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A new approach to evaluate aging effects on human oocytes: Fourier transform infrared imaging spectroscopy study
A new approach to evaluate aging effects on human oocytes: Fourier transform infrared imaging spectroscopy study Giorgia Gioacchini, Ph.D.,a,b Elisabetta Giorgini, Ph.D.,b Lisa Vaccari, Ph.D.,c Paolo Ferraris, Ph.D.,c Simona Sabbatini, Ph.D.,d Veronica Bianchi, Ph.D.,a Andrea Borini, Ph.D., M.D.,a and Oliana Carnevali, Ph.D.b a Tecnobios Procreazione, Centre for Reproductive Health, Bologna; b Dipartimento di Scienze della Vita e dell'Ambiente, Politecnica delle Marche, Via Brecce Bianche, Ancona; c SISSI Beamline, Elettra Synchrotron Light Laboratory, Universita Politecnica delle Trieste; and d Dipartimento di Scienze, Ingegneria dei Materiali, Ambiente e Urbanistica, Universita Marche, Ancona, Italy
Objective: To characterize from a vibrational point of view the alterations caused by aging on human oocytes. Design: Reproductive biology. Setting: Private assisted reproductive technology clinic, synchrotron beam line, and university infrared laboratory. Patient(s): Twenty women of different ages (30 2 and 39 2 years) selected on the basis of detailed inclusion criteria and submitted to controlled ovarian stimulation according to a specific protocol. Intervention(s): Collection of 68 supernumerary oocytes that were not used during the IVF cycle from the above cited consenting patients. Main Outcome Measure(s): Focal Plane Array Fourier transform infrared (FTIR) analysis of human oocytes. Result(s): Specific spectral differences were highlighted in the two experimental groups of oocytes. In particular, in oocytes of 39-yearold women, the occurrence of peroxidative processes and a decrease in the amount of carbohydrates were observed, together with alterations in the phospholipid membrane, proteic pattern, and nucleic acids content. Conclusion(s): For the first time, FTIR spectroscopy was applied to human oocytes, leading to strong evidence of damage from aging in the gametes of mature women, which could be related to a decline in reproductive function. All the information obtained may be considered useful to improve the scientific knowledge on huUse your smartphone man reproduction and to exploit new strategies for detecting oocyte aging. (Fertil SterilÒ to scan this QR code 2014;101:120–7. Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Human oocytes, aging damage, vibrational spectroscopy, FPA FTIR Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gioacchinig-aging-human-oocytes-spectroscopy/
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o date, the problem of infertility affects about 10%–15% of couples, and the number of women who need assisted reproductive technology (ART) to achieve pregnancy is constantly increasing (1–3). However, despite the remarkable progress achieved by
research in the last 30 years, the success rates are still unsatisfactory (25%–30%) (4–7). For a complete diagnosis, both male and female factors have to be taken into account. In women, even if oocyte quality depends on multiple biological processes, many of which can
Received June 5, 2013; revised September 3, 2013; accepted September 9, 2013; published online October 17, 2013. G.G. has nothing to disclose. E.G. has nothing to disclose. L.V. has nothing to disclose. P.F. has nothing to disclose. S.S. has nothing to disclose. V.B. has nothing to disclose. A.B. has nothing to disclose. O.C. has nothing to disclose. The study was supported by funds from the Polytechnic University of Marche (to O.C. and E.G.). Reprint requests: Oliana Carnevali, Ph.D., Dipartimento di Scienze della Vita e dell'Ambiente, Uni Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (E-mail: o.carnevali@ versita univpm.it). Fertility and Sterility® Vol. 101, No. 1, January 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.09.012 120
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be assessed only at the molecular level, the biological capability to reproduce is primarily related to age (8, 9). During oogenesis, in which mRNA, proteins, lipids, and carbohydrates are stored, oocyte competence, defined as the ability of an oocyte to be fertilized and to develop to the blastocyst stage, is progressively achieved (10–12). In this light, a complete understanding and elucidation of the oocyte macromolecular structure in terms of composition and structure of the principal biomolecules appears useful to better evaluate the correlation between oocyte competence and aging.
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Fertility and Sterility® Fourier transform infrared (FTIR) spectroscopy is a fast, label-free analytical technique, widely used for investigating functional groups, bonding types, and molecular conformations of the most relevant biological molecules (13, 14). It allows us to examine the interaction between matter and electromagnetic radiation with an appropriate continuous range of frequencies. In particular, in the mid-IR region (4,000–400 cm1), fundamental vibrations, mainly stretching and bending of chemical bonds, can be detected. The coupling of IR spectrometers with visible microscopes has led to the successful use of this technique for many diagnostic purposes in the life sciences, allowing the detection of subtle biochemical changes caused by specific pathologies, such as tissues affected by tumoral pathologies and stem cells at various differentiation steps (15–18). FTIR microspectroscopy requires thin sections of samples (monolayer cell cultures or 5 mm thick tissues from surgical resection) on which IR maps are acquired on previously selected zones (1,000 1,000 mm2). The spatial resolution is limited only by diffraction, the area of each pixel of the map reaching up to 2.56 2.56 mm2. This allows researchers to characterize specific subcellular details, giving the possibility to mutually relate the vibrational local features with the morphology of the different compartments of the sample (19, 20). Owing to the large number of spectral data collected in a single map, several programs have been developed for performing multivariate statistical analysis to highlight similarities and differences in a simple readout format (21, 22). Wood and collaborators applied IR and Raman spectroscopies to study mouse oocytes at different maturational stages, leading to the vibrational characterization of oocyte molecular architecture for the first time (23). Some vibrational studies have also been carried out on zebrafish oocytes to analyze the modification of their biochemical composition during oogenesis and, further, to improve competence by administration of melatonin and probiotic (19, 20, 24). By considering these results, to develop knowledge on human reproduction and to complement morphological and molecular techniques currently applied in oocyte quality evaluation, we decided to exploit Focal Plane Array (FPA) FTIR imaging to identify appropriate spectral biomarkers related to the aging of human female gametes.
