1 Biologie de la ReproductionLaboratoire FIV, 2 INSERM E0018, Laboratoire de Biochimie et Biologie Moléculaire and 3 Service de Gynécologie Obstétrique (UF Médecine de la Reproduction), Centre Hospitalier Universitaire d'Angers, 4, rue Larrey, F-49033 Angers cedex 01, France
4 To whom correspondence should be addressed. Email: pamaypanloup{at}chu-angers.fr
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Abstract |
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Key words: IVF/mitochondrial DNA/oocyte quality/ovarian insufficiency
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Introduction |
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During follicular growth, the number of oocyte mitochondria rises from 10 000 to
200 000 (Jansen and de Boer 1998
). The functional status of the mitochondria contributes to the quality of oocytes and probably plays an important role in the process of fertilization and embryo development (Cummins, 2004
). In particular, the developmental potential of the embryo and the outcome of IVF have been shown to be related to the ATP content of human oocytes (Van Blerkom et al., 1995
). In addition to their role in energetic conversion, mitochondria participate in numerous other essential cell functions such as the regulation of apoptosis, calcium homeostasis, FeS protein synthesis, and pyrimidine and haem synthesis (Delbart, 2000
). In an earlier report, we found a significant reduction in mitochondrial DNA (mtDNA) content in cohorts of oocytes examined after failure of an IVF procedure (Reynier et al., 2001
). We hypothesized that a defect in mitochondrial biogenesis together with insufficient mitochondrial mass may have led to defective oocyte maturation and hence to the poor outcome of IVF.
Mitochondria are frequently organized in dynamic interconnected networks, making the precise count of mitochondria technically difficult (Rube and van der Bliek, 2004). However, the quantification of mtDNA by means of real-time PCR provides a good evaluation of the mitochondrial mass in single cells, such as oocytes, because of the sensitivity of the method and the high correlation between the quantity of mtDNA and the mitochondrial mass (Wu et al., 2002
).
In order to investigate the relationship between oocyte quality and the mitochondrial mass, we determined the mtDNA content of oocytes in the context of the two main ovarian disorders, ovarian dystrophy and ovarian deficiency, as compared with the normal ovarian profile. We used real-time quantitative PCR to determine the mtDNA content of 116 oocytes obtained from 47 women undergoing IVF. All these oocytes had been discarded during ICSI procedures since they lacked the first polar body.
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Materials and methods |
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Group 1 consisted of 39 oocytes collected from 14 patients presenting a normal ovarian profile defined on day 3 of a spontaneous cycle by hormonal assay interms of FSH <5 IU/l, LH <6 IU/l, estradiol <45 pg/ml and inhibin B > 45 pg/ml; and by ovarian ultrasonography interms of >3 antral follicles per ovary.
Group 2 consisted of 47 oocytes collected from 16 patients presenting a profile of ovarian dystrophy defined on day 3 of a spontaneous cycle by hormonal assay as an FSH/LH ratio <1 with FSH <5 IU/l; and/or by ovarian ultrasonography as higher than the normal number (>10) of small follicles (28 mm in diameter) encircling the ovarian cortex like a string of pearls, with or without the increase of stroma. All patients except one presented normal androgen levels. In this group, 10 patients had an FSH/LH ratio <1 with a normal FSH value. Six of these 10 patients had an increased number of peripheral antral follicles. The other six patients had a normal FSH/LH ratio but all showed signs of ovarian dystrophy on ultrasonographic examination.
Group 3 consisted of 30 oocytes collected from 17 patients presenting a profile of ovarian insufficiency defined on day 3 of a spontaneous cycle by hormonal assay: FSH >8 IU/l (according to our laboratory norms) and/or by ovarian ultrasonography: <4 antral follicles per ovary. Fifteen of the 17 patients presented high basal FSH levels (>8 IU/l) on day 3. Among them, seven presented a low antral follicle count (<4 follicles per ovary). The two remaining patients presented normal FSH levels but elevated estradiol levels and low antral follicle counts.
As expected, the mean age of women was significantly higher in the group with ovarian insufficiency, group 3 (35.4±4.1 years), than in the group with a normal ovarian profile, group 1 (31.0±2.4 years) (MannWhitney: P=0.001). There was no significant age difference between the normal ovarian profile group and the multifollicular ovarian profile group, group 2 (30.3±3.2 years).
The average number of recombinant FSH IU used for the stimulation was significantly different for the three groups: 14x75 IU for the group with ovarian dystrophy, 22x75 IU for the group with the normal ovarian profile, and 39x75 IU for the group with ovarian insufficiency (KruskallWallis: P<0.0001).
The estradiol peak level was not statistically different between the three groups.
The number of oocytes retrieved was lower in the ovarian insufficiency group than in the other two groups (KruskallWallis: P=0.03).
