Adenosine Triphosphate Content in Human Unfertilized Oocytes, Undivided Zygotes and Embryos Unsuitable for Transfer or Cryopreservation

Transcription

1 The Journal of International Medical Research 2012; 40: Adenosine Triphosphate Content in Human Unfertilized Oocytes, Undivided Zygotes and Embryos Unsuitable for Transfer or Cryopreservation J ZHAO AND Y LI Reproductive Medicine Centre, Xiangya Hospital, Central South University, Changsha, Hunan, China OBJECTIVE: To investigate the relationship between adenosine triphosphate (ATP) content and developmental potential in human oocytes, zygotes and embryos. METHODS: ATP content was determined using a bioluminescence assay in 72 unfertilized oocytes, 26 undivided zygotes and 70 embryos unsuitable for transfer or cryopreservation, obtained from 52 women undergoing in vitro fertilization and embryo transfer (IVF ET). RESULTS: The mean ATP content increased with development: zygotes had a significantly higher ATP content than oocytes, and embryos had a significantly higher ATP content than both zygotes and oocytes. Within the embryo group, the ATP content was significantly higher in polypronuclear embryos than in inferior embryos with substantial fragmentation. When analysed in relation to IVF ET outcome, the ATP content of oocytes and embryos from women who became pregnant was significantly higher than in oocytes and embryos from those who did not become pregnant. CONCLUSIONS: Reduced ATP content may be related to fertilization failure, arrested division and abnormal embryonic development. The differences observed in oocyte and embryo ATP content between women who became pregnant and those who did not suggests that mitochondrial function is correlated with individual fertility. KEY WORDS: UNFERTILIZED OOCYTE; EMBRYO; ZYGOTE; ADENOSINE TRIPHOSPHATE CONTENT; DEVELOPMENTAL POTENTIAL Introduction A mature oocyte is essential for fertilization, which is a vital element in the outcome of in vitro fertilization and embryo transfer (IVF ET). Although the presence of a clear first polar body indicates nuclear maturation, maturation may not be complete because the oocyte cytoplasm might still be immature. 1,2 Mitochondria contain their own specific genome: a circular double-stranded 16.6 kb DNA molecule encoding 13 essential subunits of the respiratory chain complexes. 3 Mitochondria are the major source of energy in eukaryotic cells, producing adenosine triphosphate (ATP) via oxidative phosphorylation and the citric acid cycle. ATP is necessary for many cellular functions 734

