1 Reproductive Medicine, Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Göteborg, Sweden, 2 The Fertility Clinic, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 3 Fertility Center, Carlanderska Hospital, Göteborg, Sweden and 4 Novo Nordisk A/S, Copenhagen, Denmark
5 To whom correspondence should be addressed at: Reproductive Medicine, Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. Tel: +46 31 3421000; Fax: +46 31 418717; Email: christina.bergh{at}vgregion.se
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Abstract |
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Key words: aneuploidy/chromosomal abnormality/follicular-fluid meiosis activating sterol MAS/human/oocytes
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Introduction |
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Follicular Fluid-Meiosis Activating Sterol (FF-MAS) is a C-29 oxysterol occurring naturally in the biosynthetic pathway between lanosterol and cholesterol and is found in high concentrations in the follicular fluid in the ovaries in mammals including humans (1.6 µM) (Byskov et al., 1995
, 2002
). It belongs to a class of sterols that possess meiosis inducing activity and has been synthetically manufactured, identical to the naturally occurring sterol found in human follicular fluid (Gröndahl et al., 1998
; Murray et al., 2000
,2002
). Earlier studies in mice and rabbits have shown that FF-MAS is generated endogenously as a consequence of the FSH and the LH surge (Baltsen, 2001
; Gröndahl et al., 2003
). Addition of FF-MAS to the culture media has been shown to induce resumption of meiosis in mouse oocytes in vitro in a dose-dependent manner (Gröndahl et al., 1998
). Furthermore, in vitro matured mouse oocytes exposed to FF-MAS have been found to have a higher fertilization rate and an increased embryo development than both in vitro spontaneously matured oocytes and in vivo matured oocytes (Hegele-Hartung et al., 1998
). Exposure of mouse oocytes to FF-MAS has been found to result in normal appearance of spindle and chromosome alignment (Hegele-Hartung et al., 1999
). In a study of 31 in vitro maturation cycles, culture of immature human oocytes in FF-MAS supplemented media was found to significantly increase nuclear maturation (Gröndahl et al., 2000
). Cumulus expansion was also found to be more pronounced in presence of FF-MAS. Furthermore, FF-MAS mediated matured oocytes were found to have a significantly increased degree of cytoplasmic maturation and increased synchrony between nuclear and cytoplasmic maturation (Gröndahl et al., 1999
). It has also been found that MAS had positive effects on immature human oocytes retrieved both from unstimulated PCO patients and from patients undergoing ICSI (Cavilla et al., 2001
). A recent parallel study (Loft et al., 2004
) with a similar design as the present study investigated the effect of FF-MAS on cumulus enclosed oocytes. In that study, as in the present, FF-MAS was dissolved in ethanol and an ethanol control was included. Ethanol in the Loft et al. study caused a significant increased chromosomal abnormality rate at the blastomere level, as well as impaired embryo development. No further negative effects were noted for FF-MAS as such.
In the present study we wanted to investigate the possible influence of FF-MAS without the interaction by cumulus cells. Therefore denuded oocytes were used in this study. A further aim was to investigate if FF-MAS could increase the synchrony between nuclear and cytoplasmic maturation in the second part of meiosis which might result in improved embryo development, and therefore only metaphase II oocytes were used.
The primary objective of the present study was to assess the effect on the chromosomal abnormality rate in pre-embryos by adding one of two different concentrations of FF-MAS to the oocyte culture media, 4 h prior to fertilization and again for 20 h post fertilization, compared to two control groups (inert control and vehicle control). As secondary endpoint, the effect of FF-MAS on cleavage rate and embryo quality was assessed.
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Methods |
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Patients
The inclusion criteria were indication for IVF or ICSI treatment, female age between 25 and 37 years (both inclusive) and regular menstrual cycles. Furthermore, a minimum of 6 (Denmark) to 10 (Sweden) aspirated oocytes was required for the patients own treatment after donation.
Exclusion criteria were any known medical condition or genetic disorder which could preclude IVF/ICSI treatment, or could interfere with the interpretation of the results of the study, and the use of any investigational drug within 30 days prior to oocyte retrieval.
