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Progress toward “in vivo virtual histology” of ovarian follicles and corpora lutea by ultrasound biomicroscopy
Progress toward ‘‘in vivo virtual histology’’ of ovarian follicles and corpora lutea by ultrasound biomicroscopy Pilar Pallares, M.V.D.,a Claudia Letelier, M.V.D.,b,c and Antonio Gonzalez-Bulnes, Ph.D.b a Unidad de Animalario, CNIC, Madrid, Spain; b Departamento de Reproduccion Animal, INIA, Madrid, Spain; and c Instituto de Ciencia Animal y Tecnologia de Carnes, Universidad Austral de Chile, Valdivia, Chile
High-frequency ultrasound imaging (40 MHz; ultrasound biomicroscopy) provides reliable ex vivo visualization of cellular components both in follicles and corpora lutea. In the near future and after logical technical evolution it may be useful for in vivo intraoperative evaluation of ovaries in large mammals and humans. (Fertil Steril 2009;91:624–6. 2009 by American Society for Reproductive Medicine.) Key Words: Ovary, follicle, corpus luteum, ultrasonography
Ultrasound biomicroscopy (UBM) is a pulse-echo method developed for experimental studies in mice (1), providing ultra-high-frequency (30–55 MHz) and hence micro-imaging resolution (30–50 mm). We hypothesized that, in the ovary, UBM would allow evaluation of the micro-texture of follicles and corpora lutea, which is indicative of their biologic function over time (2), with resolution near to that of conventional histology.
Thus, we compared UBM and histologic imaging. In vivo UBM was not our goal because, given the high frequency of the probes, the depth of scanning is limited (5–10 mm). Hence we performed ex vivo imaging of sheep and cow ovaries collected in a local abattoir. After embedding the ovaries in ultrasound gel to eliminate air and improve the image, UBM was performed with a Vevo 770 (Visual Sonic, Toronto, Canada) equipped with a 40-MHz probe. For histologic
FIGURE 1 Imaging of a corpus luteum by ultrasound biomicroscopy (left) and histology (right). It is possible to differentiate the outlines and organization of the luteal cells and the presence of a central cavity.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
Received January 23, 2008; revised March 10, 2008; accepted May 9, 2008. P.P. has nothing to disclose. C.L. has nothing to disclose. A.G.-B. has nothing to disclose. This study was developed under a collaborative project (CC07-018) n Centro Nacional de Investigaciones Cardiovascubetween Fundacio n y lares Carlos III (CNIC) and Instituto Nacional de Investigacio Tecnologıa Agraria y Alimentaria (INIA). The CNIC is supported by the
624
Spanish Ministry of Health and Consumer Affairs and the Pro-CNIC Foundation; the INIA is supported by the Spanish Ministry of Education and Science Affairs; there was no other outside funding. n Centro Nacional de Reprints requests: Pilar Pallares, M.V.D., Fundacio Investigaciones Cardiovasculares Carlos III, Melchor Fernandez Almagro 3, 28029 Madrid, Spain (FAX: 34-91-453-12-65; E-mail: [email protected]).
Fertility and Sterility Vol. 91, No. 2, February 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.05.033
FIGURE 2 Differentiation, by ultrasound biomicroscopy (left) and histology (right), of the limits between antrum, granulosa (GC), and theca cells (TC) and surrounding tissue in a preovulatory follicle.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
FIGURE 3 Imaging, by ultrasound biomicroscopy (left) and histology (right), of the cumulus–oocyte complex (COC) in the follicular fluid.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
imaging the ovaries were fixed in Bouin’s solution, embedded in paraffin, and after sectioning (4 mm), stained with hematoxylin and eosin. Figure 1 illustrates the feasibility of UBM for imaging a corpus luteum with cavity. Ultrasound biomicroscopy also provided good visualization of follicles, making it possible to differentiate the outlines between antrum, granulosa and theca cells, and surrounding tissue (Fig. 2) and the presence of the cumulus–oocyte complex in the follicular fluid (Fig. 3). From these results, and adapting transducers for intraoperative imaging by laparoscopy or laparotomy, UBM would be Fertility and Sterility
useful for in vivo ovarian evaluation in living large mammals and humans. Clinical safety would be similar to that with conventional ultrasonography (5–8-MHz probes) because harmful effects of ultrasound have been more related to exposures of >30 minutes and at frequencies of <3.5 MHz (3, 4).
REFERENCES 1. Foster FS, Zhang MY, Zhou YQ, Liu G, Mehi J, Cherin E, et al. A new ultrasound instrument for in vivo microimaging of mice. Ultrasound Med Biol 2002;28:1165–72.
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2. Eramian MG, Adams GP, Pierson RA. Enhancing ultrasound texture differences for developing an in vivo ‘‘virtual histology’’ approach to bovine ovarian imaging. Reprod Fertil Dev 2007;19:910–24. 3. Bertuglia S, Giusti A, Picano E. Effects of diagnostic cardiac ultrasound on oxygen free radical production and microvascular perfu-
626
Pallares et al.