2 including motility, maintenance of homeostasis and regulation of cell survival. 4,5 The intrinsic quality of an oocyte is the key factor determining its developmental competence. 6 The quantity and functional status of mitochondria contribute to the quality of the oocyte and play important roles in fertilization and embryo development. 7 Reduced efficiency of mitochondrial respiration in the oocyte has been shown to be related to poor embryo development. 8,9 The aim of this study was to measure the ATP content of human oocytes, zygotes and embryos that were developmentally unsuitable for intrauterine transfer, in order to examine the relationship between ATP content and female infertility. Subjects and methods STUDY POPULATION Samples for ATP analysis were provided by consecutive women undergoing IVF ET at Xiangya Hospital, Central South University, Changsha, China, between September and December The subjects included in this study formed a subset of a large and diverse group, and were selected for inclusion according to the following criteria: (i) years old; (ii) bilateral tubal obstruction or resection without male factor involvement; (iii) secondary infertility; (iv) identical protocol for ovarian stimulation; (v) production of two or three morphologically high-quality embryos with 4 8 cells for transfer. Subjects with secondary infertility were chosen in order to exclude fertilization failure as the cause of infertility. The study protocol was approved by the Institutional Review Board and the Ethics Committee of Xiangya Hospital, Changsha, China. The study was conducted in accordance with the Declaration of Helsinki, as revised in All participants provided written informed consent. OVARIAN STIMULATION AND IVF ET All subjects underwent an ovulation induction cycle involving a 7-day gonadotrophin-releasing hormone agonist protocol. 10 Subjects received 0.1 mg/day gonadotrophin-releasing hormone agonist (triptorelin; Ferring Arzneimittel, Kiel, Germany) subcutaneous injection for 7 days, initiated on day 2 of the menstrual cycle, and 150 IU/day follicle-stimulating hormone (FSH; urofollitropin; Livzon Pharmaceutical Group, Zhuhai, China) intramuscular injection from day 3 of the menstrual cycle until human chorionic gonadotrophin (hcg) administration. The FSH dose was adjusted after 3 days according to the ovarian response. When two follicles reached 18 mm in diameter, as determined by ultrasonography, IU hcg (Profasi, Merck Serono, Geneva, Switzerland) was administered by intramuscular injection. Ultrasound-guided transvaginal oocyte retrieval was performed h after hcg injection. The oocytes were fertilized by incubation with sperm for 4 h in 0.9 ml Earle salt solution (Sigma, St Louis, MO, USA) supplemented with 1 mm pyruvate (Sigma), 50 U/ml penicillin, 50 µg/ml streptomycin (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) and 13% heat-inactivated (56 C for 30 min) maternal serum at 37 C with 92% humidity and 5% carbon dioxide in air. The culture medium was changed after 24 and 48 h. A maximum of three embryos with the best morphological appearance were transferred to the uterus of the donor, 2 3 days after oocyte retrieval. The outcome of the IVF cycle (pregnant or not pregnant) was recorded for each subject. SAMPLE COLLECTION Samples discarded during the IVF ET procedure were collected for the present study. These included oocytes that remained 735

3 unfertilized after 48 h of IVF, undivided zygotes with arrested division 48 h after fertilization and embryos that were unsuitable for transfer or cryopreservation 72 h after fertilization, including those produced following polypronuclear fertilization (polypronuclear embryos) and those showing substantial fragmentation (inferior embryos). DETERMINATION OF INTRACELLULAR ATP CONTENT Each sample was placed in a sterile tube with 50 µl of ultrapure water and stored at 80 C until analysis. The ATP content was determined by measuring the luminescence generated in an ATP-dependent luciferin luciferase bioluminescence assay (Bioluminescent Somatic Cell Assay Kit, Sigma) using a Berthold Lumat LB 9501 luminometer (Berthold Technologies, Bad Wildbad, Germany) according to the method described previously 11 and the manufacturers recommendations. A standard curve including 10 ATP concentrations from 0 to mol/µl was generated for each series of analyses, and the ATP content of each sample was calculated using the formula derived from the linear regression of the standard curve. All samples of each type were analysed simultaneously in order to reduce potential variability. STATISTICAL ANALYSES Data were expressed as mean ± SE (range). The nonparametric Wilcoxon t-test was used for between-group comparisons of ATP content. Statistical analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows. A P-value < 0.05 was considered to be statistically significant. Results The study collected 72 unfertilized oocytes from 35 women, 26 undivided zygotes from 18 women and 70 embryos from 38 women, from a total of 52 participants. The 70 embryos included 41 inferior and 29 polypronuclear embryos. The mean ATP content in the three sample groups is given in Table 1. Embryos had a significantly higher ATP content than both zygotes and oocytes (P < 0.01). In addition, the mean ATP content in zygotes was significantly higher than that in oocytes (P < 0.01) (Table 1). ATP content was significantly higher in polypronuclear embryos compared with inferior embryos with substantial fragmentation (3.17 ± and 2.88 ± pmol/embryo, respectively; P < 0.01). The ATP content of unfertilized oocytes and embryos unsuitable for transfer was analysed in relation to the outcome of IVF ET. The ATP content of oocytes and TABLE 1: Adenosine triphosphate (ATP) content of human unfertilized oocytes, undivided zygotes and embryos unsuitable for transfer or cryopreservation following controlled ovarian hyperstimulation and in vitro fertilization Organisms n ATP content (pmol/sample) Unfertilized oocytes ± ( ) Undivided zygotes ± ( ) a Embryos unsuitable for transfer or cryopreservation ± ( ) a,b Data presented as mean ± SE (range). a P < 0.01 compared with unfertilized oocytes and b P < 0.01 compared with undivided zygotes; nonparametric Wilcoxon t-test. 736