Altogether, 243 women donated 353 oocytes for this trial. No oocytes exposed to FF-MAS nor the control group were transferred to the women.
Demographics of the women donating oocytes are given in Table I. No significant differences were found between groups. The average age of the women was 32 years. Male factor (61%) and tubal factor (32%) were the most frequent infertility reasons. Most of the patients (71%) had been infertile between 25 years. Of the trial population, 54% had previously undergone assisted reproduction treatment.
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Treatment and donation
Long protocols using down-regulation with GnRH (Synarela, Searle; Suprecur, Aventis, Pharma) for at least 14 days, as well as short protocols using GnRH antagonists (Orgalutran, Organon; Cetrotide, Serono) were allowed. Patients were stimulated with a recombinant FSH (Puregon, Organon; Gonal-F, Serono), with an average daily dose between 100300 IU. At the time of hCG (10 000 IU Profasi, Serono; Pregnyl, Organon) administration at least three follicles of 17 mm or more were registered by ultrasound.
Oocyte aspiration was performed according to each clinics' normal procedure. During aspiration, follicular fluid aspirated together with the donated oocytes was collected when possible and subsequently analysed for concentration of FF-MAS.
Depending on the number of oocytes the patients wanted to donate, the selection procedures was the third aspirated oocyte from the first ovary, the third aspirated oocyte from the second ovary, and then oocytes randomly chosen among the remaining oocytes. The oocytes were randomized to treatment group within 90 min after aspiration.
The oocytes were inseminated by intracytoplasmic sperm injection (ICSI) using donor sperm from one of the same three sperm donors of proven fertility and normal karyotype (Cryos International Sperm Bank Aarhus, Denmark) (in three cases the oocytes were fertilized using the husband's sperm sample).
Cell culture and embryo evaluation
After aspiration, the cumulus enclosed oocytes were denuded and metaphase II oocytes (defined by extrusion of the first polar body) were randomly assigned to an oocyte culture system containing standard IVF media containing 10 mg/ml human serum albumin (IVF-20, Vitrolife, Gothenburg, Sweden) and one of the following treatments: FF-MAS 5 mM, FF-MAS 20 mM, ethanol 0.2% (vehicle) or water for injection (inert). The exposure time was 4 h prior to ICSI and 20 h post ICSI. Due to the lipophilic nature of the FF-MAS molecule the test solutions were dissolved in ethanol, resulting in a final concentration of 0.2% ethanol.
Denudation was performed by use of 80 IU/ml hyaluoronidase (Hyase,Vitrolife, Gothenburg, Sweden). Culture was performed in 4-well dishes (Nunc, Roskilde, Denmark). Insemination was performed by ICSI 4±1 h after randomization. After ICSI the oocytes were cultured for another 20 h in their respective treatment. The oocytes were checked for number of pronuclei (0, 1, 2, 3, >3) at 20 h post ICSI. An embryo was considered fertilized if, at 20 or 26 h, two or three pronuclei or one pronucleus, together with two polar bodies, were observed. Embryo evaluation was performed at 26, 44, 50 and 68 h (±1 h) after ICSI. At these time points, the embryos were scored for cell number, degree of fragmentation (no fragmentation, 010%, 1120%, 2150% or >50% fragmentation), localization of fragments (local or dispersed), equally or unequally sized blastomeres and cytoplasmic appearance (homogeneous or dark/granulated/vacuolated). Furthermore, high resolution digital images were stored at each time point using the FertiGrab system (Image House Medical A/S, Copenhagen, Denmark) for reassessment procedures.
In order to minimize the inter-clinical variation, the same batches of culture media, utensils and donor sperm were used at all clinics. Furthermore, morphological evaluation of the embryos was performed as a consolidated reassessment by three embryologists based on high resolution computer-based picture recording of the oocytes and embryos.