MATERIALS AND METHODS The study, approved by the Internal Review Board of Tecnobios Procreation, was carried out on human supernumerary oocytes in December 2012. Owing to legal restrictions, in Italy not all oocytes are inseminated, just the number necessary to optimize the patient's outcome for the cycle.
Sample Preparation Twenty dedicated consenting patients who were enrolled in an IVF program were selected on the basis of specific inclusion criteria (regular ovulatory menstrual cycles, FSH <10 IU/L on day 3 of the menstrual cycle, nonsmokers, and with no ovarian infertility diagnosis). The patients signed an informed consent to participate in the project research that VOL. 101 NO. 1 / JANUARY 2014
included the donation of their supernumerary oocytes. Controlled ovarian stimulation was induced according to a long protocol using leuprorelin (Enantone, Takeda), and rFSH (Gonal-F, Serono; or Puregon Organon). HCG at 10,000 IU (Gonasi, Amsa) was administered when one or more follicles reached a diameter of 23 mm (25). At the end of the treatment, 68 supernumerary metaphase II (MII) oocytes were collected and distributed into two groups on the basis of the donors' age: group A, 30 2 years old; group B, 39 2 years old (Table 1A). The cutoffs for young (group A) and old (group B) patients were, respectively, %32 and R37 years old. All the oocytes used for this project showed normal morphological features and clear and homogeneous cytoplasm with no saccuoles of endoplasmic reticulum or vacuoles. Poor-quality eggs were discarded to avoid bias in the interpretation of the results. None of the oocytes were inseminated or used to produce embryos. For the vibrational analysis, each oocyte was deposed ‘‘as it is’’ on a silicon support (a dish with a diameter of 10 mm and a thickness of 1 mm, transparent to IR radiation) and submitted to the same treatment of air drying for 30 minutes; no fixatives were used. The spectroscopic analysis was carried out within 10 hours. Previous experiments carried out in our laboratories showed that oocytes prepared as above were stable in time, providing homogeneous and reliable data sets.
FTIR Measurements and Data Analysis FTIR measurements were carried out at the IR beamline SISSI, ELETTRA synchrotron, by using a Bruker VERTEX 70 interferometer coupled with a Hyperion 3000 Vis-IR microscope and equipped with a liquid nitrogen cooled bidimensional FPA detector (area size 64 64 pixels; OPUS 6.5 software package, Bruker Optics). For each sample, the spectroscopic acquisition took only a few minutes. First, the visible image of each oocyte was acquired with a 15 condenser/objective (pixel size 2.56 mm). Then the IR map was acquired in transmission mode by using the FPA detector, which allowed us to simultaneously obtain groups of 4,096 spectra (128 scans for each detector pixel with a spectral resolution of 4 cm1). During all measurements, the microscope camera was purged with nitrogen to avoid atmospheric contamination (H2O and CO2). All of the IR maps were compared with background spectral images taken on regions of clean silicon windows. From each IR map, by using Opus 6.5 software, the total absorbance cartogram, representing the total intensity of the IR absorption, was generated by integrating within 1,800–1,000 cm1. For each oocyte, the average spectrum was obtained by merging 1,024 spectra (no representative spectra were excluded). Average spectra were two points baseline linear fitted in the spectral range 4,000–900 cm1 and vector normalized (26). All the oocytes belonging to the same experimental group showed vibrational spectra with similar profiles, thus giving reliable and reiterative results. Second Derivative (DII, 9-point smoothing) and Peak Fitting (Gaussian algorithm) procedures were adopted to determine the right position and absorbance of bands. By 121
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TABLE 1 A. Age of patients and number of donated oocytes. Group A
B
Woman
Age
No. of oocytes
W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20
30 29 31 30 31 28 30 32 38 39 39 38 40 37 41 38 39 38 39 38
3 4 3 3 4 3 4 3 3 4 4 3 4 3 4 4 3 3 4 2
B. Meaningful vibrational modes together with corresponding chemical features and biological processes. Biological aspects
Meaningful bands (cmL1)
Biochemical features
Biological processes
Plasma membrane
2,925/2,958 (nasym CH2/nasym CH3) 3,013/2,958 (n ¼CH/nasym CH3) 1,747/1,735 (1,741/1,725) (nC ¼ O transgauche) 1,556 2 (bstructures) 1,541 3 (aþ random structures) 1,518 1 (tyrosine) 1,717 (1,710) (nC ¼ O of thymine) 921 (925) (Z-DNA) 1,125 (nC-O ribose) 998 (uracil ring) 2,852/2,958 (nsym CH2/nasym CH3) 1,045/1,081 (nC-O /nsym PO2) 1,158/1,172 (nC-O H-bonded/non H-bonded proteins)
Lipid saturation level Lipid unsaturation level Different alkyl chains conformations, increase of the H-bond network Increase of the random structures
Increase of fatty acyl chains peroxidation
Proteic pattern Nucleic acids
Metabolism
More permeable membranes
A lower amount of tyrosine Increase of the H-bond network Z DNA RNA
Different proteic pattern. Proteins degradation Decreased transducing signals Modified DNA conformation Epigenetic effect RNA degradation
Alkyl chain length Carbohydrates and phosphates Substitution of C-OH with C-OP
Alterated lipid synthesis Increased carbohydrates consumption Phosphorylative process
Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
using GRAMS/AI 7.02 (Galactic Industries, Inc.), Peak Fitting was performed on average spectra interpolated in two different spectral ranges (1,780–1,470 cm1 and 1,180–900 cm1); the spectra were two points baseline linear fitted. To identify the underlying component bands, the number of peaks together with their center values were carefully individuated according to DII results and fixed before running the iterative process, to obtain the best reconstructed curve (residual close to zero). Mean values of area and wavelength were purchased for each component peak. The representative bands taken into account are reported in Table 1B, together with their spectral assignments; their attribution was done according to the literature. The percentage of band areas and ratios are presented as mean SD. Student's t-test was used for comparison between the two experimental groups, using a statistical software package, Prism5 (Graphpad Software, Inc.) with significance set at P< .05. To evaluate the distribution of the principal biocomponents (lipids, proteins, and nucleic acids), false color images were calculated for each oocyte by integrating the 122
IR maps under the following spectral regions: 3,100–2,800 cm1 (CH stretching modes), 1,580–1,480 cm1 (Amide II), and 1,300–900 cm1 (phosphate and carbohydrate moieties; HyperSpec software).
