates of oocytes according to FSH concentration. FSH Oocytes Baseline P-value concentration (n) respiration (miu/ml) rate (nl O 2 /h) a (0.016) (0.031) (0.019) NS (0.021) NS a Values are mean (SE). Figure 4. The mean baseline respiration rates (BRR) plotted against FSH concentration. All oocytes in each group were used. Table 4. Baseline respiration rates of day 0 (D0) oocytes according to the age of the patient. Patient D0-GV D0-MI age (years) n Baseline n Baseline respiration rate respiration rate (nl O 2 /h) a (nl O 2 /h) a < (0.014) (0.016) (0.008) (0.014) (0.012) b (0.021) > b (0.098) b (0.013) a Values are mean (SE). b P < GV = germinal vesicle stage; MI = immature metaphase I. Figure 5. The effect of overall patient prognosis, high or low, based on age, previous cycles and FSH concentration was plotted against baseline respiration rate (BRR). The mean, maximum and minimum for each group are indicated, and the overlap at the lower end of high prognosis patients and the upper end of low prognosis patients. at the more mature stage, MI versus GV, which may be indicative of the BRR of MII oocytes on D0: as age increased, BRR decreased. Inter- and intracohort differences For all oocytes with BRR, the patients were grouped into high versus low prognosis categories. High prognosis female patients were <38 years of age, had no severe ovarian disease (e.g. endometriosis, PCOS), BMI <35, cycle 1 or 2, and had at least three oocytes studied for base respiration measurements. Low prognosis patients included those of age >37 years, any age with ovarian disease, on cycle >2 with no pregnancy, but also with at least three oocytes for study. The average BRR and the spread of BRR were ascertained for each group. The data are presented in Figure 5. Within each group and within each patient there were differences between oocytes. However, oocytes from high prognosis patients had less of a 467

8 468 spread (minimum/maximum and range) of BRR than those with low prognosis. High prognosis patients had a mean BRR that was higher than that of the low prognosis patients. These data also indicate that there are differences in oocyte respiration within a cohort, and that more oocytes in high prognosis patients have high rates than in low prognosis patients. An interesting result is that within the cohort of oocytes from low prognosis patients, there are some oocytes that have BRR equivalent to ranges in the high prognosis patients (see Figure 5). These data indicate that the majority, but not all, oocytes from low prognosis patients have low BRR, but those with higher BRR, in the range of high prognosis patients, can be identified using this technique. These data indicate that oocyte respiration may be an effective tool in initial oocyte screening for increased success in assisted reproduction. Discussion The data presented here provide the first report of validated measurements of human oocyte respiration rates in a real-time, non-invasive system. Data have been presented previously on 14 oocytes, using a micro-spectrophotometric system (Magnusson et al., 1986). However, this method did not allow recovery of the oocytes, it was invasive, and the data were generated at much reduced oxygen concentrations compared with the current study, which used environmental oxygen concentration. Notably, the rates measured in that study (0.53 nl O 2 /h on D0 and 0.35 nl O 2 /h on D1 oocytes) were equivalent to the data presented in the current study, indicating that the measurements are real. Further, the current data were gathered from non-clinical samples in stimulated cycles, whereas the previous report was on unstimulated cycles. As the rates are equivalent, it could indicate that overall oocyte respiration is measurable and real, and not affected by stimulation. It is also significant that both studies show repeatable differences between oocytes at differing developmental stages. Within the current study this is also true for oocytes with documented developmental capacity, and oocyte origin and fate. With refinement and controlled clinical trials, oocyte respiration may become a viable and useful tool for selection prior to fertilization, as an adjunct to the current cleaving embryo morphology parameters currently in use. Respiration