Workshop 5: Inter-Relações Da Reprodução Humana e Veterinária

Workshop 5:
Inter-relaes da reproduo humana e veterinria
The interface between human and animal reproduction
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Acta Scientiae Veterinariae . 38(Supl 2): s393-s413, 2010.

ISSN 1678-0345 (Print) ISSN 1679-9216 (Online)
Human Assisted Reproduction

Paulo Serafini1,2*
ABSTRACT
Background: Infertility is defined as the failure to conceive after one year of unprotected intercourse, and this time has been lowered to 6 month if the female partner is older than 35 years. Infertile couples are offered to low and high complexity treatments are available according to their cause of infertility. Low complexity treatments comprise timed intercourse, intrauterine insemination with or without controlled ovarian stimulation. High complexity treatments comprise standard in vitro fertilization (IVF) and IVF by intracytoplasmatic sperm injection (ICSI). Additionally to these treatments, infertile patients might be benefited by accessory techniques such as preimplantational genetic screening and diagnosis. These techniques aim to detect the most common human aneuploydies related to abortion and chromosomal syndromes; and to identify embryos carriers of genetic diseases like talassemia, Huntingtons disease, fragile-X syndrome among others. Human assisted reproduction is a dynamic branch of Medicine performed by several medical specialists from gynecologists to surgeons, and professional as nurses, biologists and veterinarians. Review: Initially, human infertility was treated by intracervical insemination due to the risk of endometrial insemination and the occurrence of pelvic inflammation, but the development of better insemination techniques and instruments led to the report of a safe intrauterine insemination method in 1974. However, insemination was restricted to oligozoospermia or cervical factors and it was not able to overcome ovarian and tubal infertility factors, neither
severe male infertility factor. The range of infertility treatment was tremendously broadened in 1978 with the report of the first IVF baby birth. Since then infertile couples due to tubal factors, ovarian and moderate male infertility factors started to be treated with IVF. Several changes in embryo culture conditions and instruments for IVF were developed and the technology was world wide spread. The first IVF baby was a girl born in 1984 in Brazil. Even though, some ovarian conditions and severe male factor infertility couldnt be treated until 1992. This landmark was the report of the first babies originated by in vitro fertilization with ICSI. In parallel to those scientific and medical evolutions, drugs and new protocols of controlled ovarian stimulation increased the number of oocytes available for fertilization and, consequently, the number of exceeding embryos. These spare oocytes and embryos required the development of cryopreservation techniques for long term storage. Slow rate freezing and vitrification of embryos were reported approximately 25 years ago, but just recently vitrification of oocytes and embryos became the first option for fertility preservation and/or embryo storage in humans. Preimplantational genetic screening and diagnosis were complementary techniques developed during the same period.These tests require precise and meticulous micromanipulation techniques and skills for the obtainment of a single blastomere with intact genetic material for screening of aneuploydies by fluorescent in situ hybridization or the detection of a deleterious allele by single cell PCR. New drugs, protocols, instruments and techniques for Human Assisted Reproduction are still under development in many Universities, infertility clinics and private companies aiming to increase the efficiency of infertility treatments.
Conclusion: Human assisted reproduction is constantly evolving with the development of more precise diagnostic tests and with the improvement of clinical approaches to infertility and better conditions in embryology laboratories and this constant evolution is the result of the synergic interaction of clinical infertility specialists with researchers of different backgrounds.
Keywords: Assisted reproduction, in vitro fertilization, IVF, intracytoplasmatic sperm injection, ICSI, human reproduction.
Huntington Medicina Reprodutiva, So Paulo, Brazil; 2Centro de reproduo humana Governador Mrio Covas do Hospital das Clnicas da Faculdade de Medicina da Universidade de So Paulo*Corresponding authors address: Huntington Medicina Reprodutiva, Av. Repblica do Lbano, 529, So Paulo, SP, Brazil, Postal code: 04501-000, Phone: 5511 30596122, Fax: 5511 30596100, e-mail: pserafini@huntington.com.br
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Workshop 5: Inter-relaes da reproduo humana e veterinria. .
Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413
I. INTRODUCTION II. HISTORICAL OVERVIEW OF HUMAN ASSISTED REPRODUCTION III. PERSPECTIVES FOR HUMAN ASSISTED REPRODUCTION IV. CONCLUSIONS
I. INTRODUCTION
Infertility is a disease defined as the failure to conceive after one year of unprotected intercourse, and this time has been lowered to 6 month if the female partner is older than 35 years [1]. Approximately, 10% of the couples in reproductive age might require infertile care [2]. The last report on assisted human reproduction in Europe indicated that 359,110 treatments were performed in a population of 422.5 million (850 treatments/million) [3]; in Latin America, 34,102 assisted reproduction treatments were performed in 2007 and 42.3% of those were carried out in Brazil [4]. Treatments of assisted reproduction are indicated according to the infertility factor of the couple. Male infertility factors comprise a myriad of malfunctions from retrograde ejaculation to impaired production or release of sperm such as in oligozoospermia, teratozoosperia, asthenozoospermia, and obstructive and non-obstructive azoospermia [5]. Conformably to male infertility, several diseases are enrolled in female infertility. Didactically, female infertility can be classified as ovarian/ovulatory factors, tubal factors, uterine factors and endometriosis [6-8]. Male, female and the combination of these infertility factors may be treated with Human Assisted Reproduction of low and high complexity according to the patients needs (Table 1)[8-9]. Low complexity treatments comprise timed intercourse, intrauterine insemination with or without controlled ovarian stimulation. High complexity treatments comprise standard in vitro fertilization (IVF) and IVF by intracytoplasmatic sperm injection (ICSI). Additionally to these treatments, infertile patients might be benefited by techniques such as preimplantational genetic screening and diagnosis.
II. HISTORICAL OVERVIEW OF HUMAN ASSISTED REPRODUCTION

The treatment of infertility has been recorded since the late years of the 18th Century; at that time, by archaic techniques of artificial insemination. Despite its availability of for more than 200 years, artificial insemination remained controversial in many countries until the 1970s [10]. Modern artificial insemination consisted in the intracervical delivery of prepared semen to minimize the risk of occurrence of pelvic inflammation. Nevertheless, since 1974 the use of a of a safe intrauterine insemination methodhas replaced intracervical alternative [11]. Intrauterine artificial insemination was restricted to oligozoospermia or cervical factors [12]; however, it was not able to overcome ovarian and tubal infertility factors, neither severe male infertility factor. In vitro fertilization is the appropriate treatment for these conditions, and despite of the publication of attempts of in vitro fertilization of animal oocytes in the begging of the 20th Century, the fertilization of human oocytes was first reported in 1969 [13]. Several developments in embryo culture techniques were developed and in 1978 the birth after of the first IVF baby was annunciated and it broadened the range of treatable infertility causes; thence, infertile couples due to tubal, ovarian and moderate male infertility factors could experience parenthood through IVF [14].
In vitro fertilization treatment became worldwide spread and the first IVF baby born in Brazil was in 1984. The word technology industry took a giant leap and physicians and scientist increased performances, established treatments with both higher quality standards and have observed an even increasing pregnancy and birth rates. In 1992, Palermo and colleagues [15] reported a births of babies originated by in vitro fertilization with intracytoplasmatic sperm injection assuring that man that produced spermatozoa despite ejaculation could become parents.
In parallel to those scientific and medical developments, drugs and new protocols of ovulation induction increased the number of oocytes available for fertilization and, consequently, the number of exceeding embryos.
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These spare oocytes and embryos required the development of cryopreservation techniques for long term storage. Successful slow rate freezing of embryos were reported approximately 25 years ago [16]. Recently vitrification of oocytes and embryos became the first option for fertility preservation for female gametes and embryos storage in humans [17-18]. In fact, vitrification of oocytes has provided quality oocytes at warming resulting in pregnancy rates similar to those obtained with fresh oocytes. This methodology has been chosen in Spain as the main storage method for oocytes in an ovodonation program [19-20] Preimplantation genetic screening and diagnosis were complementary techniques developed during the same period [21].These tests require precise and meticulous micromanipulation techniques and skills for the obtainment of a single blastomere with intact genetic material for screening of aneuploydies using fluorescent in situ hybridization or the detection of a deleterious allele by single cell PCR [21]. Preimplantation genetic diagnosis is particularly interesting for the prevention of allelic disorders such as thalassemia, sickle cell anemia, cystic fibrosis, Duchennes Muscular Distrophy, among several others [22-24].