RESULTS The study aimed to achieve molecular information on the biochemical modifications induced by aging on human oocytes by using FPA FTIR imaging spectroscopy. The spectroscopic analysis was carried out on a meaningful number of supernumerary metaphase MII oocytes, which were donated by consenting patients enrolled in IVF programs and divided into two experimental groups according to age (group A, 30 2 years old; group B, 39 2 years old; Table 1A). For all oocytes, the visible images and the IR maps were acquired, from which the total absorption cartograms, representing the total intensity of the IR absorption, were calculated, integrating between 1,800 and 1,000 cm1 (Fig. 1A, 1B). To localize the distribution of the principal VOL. 101 NO. 1 / JANUARY 2014
Fertility and Sterility®
FIGURE 1
(A) Photomicrographs and (B) total absorbance cartograms, reconstructed by integrating under 1,800–1,000 cm1, of group A and B oocytes. False color images were integrated in the following spectral ranges: (C) 3,100–2,800 cm1 (CH2 and CH3 stretching modes), (D) 1,580–1,480 cm1 (Amide II), and (E) 1,300–900 cm1 (phosphates and carbohydrates moieties). (F) Average spectra of group A and B oocytes in the spectral range 4,000–900 cm1. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
biocomponents inside the oocytes, false color images (obtained by using HyperSpec software package) were elaborated, showing a different distribution of proteins (inner zone), nucleic acids (on the edge), and lipids (external zone) in both A and B oocytes. For each experimental group, a representative spectrum was extracted (called A and B average spectra; Fig. 1F). By carefully analyzing them and by comparing them to each VOL. 101 NO. 1 / JANUARY 2014
other, meaningful spectral changes were found, above all in the CH stretching region (3,100–2,800 cm1), in the proteic pattern (1,780–1,470 cm1), and in the nucleic acids spectral region (1,180–900 cm1). Specific band areas and band area ratios, which could be considered as spectral markers for a decline in oocyte quality with aging, were also calculated and the numerical variations analyzed (Table 1B; Fig. 2). 123
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FIGURE 2
ν
ν
ν
β–
ν
ν
ν
ν
α
/
Numerical variations of the following band area values for the A and B experimental groups: (A) nasym CH2/CH3; (B) n ¼CH/nasym CH3; (C) nsym CH2/ nasym CH3; (D) 1,556 2 cm1 (b-sheet); (E) 1,541 3 cm1 (helical-random components); (F) 1,518 1 cm1 (tyrosine); (G) nC¼O (trans)/nC¼O (gauche); (H) 1,045/1,081 (nC-O carbohydrates/nsym PO2); (I) 1,158/1,172 (nC-O H-bonded carbohydrates/nC-O non-H-bonded proteins). Data are presented as mean SD. Student's t-test was used for comparison between the two experimental groups, using the statistical software package Prism5 (GraphPad Software, Inc.), with significance set at P<.05. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
Plasma Membrane To analyze plasma membrane structure, particular attention was given to the spectral region 3,050–2,800 cm1. The bands at 2,958 and 2,924 cm1 (nasym CH3 and CH2), 2,872 and 2,852 cm1 (nsym CH3 and CH2), usually attributed to amino acids side chains and to plasma membrane alkyl chains (23), did not shift their position and appeared sharp and clearly defined, suggesting well-arranged alkyl chains with few rotamers in both experimental groups (27). The band at 3,013 cm1 (n ¼ CH) is usually considered a marker of peroxidative processes (28, 29); by analyzing the numerical variations of the band area ratios 2,925/2,958 (lipids saturation level) and 3,013/2,958 (lipids unsaturation 124
level; Fig. 2A, 2B), a higher amount of peroxidized fatty acyl chains was found in B oocytes from older patients. This could be explained by considering that the higher amount of polyunsaturated fatty acids present in the oocyte membrane could easily react with reactive oxygen species and induce oxidative stress. After the Peak Fitting procedure was applied on A and B average spectra in the range 1,780–1,470 cm1, the band at 1,740 cm1, usually attributed to diacyl lipids, turned out to be composed of two different absorptions, originating from the stretching of the two ester carbonyl groups, which were adjacent to C-C bonds with trans or gauche conformations (13). In group A oocytes, these two bands were respectively found at 1,747 and 1,735 cm1, while in group B VOL. 101 NO. 1 / JANUARY 2014
Fertility and Sterility® oocytes, they shifted to lower frequencies (1,741 and 1,725 cm1). These findings suggest that in oocytes from older patients, a stronger H-bond network is present, which could result in a more permeable phospholipid membrane. Modifications in alkyl chain conformations may also be present owing to the different values in the absorbance band ratios 1,747/1,735 (group A) and 1,741/1,725 (group B; Fig. 2G).
Proteic Pattern The vibrations of adjacent peptide groups are strongly influenced by each other, as well as by the network of hydrogen bonding (13). In this light, it appeared useful to study the bands at 1,658 (Amide I) and 1,549 (Amide II) cm1, which are sensitive to proteins' structural conformation (19, 27). Owing to a certain degree of saturation in the Amide I region, the study was carried out only on the Amide II moiety, which, after the Peak Fitting procedure, turned out to be composed of three different bands: 1,556 2 (a-helix and random coil components), 1,541 3 (bsheet structures), and 1,518 1 (tyrosine) cm1. The percentage amounts of the areas of the above cited bands were calculated (Fig. 2D–2F): in B oocytes, an increase in random coil structures was observed, suggesting a modified proteic pattern with a higher level of protein degradation. A decline in B oocyte quality was also pinpointed by the drastic decrease of the band at 1,518 cm1, which is attributable to tyrosine (18). In fact, tyrosine kinase proteins are cytoplasmic enzymes targeted at the plasma membrane or at the actin cytoskeleton, which play an essential role in fertilization and zygote development, acting to transduce signals from external stimuli (30). In mammals, there is evidence that kinases are important for oocyte maturation and for the maintenance of the cytoskeleton of the mature oocyte (31–34).