measurements can potentially provide two sets of information regarding the oocyte or embryo. First, the activity of the mitochondria, and perhaps the actual mitochondrial load present in the oocyte (Cummins, 2002; Dumollard et al., 2006, 2007). As the mtdna load and actual numbers of mitochondria counted in oocytes have been shown to differ so widely between oocytes and between reports, this may be a real phenomenon of oocyte growth and maturation, with only those that meet a threshold level being able to sustain development post-fertilization (Steuerwald et al., 2000; Cummins, 2002; Dumollard et al., 2007; May-Panloup et al., 2007; Shoubridge and Wai, 2007). Secondly, respiration produces ATP, thus a measurement of respiration could extrapolate to the amount of available ATP, without which fertilization and subsequent development are compromised (Dumollard et al., 2004). It is well established that mitochondria are the cell powerhouse, including within the oocyte, providing the bulk of ATP required to sustain development (Ernster and Schatz, 1981). The primordial oocyte has as few as 10,000 mitochondria, which begin to replicate during maturation. The number are set at the final stage, MII, but these numbers are vast, in the region of 900,000 (May-Panloup et al., 2005). Replication of mitochondria will not resume until later in embryo development (Cummins, 2002; Dumollard et al., 2006, 2007). Other cells that require large amounts of ATP, such as muscle cells, have far fewer mitochondrial DNA than oocytes. Cells that are relatively non-reliant on ATP, such as neurons, have even fewer (Steuerwald et al., 2000). As embryo development is directly related to the health and development of the gametes, the mitochondrial content of the developing oocyte could be a key factor in oocyte quality, a key to final embryo quality. If an oocyte has too few mitochondria, it may not be able to complete fertilization, or might be unable to sustain full development after fertilization, due to a lack of ATP. Studies have shown that oocytes in standard IVF cases from non-male factor patients that fail to fertilize, or where the fertilization rate was low, have a lower mtdna load than oocytes that achieved adequate fertilization (Reynier et al., 2001; May-Panloup et al., 2007). The data presented here were in agreement for base respiration rates versus fertilization rates. Mature oocytes from non-male factor patients showed significantly reduced respiration rates when there was complete fertilization failure or very low fertilization rates. The graphs mimicked those obtained from total mtdna analysis. In the current respiration experiments, oocytes that failed to fertilize after ICSI (male factor cases) did not show reduced rates of respiration. Reduced respiration rates could be due to low levels of active mitochondria or, coupled with the data on mtdna, could indicate overall low mitochondria count. Taken together, these data indicate that not all fertilization failure is sperm related; the technique described here may be a means of elucidating these cases. The findings of lowered BRR with both increasing age and FSH may be explained by ageing or depletion of mitochondria (Balaban et al., 2005) intracellular redox potential (Van Blerkom, 2004; Dumollard et al., 2007) and homeostasis (Duchen, 2000). Oocyte respiration could become a screening tool for older women and/or where FSH is increasing (see low and high prognosis patient data above). Within any patientspecific cohort, two morphologically identical MI oocytes could have very different respiration rates, which may indicate mitochondrial activity and general health and ATP load. The spread was less in high prognosis patients, which may explain the higher pregnancy rates for these patients; more oocytes are competent and lead to competent embryos. Using respiration-rate information prior to insemination may help to generate embryos with the highest potential for implantation in the low prognosis patients. If an oocyte has a higher BRR (>0.5 nl O 2 /h) in a 40-year-old woman it may indicate greater potential and development than one with a BRR of <0.5, closer to 0.4 nl O 2 /h. This could be a good screening tool for women >35 years of age, especially considering the cohort details.