Fixation
At 70 h (±2 h) post ICSI, all blastomeres from the embryos were fixated individually by dissolving the zona pellucida in pronase, 5 mg/ml (Sigma, St Louis, MO), followed by incubation in Ca2 + /Mg2+ free medium (EB-10, Vitrolife, Gothenburg, Sweden) until segregation of the individual blastomeres. The nucleus/nuclei from each blastomere was then fixated on silinized coated slide (Oncor Gathersburg, USA, cat. no. S1308) in a HCL/Tween-20 solution (0.01 N/0.1%). After fixation, the slides were left to dry until the following morning, then dehydrated in PBS followed by 709099% ethanol series and packed in containers with silica gel and stored at 20°C until shipment for analysis.
Cytogenetic analysis and evaluation
Fluorescence in situ hybridization (FISH) analysis for chromosomes 13, 16, 18, 21, 22, X and Y were performed at an independent laboratory (Quest Diagnostics Inc. San Juan Capistrano, CA).
The FISH analysis was performed by sequential hybridization using Vysis MultiVysion PB and CEP X/CEP Y (alpha satellite) (Vysis, Downers Grave, IL). Locus specific probes were used for the chromosomes 13, 21, 22 and centromere probes for the chromosomes 16, 18, X and Y.
The evaluation of the cytogenetic results was performed as a consolidated evaluation by three embryologists, together with a geneticist from Quest Diagnostics Inc.
The cytogenetic evaluation included documentation of the number of nuclei in each blastomere and classification of each blastomere as normal, abnormal or not-classifiable for the tested chromosomes. Furthermore, based on the finding in the blastomeres, each embryo was classified as either uniformly normal, uniformly abnormal, mosaic ploid, mosaic aneuploid, mosaic chaotic or non-classifiable (Table II). An overall classification as normal, abnormal or non-classifiable was applied to each embryo. Non-classifiable blastomeres/embryos were mainly caused by problems with penetration of the probes. A genetically normal embryo was defined for this study as an embryo with at least 50% of its analysed cells having nuclei with a normal constitution concerning these probes.
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Source of FF-MAS
FF-MAS was pharmaceutically generated and final formulated by Medicinal Chemistry, Novo Nordisk A/S according to published and patented synthesis methods (Murray et al., 2002).
Statistics
No women were excluded from the analyses of demographic data. Oocytes were sequentially assigned to one of the four treatment groups. This procedure was regarded as random. In a few cases specific observations were considered censored observations: (i) oocytes/pre-embryos lost during the trial due to technical problems were excluded from the analyses at subsequent time points; and (ii) oocytes/pre-embryos that, based on the follicular fluid MAS concentration in the culture media, were classified as non compliant to treatment allocation were excluded from all analyses. The primary analysis group consisted of all oocytes randomized, minus those censored. In total, 17 oocytes/pre-embryos were censored.
The primary endpoint of the study was the incidence of human pre-embryos with numerical chromosomal abnormalities. Assessments were made for chromosome X, Y, 13, 16, 18, 21 and 22 in pre-embryos at 70±2 h post ICSI, using multi-colour FISH. Chi-square and Fisher exact testing were used to compare the frequency of uniformly normal pre-embryos. The percentage of pre-embryos categorized as normal and abnormal was compared among groups, using Odds ratios (with 95% confidence intervals; CI).
The secondary endpoints were the incidence of 2 cells pre-embryos at 26 h post ICSI, the incidence of
4 cell pre-embryos at 44 h post ICSI, the incidence of
4 cell pre-embryos of good quality at 44 h post ICSI (defined as pre-embryos with no fragmentation or <10% fragmentation, equally sized blastomeres and homogeneous cytoplasm), the incidence of
6 cells pre-embryos at 68 h post ICSI and the incidence of
6 cell pre-embryos of good quality at 68 h post ICSI. Odds ratios (with 95% CI) were provided for comparison against the inert control group.
To measure the degree of agreement between embryologists (local observation versus reassessment observation as well as among the three embryologists at reassessment), Kappa statistics were used to calculate concordance rates on cleavage stage and morphology at 68 h post ICSI.
All statistical tests were two-sided, performed at the 0.05 level of significance. The data were analysed using SAS version 6.12 (SAS Institute Inc., Cary, USA) run on a UNIX platform.