Nucleic Acids The spectral range 1,180–900 cm1, usually related to the vibrations of the principal functional groups present in nucleic acids, was carefully investigated by the Peak Fitting procedure (Fig. 3). According to DNA moieties, in B oocytes, the band at 968 cm1 (nCC of DNA backbone) appeared unchanged in intensity and position, while the one at 921 cm1 (Z-DNA) increased and shifted to 925 cm1 (35); these findings suggest an epigenetic effect in oocytes from women ages 39 2 (36). The degree of DNA methylation is an important factor for correct embryo development (37). In fact, during oogenesis, genomic methylation imprints are of particular concern, since they are established throughout oocyte growth and are not subject to the global demethylation that occurs postfertilization (38). Methylation of CpG-rich DNA tracts greatly promotes the transition from B to Z conformations (39). An increase in H-bonded C¼O bonds was also suggested by the shift of the band related to nC¼O of thymine from 1,717 to 1,710 cm1 (18, 40). In B oocytes, some processes related to RNA degradation were also present as confirmed by the decrease in intensity of the bands at 1125 (nC-O ribose) and 998 (uracil ring) cm1 (40, 41).
Oocyte Metabolism In B oocytes, changes in metabolic processes were also detected, as evidenced by the numerical variations of the following band area ratios: [1] 2,852/2,958, nsym CH2/nasym CH3 (Fig. 2C), modifications in lipid synthesis (42, 43); [2] 1,045/1,081, nC-O carbohydrates/nsym PO2 (Fig. 2H), increase in carbohydrates consumption (44, 45); [3] 1,158/ 1,172, nC-O H-bonded proteins/nC-O non-H-bonded proteins (Fig. 2I), and the occurrence of a phosphorylative process (46).
FIGURE 3
Peak Fitting in the spectral range 1,180–900 cm1 for group A and B oocytes. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
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DISCUSSION This study represents the first spectroscopic approach to the analysis of human oocytes. The decision to exploit FPA FTIR imaging spectroscopy to investigate the biochemical processes involved in human gametes aging was due to the positive results previously obtained with zebrafish oocytes; in fact, in that study, this spectroscopic technique was successfully applied on zebrafish oocytes, affording an insightful characterization of the biological processes involved in their maturation (19, 20, 24). In humans, the ovarian hormonal stimulation protocols for IVF go beyond the natural and physiological selection process, which usually takes place during the development and maturation of a single oocyte. This hormonal practice leads to the maturation of numerous oocytes, often with compromised quality; the rescue of the follicles, which naturally would not have developed, may impair egg quality (47). In this light, the possibility of selecting only goodquality female gametes plays a crucial role in determining embryo competence and, therefore, IVF success. Recently, remarkable scientific attention has been focused on evaluating nuclear and mitochondrial genome integrity, but the transcription and translation processes, which characterize cytoplasmic maturity and competence, are still poorly understood (48). FPA FTIR imaging was performed on single oocytes to achieve an accurate analysis of female gametes at the molecular level and to better understand their macromolecular composition and distribution. The spectral analysis was carried out on well-known spectral ranges (CH2, CH3 and C¼O stretching modes, Amide II band, nucleic acids, and carbohydrates moieties), and specific spectral markers ascribable to oocytes aging were highlighted. A decline in the quality of oocytes belonging to B experimental group (women ages 39 2 years) was found, as confirmed by the following findings: [1] a more permeable plasma membrane with an increase in acyl chains peroxidation; [2] an altered proteic pattern containing a higher amount of unordered structures; [3] a decrease in signals transduction; [4] a variation in DNA conformation with an increase in the H-bond network; [5] the onset of an epigenetic effect; [6] some RNA degradation; [7] an altered lipid synthesis; [8] an increase in carbohydrates consumption; and [9] the occurrence of a phosphorylative process. The study represents the first IR approach on this specific topic, discovering meaningful macromolecular and biochemical changes in human gametes ascribable to aging. The analysis carried out on single oocytes allows researchers to simultaneously acquire important information on the composition and distribution of all the principal biomolecules (proteins, lipids, nucleic acids, and carbohydrates). In particular, a decline in oocyte quality in older patients was demonstrated by modifications in the plasma membrane, proteic pattern, composition and structure of nucleic acids, and metabolic processes. Although the results are encouraging, it is important to point out that, at the moment, the routine use of such a technique is not possible, since further statistical evidence for defining a reliable and easy protocol is necessary. 126
Acknowledgments: The authors thank Mrs. Carla Conti for her technical assistance and support and Dr. Elisabetta Damiani for her support with manuscript language editing.