Oocyte samples
The ethics committee of the University Hospital of Angers approved the plan of our study on oocytes discarded during ICSI procedures. Follicular growth was stimulated by recombinant FSH associated with a GnRH agonist. Ovulation was induced with HCG and the oocytes were retrieved by means of a transvaginal probe. Cumulus cells were then removed from each oocyte using gentle pipetting with hyaluronidase (80 IU Fertipro, Beernem, Belgium). For the ICSI procedure, only oocytes presenting their first polar body can be injected. Oocytes which had not extruded their first polar body 4 h after removal of the follicular cells [blocked at prophase I (germinal vesicle) or blocked after the prophase I state] were retained for our study. A total of 116 isolated oocytes (69 in prophase I, and 47 blocked between prophase I and metaphase II) were collected individually from 47 women over a 12 month period. The indication for ICSI had been male infertility in 42 couples (i.e. the presence of <100 000 motile spermatozoa available after sperm preparation) and previous fertilization failure in five couples. Each oocyte was placed in 50 µl of IVF-30 medium (IVF Scandinavian, Stockholm, Sweden). The oocytes were stored at 20°C until DNA extraction, performed within a month following collection.
Preparation of DNA
DNA was extracted from each oocyte by means of the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's recommendations. The DNA was bound specifically to glass fibres following the combined action of a chaotropic agent (guanidine), a detergent (Triton X-100) and the enzyme proteinase K. After washing, the silica-bound DNA was eluted with 200 µl of pre-warmed (72°C) elution buffer and maintained at 4°C. The extraction efficiency, assessed as described elsewhere (Reynier et al., 2001), was >90%.
Mitochondrial DNA quantification by real-time PCR
We used a Roche LightCycler to determine the mtDNA copy number using the LightCycler-Faststart DNA master SYBR Green 1 kit (Roche, Mannheim, Germany) as described elsewhere (Reynier et al., 2001). Briefly, 20 µl PCR mixtures were prepared as follows: 1x buffer containing 4 mmol/l MgCl2, 0.2 mmol/l dNTPs, 0.5 mmol/l of both primers (D41 and R56), SYBR Green I dye, 0.25 U of hot-start Taq DNA polymerase and 10 µl of the extracted DNA or 10 µl of standard with a known copy number. The reactions were performed as follows: initial denaturing at 95°C for 7 min and 40 cycles at 95°C for 1 s, 58°C for 5 s and 72°C for 13 s. The SYBR Green fluorescence was read at the end of each extension step (72°C). A melting curve (loss of fluorescence at a given temperature between 66 and 94°C) was analysed in order to check the specificity of the PCR product. For each run, a standard curve (log of the initial template copy number on the abscissae, and the cycle number at the crossing point on the ordinates) was generated by using five 10-fold serial dilutions (1001 000 000 copies) of the external standard. This curve allowed the determination of the starting copy number of mtDNA in each sample. All samples were tested twice. The raw data were then multiplied by 20 to calculate the total mtDNA content in each oocyte. The precision of the real-time PCR mtDNA quantification was assessed as described elsewhere (Reynier et al., 2001
). The intra- and inter-assay coefficients of variation ranged from 3.9 to 9.1% and from 9.3 to 12.7%, respectively.
Statistical analysis
Since the distribution of the variables analysed was non-Gaussian, all comparisons were made using the non-parametric MannWhitney and KruskalWallis U-tests. Differences were considered significant at P<0.05. Statistical analysis was performed with SPSS software, version 10.1 (SPSS, Chicago, IL)
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Results |
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Discussion |
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In the present study we have attempted to examine the relationship between oocyte mtDNA content and female infertility. To determine the best protocol for ovarian stimulation and oocyte collection, candidates for IVF are classified according to the results of a set of clinical, biological and ultrasound investigations (Hazout, 1999). The three main categories correspond to women with a normal ovarian profile, an ovarian dystrophy profile and an ovarian insufficiency profile. The precise pathophysiological mechanisms responsible for ovarian dystrophy and ovarian insufficiency are unknown. However, the quality, the fertilization and the cleavage rates of oocytes are impaired in patients with ovarian dystrophy (Dor et al., 1990
; Aboulghar et al., 1997
; Engmann et al., 1999
; Plachot et al., 2003
). Similarly, ovarian insufficiency is associated with cycle cancellation, poor oocyte quality and decline of IVF success rates (Pellicer et al., 1987
; Jenkins et al., 1991
) resulting from diminished ovarian reserve at the qualitative and quantitative levels (Nasseri et al., 1999
; Bancsi et al., 2002
).