Sample size
With a significance level of 0.05 and power of 0.90, a sample size of 56 pre-embryos per group was needed to detect an increase in aneuploidy rate from 30% in the control group to 60% in the test groups. Based on the design with four treatment groups, a total of 224 pre-embryos were to be evaluated for chromosomal abnormalities. With the assumption that 50% of all retrieved oocytes would cleave, it meant that 448 oocytes were to be recruited in total. Based on a donation rate of 12 oocytes per woman this would require a population of 300 IVF/ICSI patients.
An interim analysis was made after 3 months of enrolment with the primary objective to assess any increase in incidence of pre-embryos with chromosome abnormalities in the FF-MAS groups. The interim statistical analysis was made on data from the 173 oocytes randomized to the trial by 30 June 2000. The results were available on 31 Oct 2000 and based on the conclusive results of the interim analysis, the trial was discontinued. However, at that time point a total of 345 metaphase II oocytes had been randomized, yielding a total of 239 pre-embryos with FISH results. Thus, the study was run in accordance with the primary sample size calculation.
Ethical approval
Approvals from The Danish and Swedish Ethical Committee were obtained. Written informed consent was obtained from both partners. The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP).
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Results |
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Cytogenetic evaluation of blastomeres
A total of 855 blastomeres derived from 239 pre-embryos underwent FISH analysis. The number of normal, abnormal, mononucleated and multinucleated blastomeres is shown in Table IV. The percentage of blastomeres categorized as abnormal was 48% in the inert control group, 39% in the vehicle control group, 59% in the FF-MAS 5 µM group and 71% in the FF-MAS 20 µM group. The percentage of blastomeres categorized as abnormal was significantly higher in the FF-MAS 5 µM and FF-MAS 20 µM groups compared to the inert control group and also compared to the vehicle control group (Table IV). When comparing the pooled FF-MAS groups vs the pooled control groups the percentage of abnormal blastomeres was significantly higher in the FF-MAS groups.
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Fertilization, cleavage rate and pre-embryo quality
A total of 345 metaphase II oocytes underwent ICSI. At 20 h post ICSI 76% had fertilized and at 26 h the fertilization rate had increased to 78%. The fertilization rates at 20 and 26 h post ICSI were not significantly different among groups (Table V).
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The frequency of pre-embryos with two cells or more (overall and of good quality) at 26 h is summarized in Table VI. No statistical differences were found between groups.
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At 68 h post ICSI, the percentage of pre-embryos with six cells or more (overall and of good quality) was significantly lower in each of the FF-MAS groups compared to the inert control group. There were no significant differences between the inert and vehicle control groups.
Pre-embryo morphology (fragmentation, distribution of fragments, uniformity of blastomere size and cytoplasm homogeneity)
Fragmentation was graded as follows: 0%, >0 to 10%, 1120%, >20 to
50% or >50% at 26, 44, 50 and 68 h post ICSI. Although at 68 h the percentage of pre-embryos with a fragmentation grade of 010% appeared to be consistently lower in the FF-MAS groups compared to the control groups, no statistical differences were found. The distribution of fragments as locally or dispersed was assessed with no significant differences between groups at 68 h.
A significant difference in the distribution of equally and unequally sized blastomeres was found. Among 6 cells pre-embryos at 68 h, 42 and 54% of the blastomeres were equally sized in the inert and vehicle control groups, respectively, while this was the case for 13 and 18% of the blastomeres in the FF-MAS 5 µM and 20 µM groups, respectively.
Appearance of the cytoplasm was recorded as either homogeneous or dark/granulated/vacuolated and significant differences were found between groups. At 68 h, the percentage of pre-embryos with homogeneous cytoplasm was 74 and 70% in the control groups and 3828% in the FF-MAS groups.
As stated in Statistics the Kappa value between embryologists with respect to cleavage and pre-embryo morphology at 68 h post ICSI was calculated. Overall the concordance rate for local observations versus reassessment observations was high. The highest degree of agreement was noted for cleavage rate, where the Kappa value ranged from 0.93 to 0.95. The concordance rates among the three embryologists were also high, the Kappa value for cleavage rate ranging from 0.87 to 0.90 and for fragmentation ranging from 0.64 to 0.71.