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Banyay M, Sarkar M, Graslund A. A library of IR bands of nucleic acids in solution. Biophys Chem 2003;104:477–88. Lopes FL, Fortier AL, Darricarrere N, Chan D, Arnold DR, Trasler JM. Reproductive and epigenetic outcomes associated with aging mouse oocytes. Hum Mol Gen 2009;18:2032–44. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293:1089–93. Crawford JL, Kolpak FJ, Wang AH, Quigley GJ, van Boom JH, van der Marel G, et al. The tetramer d(CpGpCpG) crystallizes as a left-handed double helix. Proc Natl Acad Sci USA 1980;77:4016–20. Temiz NA, Donohue DE, Bacolla A, Luke BT, Collins JR. The role of methylation in the intrinsic dynamics of B-and Z-DNA. PLoS ONE 2012;7:e35558. Dovbeshko GI, Gridina NY, Kruglova EB, Pashchuk OP. FTIR spectroscopy studies of nucleic acid damage. Talanta 2000;53:233–46. Wood BR, Tait B, McNaughton D. Fourier transform infrared spectroscopy as a method for monitoring dynamics of lymphocyte activation. Appl Spectrosc 2000;54:353–9. Bozkurt O, Severcan M, Severcan F. Diabetes induces compositional, structural and functional alterations on rat skeletal soleus muscle revealed by FTIR spectroscopy: a comparative study with EDL muscle. Analyst 2010;135:3110–9. Elibol-Can B, Jakubowska-Dogru E, Severcan M, Severcan F. The effects of short-term chronic ethanol intoxication and ethanol withdrawal on the molecular composition of the rat hippocampus by FT-IR Spectroscopy. Alcohol Clin Exp Res 2011;35:2050–62. Wong PTT, Lacelle S, Yazdi HM. Normal and malignant human colonic tissues investigated by pressure-tuning FT-IR spectroscopy. Appl Spectrosc 1993;47:1830–6. Ci YZ, Gao TY, Feng J, Guo ZQ. Fourier transform infrared spectroscopic characterization of human breast tissue: implications for breast cancer diagnosis. Appl Spectrosc 1999;53:312–5. Conti C, Ferraris P, Garavaglia MG, Giorgini E, Rubini C, Sabbatini S, et al. Microimaging FTIR of head and neck tumors. IV. Microsc Res Tech 2009; 72:67–75. Tilly JL. Commuting the death sentence: how oocytes strive to survive. Nat Rev Mol Cell Biol 2001;2:838–48. Jones KT, Lane SIR. Chromosomal, metabolic, environmental, and hormonal origins of aneuploidy in mammalian oocytes. Exp Cell Res 2012;318:1394–9.
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Objective: To characterize from a vibrational point of view the alterations caused by aging on human oocytes. Design: Reproductive biology. Setting: Private assisted reproductive technology clinic, synchrotron beam line, and university infrared laboratory. Patient(s): Twenty women of different ages (30 2 and 39 2 years) selected on the basis of detailed inclusion criteria and submitted to controlled ovarian stimulation according to a specific protocol. Intervention(s): Collection of 68 supernumerary oocytes that were not used during the IVF cycle from the above cited consenting patients. Main Outcome Measure(s): Focal Plane Array Fourier transform infrared (FTIR) analysis of human oocytes. Result(s): Specific spectral differences were highlighted in the two experimental groups of oocytes. In particular, in oocytes of 39-yearold women, the occurrence of peroxidative processes and a decrease in the amount of carbohydrates were observed, together with alterations in the phospholipid membrane, proteic pattern, and nucleic acids content. Conclusion(s): For the first time, FTIR spectroscopy was applied to human oocytes, leading to strong evidence of damage from aging in the gametes of mature women, which could be related to a decline in reproductive function. All the information obtained may be considered useful to improve the scientific knowledge on huUse your smartphone man reproduction and to exploit new strategies for detecting oocyte aging. (Fertil SterilÒ to scan this QR code 2014;101:120–7. Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Human oocytes, aging damage, vibrational spectroscopy, FPA FTIR Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gioacchinig-aging-human-oocytes-spectroscopy/
T
o date, the problem of infertility affects about 10%–15% of couples, and the number of women who need assisted reproductive technology (ART) to achieve pregnancy is constantly increasing (1–3). However, despite the remarkable progress achieved by
research in the last 30 years, the success rates are still unsatisfactory (25%–30%) (4–7). For a complete diagnosis, both male and female factors have to be taken into account. In women, even if oocyte quality depends on multiple biological processes, many of which can
Received June 5, 2013; revised September 3, 2013; accepted September 9, 2013; published online October 17, 2013. G.G. has nothing to disclose. E.G. has nothing to disclose. L.V. has nothing to disclose. P.F. has nothing to disclose. S.S. has nothing to disclose. V.B. has nothing to disclose. A.B. has nothing to disclose. O.C. has nothing to disclose. The study was supported by funds from the Polytechnic University of Marche (to O.C. and E.G.). Reprint requests: Oliana Carnevali, Ph.D., Dipartimento di Scienze della Vita e dell'Ambiente, Uni Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (E-mail: o.carnevali@ versita univpm.it). Fertility and Sterility® Vol. 101, No. 1, January 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.09.012 120
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be assessed only at the molecular level, the biological capability to reproduce is primarily related to age (8, 9). During oogenesis, in which mRNA, proteins, lipids, and carbohydrates are stored, oocyte competence, defined as the ability of an oocyte to be fertilized and to develop to the blastocyst stage, is progressively achieved (10–12). In this light, a complete understanding and elucidation of the oocyte macromolecular structure in terms of composition and structure of the principal biomolecules appears useful to better evaluate the correlation between oocyte competence and aging.
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Fertility and Sterility® Fourier transform infrared (FTIR) spectroscopy is a fast, label-free analytical technique, widely used for investigating functional groups, bonding types, and molecular conformations of the most relevant biological molecules (13, 14). It allows us to examine the interaction between matter and electromagnetic radiation with an appropriate continuous range of frequencies. In particular, in the mid-IR region (4,000–400 cm1), fundamental vibrations, mainly stretching and bending of chemical bonds, can be detected. The coupling of IR spectrometers with visible microscopes has led to the successful use of this technique for many diagnostic purposes in the life sciences, allowing the detection of subtle biochemical changes caused by specific pathologies, such as tissues affected by tumoral pathologies and stem cells at various differentiation steps (15–18). FTIR microspectroscopy requires thin sections of samples (monolayer cell cultures or 5 mm thick tissues from surgical resection) on which IR maps are acquired on previously selected zones (1,000 1,000 mm2). The spatial resolution is limited only by diffraction, the area of each pixel of the map reaching up to 2.56 2.56 mm2. This allows researchers to characterize specific subcellular details, giving the possibility to mutually relate the vibrational local features with the morphology of the different compartments of the sample (19, 20). Owing to the large number of spectral data collected in a single map, several programs have been developed for performing multivariate statistical analysis to highlight similarities and differences in a simple readout format (21, 22). Wood and collaborators applied IR and Raman spectroscopies to study mouse oocytes at different maturational stages, leading to the vibrational characterization of oocyte molecular architecture for the first time (23). Some vibrational studies have also been carried out on zebrafish oocytes to analyze the modification of their biochemical composition during oogenesis and, further, to improve competence by administration of melatonin and probiotic (19, 20, 24). By considering these results, to develop knowledge on human reproduction and to complement morphological and molecular techniques currently applied in oocyte quality evaluation, we decided to exploit Focal Plane Array (FPA) FTIR imaging to identify appropriate spectral biomarkers related to the aging of human female gametes.