We found two distinct ranges of mtDNA levels with no overlap between patients with ovarian insufficiency and those with a normal profile. Thus, ovarian insufficiency is clearly associated with specifically low mtDNA content, probably indicating abnormal mitochondrial biogenesis during oocyte growth and reflecting cytoplasmic immaturity. The range of oocyte mtDNA content in women with ovarian dystrophy, in contrast to those with ovarian insufficiency, was identical to that of women with a normal ovarian profile. It is thus likely that the poor oocyte quality associated with ovarian insufficiency and ovarian dystrophy has different molecular aetiologies. Our results suggest that in the case of ovarian dystrophy, impaired oocyte maturity is unrelated to mitochondrial biogenesis. This is corroborated by the fact that the biological effects of oocyte immaturity in ovarian dystrophy and ovarian insufficiency are quite different. During IVF procedures, the proportion of immature oocytes is significantly higher and the rate of fertilization is significantly lower in patients with ovarian dystrophy than in patients with a normal ovarian profile. Thus, although there exists a general immaturity in the cohort of oocytes in ovarian dystrophy, once fertilized, a subset of mature oocytes develop into good quality embryos (Dor et al., 1990; Hardy et al., 1995
; Engmann et al., 1999
). This is very different from the case of ovarian insufficiency, which is characterized by poor oocyte quality as well as poor embryo quality.
Our results raise the question of the functional consequences of impaired mitochondrial biogenesis observed in the oocytes of patients with ovarian insufficiency. In the embryo, the generation of ATP during the pre-compaction stages is largely dependent upon oxidative phosphorylation (Bavister and Squirrell, 2000; Thompson et al., 2000
; Van Blerkom et al., 1998
, 2000
). The variation of the ATP content of human oocytes has been associated with the developmental competence of embryos (Van Blerkom et al., 1995
; Wilding et al., 2001
). Indeed, ATP seems to be generated prior to cavitation by pre-existing oocyte mitochondrial proteins and transcripts that must be produced under the control of the maternal nuclear and mitochondrial genome during oogenesis (Cummins, 2004
). The low mitochondrial mass may be insufficient to constitute the necessary energetic reserves during follicular growth. Moreover, mitochondria are involved in many essential cellular processes other than ATP production, so that several functions, such as regulation of apoptosis, calcium homeostasis, pyrimidine and haem synthesis and other metabolic pathways, could be impaired by the low mitochondrial mass and lead to the poor outcome of IVF.
The importance of mitochondria in oocyte quality and embryo development is highlighted by the cytoplasmic transfer procedure. In animal experiments, normal developmental potential has been restored to eggs with ooplasmic deficiencies by the transfer of ooplasm from normal eggs (Flood et al., 1990; Levron et al., 1996
). In humans, inter-oocyte cytoplasmic transfer has been used in an attempt to overcome pregnancy failure due to poor oocyte quality (Cohen et al., 1997
). A relationship between mitochondrial function and developmental capacity may explain the promotion of the developmental potential of seemingly incompetent oocytes by the introduction of ooplasm aspirated from a normal oocyte. Although the precise cellular structures and macromolecules thus introduced into the oocyte are unknown, mitochondria could represent an important component. Indeed, the isolation and transfer of mitochondria between oocytes is known to increase ATP production in the recipients, with the persistence of activity in the transferred mitochondria (Van Blerkom et al., 1998
). Moreover, the microinjection of small numbers of mitochondria into mouse oocytes prevents the onset of apoptosis (Perez et al., 2000
).
In conclusion, we found that the mtDNA content was particularly low in all the oocytes retrieved from patients with ovarian insufficiency, whereas oocytes from patients with ovarian dystrophy contained normal quantity of mitochondrial genomes. We believe that ovarian insufficiency and the associated poor oocyte quality are specifically linked with impaired mitochondrial biogenesis, probably related to disorders in cytoplasmic maturation. Further research into the pathophysiological mechanisms involved should allow us to establish whether impaired mitochondrial biogenesis is the central abnormality responsible for poor oocyte quality or simply evidence of impaired oogenesis.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290, 457465.[ISI][Medline]
Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD and te Velde ER (2002) Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 77, 328336.[CrossRef][ISI][Medline]
Bavister BD and Squirrell JM (2000) Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod 15(Suppl 2), 189198.
Bell JC, Smith LC, Rumpf R and Goff AK (1997) Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro. Reprod Fertil Dev 9, 603608.[CrossRef][ISI][Medline]
Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G and Schon EA (1995) Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 57, 239247.[ISI][Medline]
Cohen J, Scott R, Schimmel T, Levron J and Willadsen S (1997) Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 350, 186187.[ISI][Medline]
Cummins JM (2004) The role of mitochondria in the establishment of oocyte functional competence. Eur J Obstet Gynecol Reprod Biol 115 (Suppl), S23S29.[CrossRef][ISI][Medline]
Delbart C (2000) Rôle des Mitochondries. In Les Mitochondries: Biologie et Incidences Physiopathologiques. Tec & Doc, Paris, pp. 6193.