FF-MAS in follicular fluid
Altogether FF-MAS was measured in samples of 272 follicles. The mean concentration of FF-MAS in follicular fluid was 545 µg/ml (1.3 µM) ranging from 137 to 2175 µg/ml (0.35.3 µM). Among follicular fluid derived from follicles of the same woman, the coefficient of correlation in FF-MAS concentration was 0.70. The FF-MAS concentration in follicular fluid was poorly correlated (P=0.15) to the volume of fluid in the follicle. The correlation between FF-MAS and age of woman, BMI, smoking (ever smoking, current smoking, amount of smoking), FSH-dose, infertility reason and duration of infertility was weak.
FF-MAS concentration was also checked for possible correlation to embryo morphology in the control group. A significant correlation between FF-MAS and unequal sized blastomeres was found where an increased FF-MAS concentration correlated to an increased rate of unequal sized blastomeres. No other significant correlations between concentration of FF-MAS and embryo development were found.
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Discussion |
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In the present study pre-embryos were classified as uniformly normal or abnormal depending on a consistent diploid or aneuploid content. Pre-embryos classified as mosaic and representing almost half of all pre-embryos, were classified as overall normal or abnormal depending on the percentage of normal blastomeres. An overall normal pre-embryo was defined as a pre-embryo with 50% of its cells having a normal diploid constitution for the chromosomes tested. This level of 50% was arbitrarily chosen but is supported by a study of Bielanska et al. (2002)
, which suggested that the developmental potential of the pre-embryo may be impaired if the majority of its cells are abnormal. Although this algorithm for categorizing mosaic pre-embryos could be discussed, it provided a systematic way of evaluation, particularly for the purpose of this study. In addition, the same algorithm was chosen in two other publications from parallel studies from the same research project (Loft et al., 2004
, Ziebe et al., 2003
).
Since the primary focus of the study was the rate of aneuploidy, the age of the participating women were within certain limits, between 25 and 37 years. The women participating in the trial were not karyotyped and the expected rate of chromosomal aberrations would be from 1 to 3%. Sperm donors were used for insemination of 99% of the oocytes and all donors had a normal karyotype and proven fertility.
After oocyte retrieval, oocytes were denuded from cumulus cells and exposed for 4 h pre ICSI and 20 h post ICSI to standard IVF media, supplemented with either water for injection (inert control), ethanol 0.2% (vehicle control) or one of two different concentrations of FF-MAS: 5 µM or 20 µM. The choice of exposure time, 4 h and 20 h had earlier been found to be optimal for maturation of denuded mouse oocytes (Gröndahl et al., 2003). Recently it was shown that FF-MAS promoted the metaphase I to metaphase II transition (nuclear maturation) as well as the the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) (Carrie et al., 2004
). However, in neither of these studies oocytes were exposed to FF-MAS post fertilization.
The justification of the MAS doses were that 5 µM FF-MAS has been found to be the minimal effective dose in mouse oocytes giving maximum response in terms of percent germinal vesicle breakdown (%GVB i.e. resumption of meiosis; Gröndahl et al., 1998) and 20 µM FF-MAS has been found in earlier studies on human immature oocytes aspirated from small follicles in PCOS patients, to significantly enhance nuclear maturation (Gröndahl et al., 2000
). The FF-MAS concentration (mean 1.3 µM, range 0.35.3 µM) found in the follicular fluid samples from this study was in line with previously published results by Baltsen and Byskov (1999)
where FF-MAS in human follicular fluid from IVF stimulated patients was assayed to
1.6 µM. The concentration 20 µM is
1020 times higher than mean levels of 1.6 µM (Baltsen and Byskov, 1999
; Byskov et al., 2002
). Since we, in this study, were interested in testing the cytogenetic safety of FF-MAS we explored the effect in the upper normal range 5 µM, and a high dose (i.e. four times the maximum level) 20 µM, in order to establish a proper safety margin.