4 untransferred embryos from women who became pregnant following IVF ET was significantly higher than those from women who did not become pregnant (P < 0.01 for both comparisons; Table 2). Pregnancy continued in eight of the 18 women who provided zygotes, 15 of the 35 who provided oocytes and 18 of the 38 who provided embryos. Discussion All mitochondria in an individual are maternally inherited. During the preimplantation stages, the number of mitochondria remains relatively constant as their replication is not initiated until after implantation. 12 The complement present in each mature oocyte at the time of fertilization, therefore, represents a fixed number that is halved with each cell division during the preimplantation stages, assuming that segregation between daughter cells is largely numerically equivalent. As the primary source of ATP, the number of mitochondria in the oocyte is likely to be an essential characteristic of the bioenergetic ability of the embryos to progress and develop normally after fertilization All the mitochondrially derived ATP-requiring activities of the early embryo (until compaction) are, therefore, dependent on the number of mitochondria present in the oocyte at metaphase II (MII). In view of the above, direct comparison of ATP content between oocytes, zygotes and embryos is relevant. In humans, the net ATP content in the mature oocyte appears to persist throughout the preimplantation stages, and this initial ATP amount may, therefore, be an important determinant of subsequent developmental potential. The mean ATP content of oocytes, undivided zygotes and embryos increased significantly with the stage of development in the present study, suggesting an association between initial ATP content and developmental potential in these samples. Mitochondrial function may influence the continued development of embryos that appear normal at the early cleavage stages, and may explain the developmental failure that can occur even with grossly normal-appearing TABLE 2: Comparison of adenosine triphosphate (ATP) content in unfertilized oocytes and embryos unsuitable for transfer or cryopreservation from women (n = 52) who underwent in vitro fertilization and embryo transfer (IVF ET), stratified according to pregnancy status Outcome of IVF ET Characteristic Pregnant Not pregnant Unfertilized oocytes, n Donors, n Donor age, years ± ± 3.83 ATP content, pmol/oocyte 1.90 ± ± a Embryos unsuitable for transfer or cryopreservation, n Donors, n Donor age, years ± ± 4.08 ATP content, pmol/embryo 3.12 ± ± a Data presented as n of participants or samples, or mean ± SE. a P < 0.01 compared with pregnant group, nonparametric Wilcoxon t-test. Data from undivided zygotes were insufficient for statistical analysis. 737