Dor J, Shulman A, Levran D, Ben-Rafael Z, Rudak E and Mashiach S (1990) The treatment of patients with polycystic ovarian syndrome by in-vitro fertilization and embryo transfer: a comparison of results with those of patients with tubal infertility. Hum Reprod 5, 816818.[Abstract]
Engmann L, Maconochie N, Sladkevicius P, Bekir J, Campbell S and Tan SL (1999) The outcome of in-vitro fertilization treatment in women with sonographic evidence of polycystic ovarian morphology. Hum Reprod 14, 167171.
FIVNAT (2003) Dossier FIVNAT 2003. Organon, Paris.
Flood JT, Chillik CF, Van Uem JF, Iritani A and Hodgen GD (1990) Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm. Fertil Steril 53, 10491054.[ISI][Medline]
Gandolfi TA and Gandolfi F (2001) The maternal legacy to the embryo: cytoplasmic components and their effects on early development. Theriogenology 55, 12551276.[CrossRef][ISI][Medline]
Gosden RG (2002) Oogenesis as a foundation for embryogenesis. Mol Cell Endocrinol 186, 149153.[CrossRef][ISI][Medline]
Hardy K, Robinson FM, Paraschos T, Wicks R, Franks S and Winston RM (1995) Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries. Hum Reprod 10, 21252135.[Abstract]
Hazout A (1999) Protocole de stimulation, maturation ovocytaire et maturation embryonnaire. In Hamamah S and Ménézo Y (eds) Ovocyte et Embryon. Ellipses, Tours, pp. 288294.
Jansen RP and de Boer K (1998) The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate. Mol Cell Endocrinol 145, 8188.[CrossRef][ISI][Medline]
Jenkins JM, Davies DW, Devonport H, Anthony FW, Gadd SC, Watson RH and Masson GM (1991) Comparison of poor responders with good responders using a standard buserelin/human menopausal gonadotrophin regime for in-vitro fertilization. Hum Reprod 6, 918921.[Abstract]
Ji YZ, Bomsel M, Jouannet P and Wolf JP (1997) Modifications of the human oocyte plasma membrane protein pattern during preovulatory maturation. Mol Reprod Dev 47, 120126.[CrossRef][ISI][Medline]
Levron J, Willadsen S, Bertoli M and Cohen J (1996) The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm. Hum Reprod 11, 12871292.[Abstract]
Nasseri A, Mukherjee T, Grifo JA, Noyes N, Krey L and Copperman AB (1999) Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy. Fertil Steril 71, 715718.[CrossRef][ISI][Medline]
Pellicer A, Lightman A, Diamond MP, Russell JB and DeCherney AH (1987) Outcome of in vitro fertilization in women with low response to ovarian stimulation. Fertil Steril 47, 812815.[ISI][Medline]
Perez GI, Trbovich AM, Gosden RG and Tilly JL (2000) Mitochondria and the death of oocytes. Nature 403, 500501.[CrossRef][ISI][Medline]
Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Tesquier A, Serkine AM, Boujenah A, Abirached F et al. (2003) [Oocyte and embryo quality in polycystic ovary syndrome]. Gynecol Obstet Fertil 31, 350354.[Medline]
Reynier P, May-Panloup P, Chretien MF, Morgan CJ, Jean M, Savagner F, Barriere P and Malthiery Y (2001) Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 7, 425429.
Rube DA and van der Bliek AM (2004) Mitochondrial morphology is dynamic and varied. Mol Cell Biochem 256, 331339.[CrossRef][ISI]
Steuerwald N, Cohen J, Herrera RJ and Brenner CA (2000) Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol Hum Reprod 6, 448453.
Thompson JG, McNaughton C, Gasparrini B, McGowan LT and Tervit HR (2000) Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. J Reprod Fertil 118, 4755.[CrossRef][ISI][Medline]
Van Blerkom J, Davis PW and Lee J (1995) ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 10, 415424.[Abstract]
Van Blerkom J, Sinclair J and Davis P (1998) Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy. Hum Reprod 13, 28572868.
Van Blerkom J, Davis P and Alexander S (2000) Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod 15, 26212633.
Wilding M, Dale B, Marino M, di Matteo L, Alviggi C, Pisaturo ML, Lombardi L and de Placido G (2001) Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 16, 909917.
Wu H, Kanatous SB, Thurmond FA, Gallardo T, Isotani E, Bassel-Duby R and Williams RS (2002) Regulation of mitochondrial biogenesis in skeletal muscle by CaMK. Science 296, 349352.
Submitted on August 10, 2004; resubmitted on October 12, 2004; accepted on November 18, 2004.