Ovarian ultrasound biomicroscopy
sion during ischemia reperfusion. Ultrasound Med Biol 2004;30: 549–57. 4. Rao S, Ovchinnikov N, McRae A. Gestational stage sensitivity to ultrasound effect on postnatal growth and development of mice. Birth Defects Res A Clin Mol Teratol 2006;76:602–8.
Vol. 91, No. 2, February 2009
High-frequency ultrasound imaging (40 MHz; ultrasound biomicroscopy) provides reliable ex vivo visualization of cellular components both in follicles and corpora lutea. In the near future and after logical technical evolution it may be useful for in vivo intraoperative evaluation of ovaries in large mammals and humans. (Fertil Steril 2009;91:624–6. 2009 by American Society for Reproductive Medicine.) Key Words: Ovary, follicle, corpus luteum, ultrasonography
Ultrasound biomicroscopy (UBM) is a pulse-echo method developed for experimental studies in mice (1), providing ultra-high-frequency (30–55 MHz) and hence micro-imaging resolution (30–50 mm). We hypothesized that, in the ovary, UBM would allow evaluation of the micro-texture of follicles and corpora lutea, which is indicative of their biologic function over time (2), with resolution near to that of conventional histology.
Thus, we compared UBM and histologic imaging. In vivo UBM was not our goal because, given the high frequency of the probes, the depth of scanning is limited (5–10 mm). Hence we performed ex vivo imaging of sheep and cow ovaries collected in a local abattoir. After embedding the ovaries in ultrasound gel to eliminate air and improve the image, UBM was performed with a Vevo 770 (Visual Sonic, Toronto, Canada) equipped with a 40-MHz probe. For histologic
FIGURE 1 Imaging of a corpus luteum by ultrasound biomicroscopy (left) and histology (right). It is possible to differentiate the outlines and organization of the luteal cells and the presence of a central cavity.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
Received January 23, 2008; revised March 10, 2008; accepted May 9, 2008. P.P. has nothing to disclose. C.L. has nothing to disclose. A.G.-B. has nothing to disclose. This study was developed under a collaborative project (CC07-018) n Centro Nacional de Investigaciones Cardiovascubetween Fundacio n y lares Carlos III (CNIC) and Instituto Nacional de Investigacio Tecnologıa Agraria y Alimentaria (INIA). The CNIC is supported by the
624
Spanish Ministry of Health and Consumer Affairs and the Pro-CNIC Foundation; the INIA is supported by the Spanish Ministry of Education and Science Affairs; there was no other outside funding. n Centro Nacional de Reprints requests: Pilar Pallares, M.V.D., Fundacio Investigaciones Cardiovasculares Carlos III, Melchor Fernandez Almagro 3, 28029 Madrid, Spain (FAX: 34-91-453-12-65; E-mail: [email protected]).
Fertility and Sterility Vol. 91, No. 2, February 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.05.033
FIGURE 2 Differentiation, by ultrasound biomicroscopy (left) and histology (right), of the limits between antrum, granulosa (GC), and theca cells (TC) and surrounding tissue in a preovulatory follicle.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
FIGURE 3 Imaging, by ultrasound biomicroscopy (left) and histology (right), of the cumulus–oocyte complex (COC) in the follicular fluid.
Pallares. Ovarian ultrasound biomicroscopy. Fertil Steril 2009.
imaging the ovaries were fixed in Bouin’s solution, embedded in paraffin, and after sectioning (4 mm), stained with hematoxylin and eosin. Figure 1 illustrates the feasibility of UBM for imaging a corpus luteum with cavity. Ultrasound biomicroscopy also provided good visualization of follicles, making it possible to differentiate the outlines between antrum, granulosa and theca cells, and surrounding tissue (Fig. 2) and the presence of the cumulus–oocyte complex in the follicular fluid (Fig. 3). From these results, and adapting transducers for intraoperative imaging by laparoscopy or laparotomy, UBM would be Fertility and Sterility
useful for in vivo ovarian evaluation in living large mammals and humans. Clinical safety would be similar to that with conventional ultrasonography (5–8-MHz probes) because harmful effects of ultrasound have been more related to exposures of >30 minutes and at frequencies of <3.5 MHz (3, 4).
REFERENCES 1. Foster FS, Zhang MY, Zhou YQ, Liu G, Mehi J, Cherin E, et al. A new ultrasound instrument for in vivo microimaging of mice. Ultrasound Med Biol 2002;28:1165–72.
625
2. Eramian MG, Adams GP, Pierson RA. Enhancing ultrasound texture differences for developing an in vivo ‘‘virtual histology’’ approach to bovine ovarian imaging. Reprod Fertil Dev 2007;19:910–24. 3. Bertuglia S, Giusti A, Picano E. Effects of diagnostic cardiac ultrasound on oxygen free radical production and microvascular perfu-
626
Pallares et al.
Ovarian ultrasound biomicroscopy
sion during ischemia reperfusion. Ultrasound Med Biol 2004;30: 549–57. 4. Rao S, Ovchinnikov N, McRae A. Gestational stage sensitivity to ultrasound effect on postnatal growth and development of mice. Birth Defects Res A Clin Mol Teratol 2006;76:602–8.
Vol. 91, No. 2, February 2009