MATERIALS AND METHODS The study, approved by the Internal Review Board of Tecnobios Procreation, was carried out on human supernumerary oocytes in December 2012. Owing to legal restrictions, in Italy not all oocytes are inseminated, just the number necessary to optimize the patient's outcome for the cycle.
Sample Preparation Twenty dedicated consenting patients who were enrolled in an IVF program were selected on the basis of specific inclusion criteria (regular ovulatory menstrual cycles, FSH <10 IU/L on day 3 of the menstrual cycle, nonsmokers, and with no ovarian infertility diagnosis). The patients signed an informed consent to participate in the project research that VOL. 101 NO. 1 / JANUARY 2014
included the donation of their supernumerary oocytes. Controlled ovarian stimulation was induced according to a long protocol using leuprorelin (Enantone, Takeda), and rFSH (Gonal-F, Serono; or Puregon Organon). HCG at 10,000 IU (Gonasi, Amsa) was administered when one or more follicles reached a diameter of 23 mm (25). At the end of the treatment, 68 supernumerary metaphase II (MII) oocytes were collected and distributed into two groups on the basis of the donors' age: group A, 30 2 years old; group B, 39 2 years old (Table 1A). The cutoffs for young (group A) and old (group B) patients were, respectively, %32 and R37 years old. All the oocytes used for this project showed normal morphological features and clear and homogeneous cytoplasm with no saccuoles of endoplasmic reticulum or vacuoles. Poor-quality eggs were discarded to avoid bias in the interpretation of the results. None of the oocytes were inseminated or used to produce embryos. For the vibrational analysis, each oocyte was deposed ‘‘as it is’’ on a silicon support (a dish with a diameter of 10 mm and a thickness of 1 mm, transparent to IR radiation) and submitted to the same treatment of air drying for 30 minutes; no fixatives were used. The spectroscopic analysis was carried out within 10 hours. Previous experiments carried out in our laboratories showed that oocytes prepared as above were stable in time, providing homogeneous and reliable data sets.
FTIR Measurements and Data Analysis FTIR measurements were carried out at the IR beamline SISSI, ELETTRA synchrotron, by using a Bruker VERTEX 70 interferometer coupled with a Hyperion 3000 Vis-IR microscope and equipped with a liquid nitrogen cooled bidimensional FPA detector (area size 64 64 pixels; OPUS 6.5 software package, Bruker Optics). For each sample, the spectroscopic acquisition took only a few minutes. First, the visible image of each oocyte was acquired with a 15 condenser/objective (pixel size 2.56 mm). Then the IR map was acquired in transmission mode by using the FPA detector, which allowed us to simultaneously obtain groups of 4,096 spectra (128 scans for each detector pixel with a spectral resolution of 4 cm1). During all measurements, the microscope camera was purged with nitrogen to avoid atmospheric contamination (H2O and CO2). All of the IR maps were compared with background spectral images taken on regions of clean silicon windows. From each IR map, by using Opus 6.5 software, the total absorbance cartogram, representing the total intensity of the IR absorption, was generated by integrating within 1,800–1,000 cm1. For each oocyte, the average spectrum was obtained by merging 1,024 spectra (no representative spectra were excluded). Average spectra were two points baseline linear fitted in the spectral range 4,000–900 cm1 and vector normalized (26). All the oocytes belonging to the same experimental group showed vibrational spectra with similar profiles, thus giving reliable and reiterative results. Second Derivative (DII, 9-point smoothing) and Peak Fitting (Gaussian algorithm) procedures were adopted to determine the right position and absorbance of bands. By 121
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TABLE 1 A. Age of patients and number of donated oocytes. Group A
B
Woman
Age
No. of oocytes
W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20
30 29 31 30 31 28 30 32 38 39 39 38 40 37 41 38 39 38 39 38
3 4 3 3 4 3 4 3 3 4 4 3 4 3 4 4 3 3 4 2
B. Meaningful vibrational modes together with corresponding chemical features and biological processes. Biological aspects
Meaningful bands (cmL1)
Biochemical features
Biological processes
Plasma membrane
2,925/2,958 (nasym CH2/nasym CH3) 3,013/2,958 (n ¼CH/nasym CH3) 1,747/1,735 (1,741/1,725) (nC ¼ O transgauche) 1,556 2 (bstructures) 1,541 3 (aþ random structures) 1,518 1 (tyrosine) 1,717 (1,710) (nC ¼ O of thymine) 921 (925) (Z-DNA) 1,125 (nC-O ribose) 998 (uracil ring) 2,852/2,958 (nsym CH2/nasym CH3) 1,045/1,081 (nC-O /nsym PO2) 1,158/1,172 (nC-O H-bonded/non H-bonded proteins)
Lipid saturation level Lipid unsaturation level Different alkyl chains conformations, increase of the H-bond network Increase of the random structures
Increase of fatty acyl chains peroxidation
Proteic pattern Nucleic acids
Metabolism
More permeable membranes
A lower amount of tyrosine Increase of the H-bond network Z DNA RNA
Different proteic pattern. Proteins degradation Decreased transducing signals Modified DNA conformation Epigenetic effect RNA degradation
Alkyl chain length Carbohydrates and phosphates Substitution of C-OH with C-OP
Alterated lipid synthesis Increased carbohydrates consumption Phosphorylative process
Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
using GRAMS/AI 7.02 (Galactic Industries, Inc.), Peak Fitting was performed on average spectra interpolated in two different spectral ranges (1,780–1,470 cm1 and 1,180–900 cm1); the spectra were two points baseline linear fitted. To identify the underlying component bands, the number of peaks together with their center values were carefully individuated according to DII results and fixed before running the iterative process, to obtain the best reconstructed curve (residual close to zero). Mean values of area and wavelength were purchased for each component peak. The representative bands taken into account are reported in Table 1B, together with their spectral assignments; their attribution was done according to the literature. The percentage of band areas and ratios are presented as mean SD. Student's t-test was used for comparison between the two experimental groups, using a statistical software package, Prism5 (Graphpad Software, Inc.) with significance set at P< .05. To evaluate the distribution of the principal biocomponents (lipids, proteins, and nucleic acids), false color images were calculated for each oocyte by integrating the 122
IR maps under the following spectral regions: 3,100–2,800 cm1 (CH stretching modes), 1,580–1,480 cm1 (Amide II), and 1,300–900 cm1 (phosphate and carbohydrate moieties; HyperSpec software).