9 The fact that there are differences between oocytes is important. May-Panloup et al. (2005), showed variations in mtdna load, from 11 to 903 K and these differences can exist even within cohorts. Similarly, the data here shows differences in BRR within cohorts and not only between oocyte sources. As embryo development is directly related to the health and development of the gametes, the mitochondrial content of the developing oocyte could be a key factor in oocyte quality, and the key to final embryo quality. To measure either mitochondria or mtdna load necessitates the destruction of the oocyte, where as respiration measurements, which document mitochondrial activity, are non-invasive. Using technology such as that presented here, it may be feasible to build an indirect profile of an oocyte s mitochondrial load and activity, of its ability to respire at a rate that can sustain fertilization and development and then to establish thresholds in controlled prospective studies and ultimately be able to use oocyte respiration rate to select oocytes with the greatest developmental competence. Ultimately, proof of concept in a human clinical programme is required by performing an internal review board approved controlled randomized trial, where oocyte respiration is used for oocyte selection, with subsequent improved development and live delivery rate. Acknowledgements This research was supported by an unrestricted research grant from Organon USA Inc., a part of the Schering-Plough Corporation. References Balaban RS, Nemoto S, Finkel T 2005 Mitochondria, oxidants, and aging. Cell 120, Cummins J 2002 The role of maternal mitochondria during oogenesis, fertilization and embryogenesis. Reproductive BioMedicine Online 4, Duchen M 2000 Mitochondria and calcium: from cell signalling to cell death. Journal of Physiology 529, Dumollard R, Duchen M, Carroll J 2007 The role of mitochondrial function in the oocyte and embryo. Current Topics in Developmental Biology 77, Dumollard R, Duchen M, Sardet C 2006 Calcium signals and mitochondria at fertilisation. Seminars in Cell and Developmental Biology 17, Dumollard R, Marangos P, Fitzharris G et al Sperm-triggered [Ca 2+ ] oscillations and Ca 2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production. Development 131, Ebner T, Moser G, Tews G 2006 Is oocyte morphology prognostic of embryo developmental potential after ICSI? Reproductive BioMedicine Online 12, Ebner T, Yaman C, Moser M et al Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Human Reproduction 15, Ernster L, Schatz G 1981 Mitochondria: a historical review. Journal of Cell Biology 91, Jansen R, de Boer K 1998 The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate. Molecular and Cellular Endocrinology 145, Lopes A, Larsen L, Ramsing N et al Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system. Reproduction 130, Madaschi C, Carvalho de Souza Bonetti T, Paes de Almeida Ferreira Braga D et al Spindle imaging: a marker for embryo development and implantation. Fertility and Sterility 88 [Epub ahead of print]. Magnusson C, Hillensjö T, Hamberger L, Nilsson L 1986 Oxygen consumption by human oocytes and blastocysts grown in vitro. Human Reproduction 1, May-Panloup P, Chretien M-F, Malthiery Y, Reynier P 2007 Mitochondrial DNA in the oocyte and the developing embryo. Current Topics in Developmental Biology 77, May-Panloup P, Chretien M, Jacques C et al Low oocyte mitochondrial DNA content in ovarian insufficiency. Human Reproduction 20, Montag M, Schimming T, Köster M et al Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles. Reproductive BioMedicine Online [Epub ahead of print]. Munné S, Alikani M, Cohen J 1994 Monospermic polyploidy and atypical morphology. Human Reproduction 9, Ottosen L, Hindkjaer J, Lindenberg S, Ingerslev H 2007 Murine pre-embryo oxygen consumption and developmental competence. Journal of Assisted Reproduction and Genetics 24, Pascale M-P, Chretien M-F, Malthiery Y, Reynier P 2007 Mitochondrial DNA in the oocyte and the developing embryo. Current Topics in Developmental Biology 77, Reynier P, May-Panloup P, Chretien M et al Mitochondrial DNA content affects the fertilizability of human oocytes. Molecular Human Reproduction 7, Shen Y, Stalf T, Mehnert C et al High magnitude of light retardation by the zona pellucida is associated with conception cycles. Human Reproduction 20, Shoubridge EA, Wai T 2007 Mitochondrial DNA and the mammalian oocyte (review). Current Topics in Developmental Biology 77, Steuerwald N, Cohen J, Herrera R, Brenner C 2000 Quantification of mrna in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT PCR. Molecular Human Reproduction 6, Van Blerkom J 2004 Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction 128, Van Blerkom J, Bell H, Henry G 1987 The occurrence, recognition and developmental fate of pseudo-multipronuclear eggs after in-vitro fertilization of human oocytes. Human Reproduction 2, Wilding M, Dale B, Marino M et al Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Human Reproduction 16, Declaration: The authors report no financial or commercial conflicts of interest. Received 1 November 2007; refereed 6 December 2007; accepted 28 April