III. PERSPECTIVES FOR HUMAN ASSISTED REPRODUCTION

Recently, we proposed several approaches for new ovulation induction methods based on the description of follicular waves in women [25] described originally by Baerwald et al. [26-27]. These protocols are currently under clinical evaluation and it is our expectation that it might enhance pregnancy rates after IVF. Additionally, remarkable modifications on embryo culture systems are forthcoming as technologies as proteomics and genomics come into place. The in vivo environment for embryo development is chemically and physically dynamic; however, embryo culture systems currently used in Human Assisted Reproduction is static [2829]. The establishment of physically dynamic conditions through tilting embryo culture media [30] or, in a more sophisticated approach, through a complex series of channels creating microfluidic stream of media [28-29] are still on progress, but they are promising technology on embryo culture techniques and they may have a great impact on pregnancy rates. Complementary techniques such as PGD are also becoming more sophisticated through the implementation of feasible microarray based comparative genomic hybridization [31]. This technique allows high resolution investigation of the embryo genome and detection of deleterious copy number variation of genes, causes of miscarriage and it still allows the determination of chromosomal numbers [32-33].
IV. CONCLUSIONS
Human Assisted Reproduction is constantly evolving with the development of more precise diagnostic tests and with the improvement of clinical approaches to infertility and better conditions in embryology laboratories and this constant evolution is the result of the synergic interaction of clinical infertility specialists with researchers of different backgrounds.
REFERENCES
1 ASRM. 2008. Practice Committee of the American Society for Reproductive Medicine: Definitions of infertility and recurrent pregnancy loss. Fertililiy and Sterility. 89(6): 1603. 2 Beurskens M.P., Maas J.W. & Evers J.L. 1995. Subfertility in South Limburg: calculation of incidence and appeal for specialist care. Nederlands Tijdschrift Geneeskunde. 139(5): 235-238. 3 Mouzon J.D., Goossens V., Bhattacharya S., Castilla J.A., Ferraretti A.P., Korsak P., Kupka M., Nygren K.G. & Nyboe Andersen A. 2006. Assisted reproductive technology in Europe: results generated from European registers by ESHRE. Human Reproduction. 112. 4 RedLARA. 2009. Registro Latinoamericano de Reproduccin Asistida. Red Latino Americana de Reproduccin Asistida. 75 p. 5 Jose-Miller A.B., Boyden J.W. & Frey K.A. 2007. Infertility. American Familiy Physician. 75(6): 849-856. 6 Bhattacharya S., Porter M., Amalraj E., Templeton A., Hamilton M., Lee A. J. & Kurinczuk J.J. 2009. The epidemiology of infertility in the North East of Scotland. Human Reproduction 24 (12): 3096-3107. 7 Ozkan S., Murk W. & Arici A. 2008. Endometriosis and infertility: epidemiology and evidence-based treatments. Annals of the New York Academy of Sciences. 1127: 92-100.
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8 Yli-Kuha A.N., Gissler M., Luoto R. & Hemminki E. 2009. Success of infertility treatments in Finland in the period 1992-2005. European Journal of Obstetrics, Gynecology and Reproductive Biology. 144(1): 54-58. 9 Hrometz S.L. & Gates V.A. 2009. Review of available infertility treatments. Drugs Today (Barcelona). 45(4): 275-291. 10 Clarke G.N. 2006. A.R.T. and history, 1678-1978. Human Reproduction. 21(7): 1645-1650. 11 Barwin B.N. 1974. Intrauterine insemination of husbands semen. Journal of Reproduction Fertility. 36(1): 101-106. 12 Da Motta E.L. & Serafini P. 2002 The treatment of infertility and its historical context. Reproductive Biomedicine Online. 5(1): 65-77. 13 Edwards R.G., Bavister B.D. & Steptoe P.C. 1969. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature. 221(5181): 632-635. 14 Steptoe P.C. & Edwards R.G. 1978. Birth after the reimplantation of a human embryo. Lancet. 312(8085): 366. 15 Palermo G., Joris H., Devroey P. & Van Steirteghem A.C. 1992. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 340(8810): 17-18. 16 Trounson A. & Mohr L. 1983. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 305(5936): 707-709. 17 Kuwayama M. 2007. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology. 67(1): 73-80. 