RESULTS The study aimed to achieve molecular information on the biochemical modifications induced by aging on human oocytes by using FPA FTIR imaging spectroscopy. The spectroscopic analysis was carried out on a meaningful number of supernumerary metaphase MII oocytes, which were donated by consenting patients enrolled in IVF programs and divided into two experimental groups according to age (group A, 30 2 years old; group B, 39 2 years old; Table 1A). For all oocytes, the visible images and the IR maps were acquired, from which the total absorption cartograms, representing the total intensity of the IR absorption, were calculated, integrating between 1,800 and 1,000 cm1 (Fig. 1A, 1B). To localize the distribution of the principal VOL. 101 NO. 1 / JANUARY 2014
Fertility and Sterility®
FIGURE 1
(A) Photomicrographs and (B) total absorbance cartograms, reconstructed by integrating under 1,800–1,000 cm1, of group A and B oocytes. False color images were integrated in the following spectral ranges: (C) 3,100–2,800 cm1 (CH2 and CH3 stretching modes), (D) 1,580–1,480 cm1 (Amide II), and (E) 1,300–900 cm1 (phosphates and carbohydrates moieties). (F) Average spectra of group A and B oocytes in the spectral range 4,000–900 cm1. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
biocomponents inside the oocytes, false color images (obtained by using HyperSpec software package) were elaborated, showing a different distribution of proteins (inner zone), nucleic acids (on the edge), and lipids (external zone) in both A and B oocytes. For each experimental group, a representative spectrum was extracted (called A and B average spectra; Fig. 1F). By carefully analyzing them and by comparing them to each VOL. 101 NO. 1 / JANUARY 2014
other, meaningful spectral changes were found, above all in the CH stretching region (3,100–2,800 cm1), in the proteic pattern (1,780–1,470 cm1), and in the nucleic acids spectral region (1,180–900 cm1). Specific band areas and band area ratios, which could be considered as spectral markers for a decline in oocyte quality with aging, were also calculated and the numerical variations analyzed (Table 1B; Fig. 2). 123
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FIGURE 2
ν
ν
ν
β–
ν
ν
ν
ν
α
/
Numerical variations of the following band area values for the A and B experimental groups: (A) nasym CH2/CH3; (B) n ¼CH/nasym CH3; (C) nsym CH2/ nasym CH3; (D) 1,556 2 cm1 (b-sheet); (E) 1,541 3 cm1 (helical-random components); (F) 1,518 1 cm1 (tyrosine); (G) nC¼O (trans)/nC¼O (gauche); (H) 1,045/1,081 (nC-O carbohydrates/nsym PO2); (I) 1,158/1,172 (nC-O H-bonded carbohydrates/nC-O non-H-bonded proteins). Data are presented as mean SD. Student's t-test was used for comparison between the two experimental groups, using the statistical software package Prism5 (GraphPad Software, Inc.), with significance set at P<.05. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
Plasma Membrane To analyze plasma membrane structure, particular attention was given to the spectral region 3,050–2,800 cm1. The bands at 2,958 and 2,924 cm1 (nasym CH3 and CH2), 2,872 and 2,852 cm1 (nsym CH3 and CH2), usually attributed to amino acids side chains and to plasma membrane alkyl chains (23), did not shift their position and appeared sharp and clearly defined, suggesting well-arranged alkyl chains with few rotamers in both experimental groups (27). The band at 3,013 cm1 (n ¼ CH) is usually considered a marker of peroxidative processes (28, 29); by analyzing the numerical variations of the band area ratios 2,925/2,958 (lipids saturation level) and 3,013/2,958 (lipids unsaturation 124
level; Fig. 2A, 2B), a higher amount of peroxidized fatty acyl chains was found in B oocytes from older patients. This could be explained by considering that the higher amount of polyunsaturated fatty acids present in the oocyte membrane could easily react with reactive oxygen species and induce oxidative stress. After the Peak Fitting procedure was applied on A and B average spectra in the range 1,780–1,470 cm1, the band at 1,740 cm1, usually attributed to diacyl lipids, turned out to be composed of two different absorptions, originating from the stretching of the two ester carbonyl groups, which were adjacent to C-C bonds with trans or gauche conformations (13). In group A oocytes, these two bands were respectively found at 1,747 and 1,735 cm1, while in group B VOL. 101 NO. 1 / JANUARY 2014
Fertility and Sterility® oocytes, they shifted to lower frequencies (1,741 and 1,725 cm1). These findings suggest that in oocytes from older patients, a stronger H-bond network is present, which could result in a more permeable phospholipid membrane. Modifications in alkyl chain conformations may also be present owing to the different values in the absorbance band ratios 1,747/1,735 (group A) and 1,741/1,725 (group B; Fig. 2G).