The FISH technique has been developed to detect chromosome abnormalities on interphase nuclei (Pinkle et al., 1986). Several studies have applied the FISH technique for aneuploidy studies in pre-embryos (Munne et al., 1994
; Harper et al., 1995
; Delhanty et al., 1997
) and scoring criteria for defining numeric abnormalities have been proposed (Munne et al., 1998
). Earlier studies have reported a frequency of aneuploidy in human pre-embryos between 25 and 71% (Trounson et al., 2001
). There are several methodological differences among studies explaining this huge variation in aneuploidy. The majority of studies report on surplus embryos which have been judged to be of lower morphological development and not suitable for transfer. Other reports have analysed samples of embryos in a preimplantation genetic diagnosis programme (Munne et al., 1993
, Munne et al., 1995
; Gianaroli et al., 1999
). Few studies (Bielanska et al., 2002
) have evaluated all blastomeres in a large sample of non selected pre-embryos and most analysis have assessed only one or two blastomeres for classification of the whole pre-embryo. The number of chromosomes analysed also varies. Most studies report evaluation of three to nine chromosomes with chromosome 13, 18, 21, X and Y most frequently investigated. The chromosomes selected for evaluation in this study were those often associated with trisomies with potential of delivery, i.e. chromosome X, Y, 13, 18 and 21 and with trisomies frequently found in spontaneous abortions i.e. chromosome 16 and 22 (Iwarsson et al., 1999). Lastly, technical aspects such as types of probes and criteria for signals could also influence the results and explain differences in results in earlier studies.
We found that in the inert control group 50.8% were overall normal according to our definition and 31.7% were uniformly normal. This is in line with earlier findings by Loft et al. (2004) where 59% of pre-embryos were overall normal and 30% were uniformly normal in the control group (Loft et al., 2004
). The observations are also in line with findings by Bielanska and co-workers et al. (2002)
and Iwarsson et al. (1999)
who found that 29.6 and 27.9% of surplus high quality embryos were uniformly normal.
When data were analysed at the blastomere level, it was apparent that the groups which had been exposed to FF-MAS had a lower number of blastomeres analyzed, probably reflecting the lower cleavage rate. Exposure to FF-MAS was associated with a lower frequency of mononucleated blastomeres and a higher frequency of multinucleated blastomeres. A high level of multinucleation has previously been shown to be associated with increased rates of aneuploidy (Kligman et al., 1996; Hardarson et al., 2001
).
When analysing pre-embryo cleavage and quality, good quality embryos were defined as those embryos with no, or <10% fragmentation, with evenly sized blastomeres and with a homogeneous cytoplasm. At all time points from 44 h and onwards, a reduced cleavage rate and percentage of good quality pre-embryos was found in both FF-MAS groups. It is known that some relationship exists between cytogenetic and morphological characteristics, i.e. unevenly sized blastomeres are frequently associated with multinucleation and aneuploidy (Hardarson et al., 2001). These results showing a poorer pre-embryo development were in contrast to the previous study on cumulus enclosed oocytes (Loft et al., 2004
) where no negative effects on pre-embryo development were noticed for the FF-MAS group while ethanol resulted in a significantly reduction in cleavage for
2-cell embryos at 26 h. In the present study, however, ethanol 0.2% did not interfere with either the aneuploidy rate or the pre-embryo development.
The reasons for these findings of an increased chromosomal abnormality rate and poorer embryo development for oocytes/embryos exposed to FF-MAS are not known. However, denuded oocytes might be more sensitive to FF-MAS than cumulus enclosed oocytes. It may also be that the exposure time for FF-MAS and/or duration of exposure was not optimal in the present study.
In summary, the denudation of cumulus enclosed oocytes and subsequent culturing of the oocytes in vitro exposing oocytes to high levels of FF-MAS at concentrations of 5 µM or 20 µM both before fertilization and 20 h after fertilization, was not beneficial for the oocytes, and we can conclude that FF-MAS used in this setting on denuded oocytes and in these concentrations increased the frequency of aneuploidy and had detrimental effects on pre-embryo quality. Further studies are underway in the CEMAS study group to establish if FF-MAS will have any beneficial effects when using cumulus-enclosed oocytes and/or using lower concentrations or other timing of exposure in human oocytes as previously seen in animal studies.
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Acknowledgements |
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Submitted on December 1, 2003; accepted on May 27, 2004.
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