5 oocytes, resulting in embryos with no evidence of nuclear or cytoplasmic disorder. A low mitochondrial mass may be insufficient to supply the energy reserves necessary to support fertilization, division and embryo development. The results of the present study were consistent with those of Van Blerkom et al., 11 who reported a mean ATP content for human MII oocytes of 1.85 ± pmol/oocyte. They found that the ATP content of normal-appearing MII oocytes differed significantly between cohorts, but a higher potential for development and implantation was associated with an ATP concentration > 2 pmol/oocyte. 11 Reduced ATP content appeared to influence embryo development in the present study. Polypronuclear embryos contained significantly more ATP than inferior embryos with substantial fragmentation. Since development is not arrested in polypronuclear embryos, indicating the absence of any cytoplasm disorder, they would be expected to have a higher ATP content. This is consistent with the findings of others who reported that the expression of eight oxidative phosphorylation mitochondrial genes in tripronucleate embryos was significantly increased, compared with embryos with arrested development. 17,18 In order to examine the relationship between ATP content and the outcome of IVF ET, the ATP content of oocytes and embryos was analysed according to the outcome after intrauterine transfer of sibling embryos. Unfertilized oocytes and untransferred embryos from women who became pregnant had a significantly higher ATP content than those from women who did not become pregnant, despite the subjects being matched for age, fertility history and ovarian stimulation protocol. Several studies have shown that the developmental potential of the embryo and the outcome of IVF are related to both the ATP content and the mitochondrial content of human oocytes. 7,19,20 A higher rate of embryonic development has been shown to be associated with higher ATP concentration, not only in humans, but also in cattle 8 and mice. 21 In conclusion, the present study demonstrated a relationship between ATP content and development potential. This may help to explain the diverse defects in developmental ability that frequently occur in humans, such as fertilization failure, arrested division and abnormal embryonic development. Assessments of ATP content prior to fertilization, using noninvasive methods such as substrate uptake or metabolite production, could have important clinical implications for gamete selection in IVF. In addition, the difference in oocyte and embryo ATP content between the women who became pregnant and those who did not suggest that mitochondrial function is correlated with individual fertility. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 22 November 2011 Accepted subject to revision 27 November 2011 Revised accepted 2 March 2012 Copyright 2012 Field House Publishing LLP References 1 Brevini TA, Cillo F, Antonini S, et al: Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes. Anim Reprod Sci 2007; 98: Combelles CM, Albertini DF: Microtubule 738

6 patterning during meiotic maturation in mouse oocytes is determined by cell cyclespecific sorting and redistribution of γ-tubulin. Dev Biol 2001; 239: Anderson S, Bankier AT, Barrell BG, et al: Sequence and organization of the human mitochondrial genome. Nature 1981; 290: Zeng HT, Ren Z, Yeung WS, et al: Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes. Hum Reprod 2007; 22: St John JC: The transmission of mitochondrial DNA following assisted reproductive techniques. Theriogenology 2002; 57: Rizos D, Ward F, Duffy P, et al: Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev 2002; 61: Santos TA, El Shourbagy S, St John JC: Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril 2006; 85: Stojkovic M, Machado SA, Stojkovic P, et al: Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod 2001; 64: Wilding M, Dale B, Marino M, et al: Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 2001; 16: Zhang JM, Li YP, Liu J, et al: Effect of 7-day gonadotropin-releasing hormone agonist protocol on IGF-II and IGFBP-4 levels in the follicular fluid. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2009; 34: [in Chinese, English abstract]. 11 Van Blerkom J, Davis PW, Lee J: ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 1995; 10: Harvey AJ, Gibson TC, Quebedeaux TM, et al: Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation. Curr Top Dev Biol 2007; 77: Shoubridge EA, Wai T: Mitochondrial DNA and the mammalian oocyte. Curr Top Dev Biol 2007; 77: El Shourbagy SH, Spikings EC, Freitas M, et al: Mitochondria directly influence fertilisation outcome in the pig. Reproduction 2006; 131: Van Blerkom J: The role of mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction 2004; 128: Van Blerkom J: Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod BioMed Online 2008; 16: Hsieh RH, Au HK, Yeh TS, et al: Decreased expression of mitochondrial genes in human unfertilized oocytes and arrested embryos. Fertil Steril 2004; 81(suppl 1): Au HK, Yeh TS, Kao SH, et al: Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos. Ann NY Acad Sci 2005; 1042: Cummins JM: The role of maternal mitochondria during oogenesis, fertilization and embryogenesis. Reprod Biomed Online 2002; 4: Chan CC, Liu VW, Lau EY, et al: Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes. Mol Hum Reprod 2005; 11: Van Blerkom J: Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc Natl Acad Sci USA 1991; 88: Author s address for correspondence Dr Yanping Li Reproductive Medicine Centre, Xiangya Hospital, 87 Xiang Ya Road, Changsha, Hunan , China. zhaojing @sina.com 739