Proteic Pattern The vibrations of adjacent peptide groups are strongly influenced by each other, as well as by the network of hydrogen bonding (13). In this light, it appeared useful to study the bands at 1,658 (Amide I) and 1,549 (Amide II) cm1, which are sensitive to proteins' structural conformation (19, 27). Owing to a certain degree of saturation in the Amide I region, the study was carried out only on the Amide II moiety, which, after the Peak Fitting procedure, turned out to be composed of three different bands: 1,556 2 (a-helix and random coil components), 1,541 3 (bsheet structures), and 1,518 1 (tyrosine) cm1. The percentage amounts of the areas of the above cited bands were calculated (Fig. 2D–2F): in B oocytes, an increase in random coil structures was observed, suggesting a modified proteic pattern with a higher level of protein degradation. A decline in B oocyte quality was also pinpointed by the drastic decrease of the band at 1,518 cm1, which is attributable to tyrosine (18). In fact, tyrosine kinase proteins are cytoplasmic enzymes targeted at the plasma membrane or at the actin cytoskeleton, which play an essential role in fertilization and zygote development, acting to transduce signals from external stimuli (30). In mammals, there is evidence that kinases are important for oocyte maturation and for the maintenance of the cytoskeleton of the mature oocyte (31–34).
Nucleic Acids The spectral range 1,180–900 cm1, usually related to the vibrations of the principal functional groups present in nucleic acids, was carefully investigated by the Peak Fitting procedure (Fig. 3). According to DNA moieties, in B oocytes, the band at 968 cm1 (nCC of DNA backbone) appeared unchanged in intensity and position, while the one at 921 cm1 (Z-DNA) increased and shifted to 925 cm1 (35); these findings suggest an epigenetic effect in oocytes from women ages 39 2 (36). The degree of DNA methylation is an important factor for correct embryo development (37). In fact, during oogenesis, genomic methylation imprints are of particular concern, since they are established throughout oocyte growth and are not subject to the global demethylation that occurs postfertilization (38). Methylation of CpG-rich DNA tracts greatly promotes the transition from B to Z conformations (39). An increase in H-bonded C¼O bonds was also suggested by the shift of the band related to nC¼O of thymine from 1,717 to 1,710 cm1 (18, 40). In B oocytes, some processes related to RNA degradation were also present as confirmed by the decrease in intensity of the bands at 1125 (nC-O ribose) and 998 (uracil ring) cm1 (40, 41).
Oocyte Metabolism In B oocytes, changes in metabolic processes were also detected, as evidenced by the numerical variations of the following band area ratios: [1] 2,852/2,958, nsym CH2/nasym CH3 (Fig. 2C), modifications in lipid synthesis (42, 43); [2] 1,045/1,081, nC-O carbohydrates/nsym PO2 (Fig. 2H), increase in carbohydrates consumption (44, 45); [3] 1,158/ 1,172, nC-O H-bonded proteins/nC-O non-H-bonded proteins (Fig. 2I), and the occurrence of a phosphorylative process (46).
FIGURE 3
Peak Fitting in the spectral range 1,180–900 cm1 for group A and B oocytes. Gioacchini. FTIR approach to human oocytes. Fertil Steril 2014.
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125
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DISCUSSION This study represents the first spectroscopic approach to the analysis of human oocytes. The decision to exploit FPA FTIR imaging spectroscopy to investigate the biochemical processes involved in human gametes aging was due to the positive results previously obtained with zebrafish oocytes; in fact, in that study, this spectroscopic technique was successfully applied on zebrafish oocytes, affording an insightful characterization of the biological processes involved in their maturation (19, 20, 24). In humans, the ovarian hormonal stimulation protocols for IVF go beyond the natural and physiological selection process, which usually takes place during the development and maturation of a single oocyte. This hormonal practice leads to the maturation of numerous oocytes, often with compromised quality; the rescue of the follicles, which naturally would not have developed, may impair egg quality (47). In this light, the possibility of selecting only goodquality female gametes plays a crucial role in determining embryo competence and, therefore, IVF success. Recently, remarkable scientific attention has been focused on evaluating nuclear and mitochondrial genome integrity, but the transcription and translation processes, which characterize cytoplasmic maturity and competence, are still poorly understood (48). FPA FTIR imaging was performed on single oocytes to achieve an accurate analysis of female gametes at the molecular level and to better understand their macromolecular composition and distribution. The spectral analysis was carried out on well-known spectral ranges (CH2, CH3 and C¼O stretching modes, Amide II band, nucleic acids, and carbohydrates moieties), and specific spectral markers ascribable to oocytes aging were highlighted. A decline in the quality of oocytes belonging to B experimental group (women ages 39 2 years) was found, as confirmed by the following findings: [1] a more permeable plasma membrane with an increase in acyl chains peroxidation; [2] an altered proteic pattern containing a higher amount of unordered structures; [3] a decrease in signals transduction; [4] a variation in DNA conformation with an increase in the H-bond network; [5] the onset of an epigenetic effect; [6] some RNA degradation; [7] an altered lipid synthesis; [8] an increase in carbohydrates consumption; and [9] the occurrence of a phosphorylative process. The study represents the first IR approach on this specific topic, discovering meaningful macromolecular and biochemical changes in human gametes ascribable to aging. The analysis carried out on single oocytes allows researchers to simultaneously acquire important information on the composition and distribution of all the principal biomolecules (proteins, lipids, nucleic acids, and carbohydrates). In particular, a decline in oocyte quality in older patients was demonstrated by modifications in the plasma membrane, proteic pattern, composition and structure of nucleic acids, and metabolic processes. Although the results are encouraging, it is important to point out that, at the moment, the routine use of such a technique is not possible, since further statistical evidence for defining a reliable and easy protocol is necessary. 126
Acknowledgments: The authors thank Mrs. Carla Conti for her technical assistance and support and Dr. Elisabetta Damiani for her support with manuscript language editing.
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