18 Smith G.D., Serafini P.C., Fioravanti J., Yadid I., Coslovsky M., Hassun P., Alegretti J.R. & Motta E.L. 2010 Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertility and Sterility (in press). 19 Cobo A., Meseguer M., Remoh J. & Pellicer A. 2010. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Human Reproduction.( in press). 20 Cobo A., Domingo J., Perez S., Crespo J., Remohi J. & Pellicer A. 2008. Vitrification: an effective new approach to oocyte banking and preserving fertility in cancer patients. Clinical & Translational Oncology. 10(5): 268-273. 21 Verlinsky Y. & Kuliev A. 1992 Micromanipulation of gametes and embryos in preimplantation genetic diagnosis and assisted fertilization. Current Opinion in Obstetrics & Gynecology. 4(5): 720-725. 22 Liu J., Lissens W., Devroey P., Liebaers I. & Van Steirteghem A. 1996. Cystic fibrosis, Duchenne muscular dystrophy and preimplantation genetic diagnosis. Human Reproduction Update. 2(6): 531-539. 23 Xu K., Shi Z.M., Veeck L.L., Hughes M.R. & Rosenwaks Z. 1999 First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia. The Journal of American Medical Association. 281(18): 1701-1706. 24 Petrou M. 2009. Preimplantation genetic diagnosis. Hemoglobin. 33 (1): S7-13. 25 Bianchi P., Serafini P., da Rocha A.M., Hassun P., da Motta E., Baruselli P. & Baracat E. 2010 Follicular Waves in the Human Ovary: A New Physiological Paradigm for Novel Ovarian Stimulation Protocols. Reproductive Sciences. (in press). 26 Baerwald A.R., Adams G.P. & Pierson R.A. 2003. A new model for ovarian follicular development during the human menstrual cycle. Fertility and Sterility. 80(1): 116-22. 27 Baerwald A.R., Adams G.P. & Pierson R.A. 2003. Characterization of ovarian follicular wave dynamics in women. Biology of Reproduction. 69(3): 1023-31. 28 Heo Y.S., Cabrera L.M., Bormann C.L., Shah C.T., Takayama S. & Smith G.D. 2010. Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Human Reproduction. 25(3): 613-22. 29 Smith G.D. & Takayama S. 2007. Gamete and embryo isolation and culture with microfluidics. Theriogenology. 68 (1): S190-195. 30 Matsuura K., Hayashi N., Kuroda Y., Takiue C., Hirata R., Takenami M., Aoi Y., Yoshioka N., Habara T., Mukaida T. & Naruse K. 2010. Improved development of mouse and human embryos using a tilting embryo culture system. Reproductive Biomedicine Online. 20(3): 358-364. 31 Basille C., Frydman R., El Aly A., Hesters L., Fanchin R., Tachdjian G., Steffann J., LeLorch M. & Achour-Frydman N. 2009. Preimplantation genetic diagnosis: state of the art. European Journal of Obstetrics & Gynecology and Reproductive Biology. 145(1): 913. 32 Perrin A., Delobel B., Andrieux J., Gosset P., Gueganic N., Petit F., De Braekeleer M. & Morel F. 2010. Molecular cytogenetic analysis by genomic hybridization to determine the cause of recurrent miscarriage. Fertility Sterility. 93(6): 2075 e3-6. 33 Simpson J.L. 2010. Preimplantation genetic diagnosis at 20 years. Prenatal Diagnosis. 30(7): 682-695.
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The Role of the Veterinarian on Human Assisted Reproduction

Andr Monteiro da Rocha1
ABSTRACT
Background: Infertility is a disease affecting 10% of the human population in reproductive age and couples experiencing difficulties to achieve pregnancy may benefit from assisted reproduction treatment. According to the Red Latinoamericana de Reproduccin Asistida, approximately 34,100 in vitro fertilization (IVF) treatments were performed in Latin America in 2007 and 42.3% (14,428) of these treatments were conducted in Brazil. Standard techniques and treatments for infertility were developed within the last 4 decades and some of them are still under technological improvements or they are frequently considered experimental or state of art technology. Usually, these treatments and techniques to overcome infertility were developed by professionals with different backgrounds including physicians, biologists, veterinarians and nurses. We aimed to review the role of veterinarians in translating animal biotechnology to human assisted procreation, developing new assisted reproduction technologies and treatments, and finally acting in clinical embryology laboratories as embryologists or supervisors. Review: Human assisted reproduction landmarks were: (i) the birth of the first in vitro fertilization (IVF) child; (ii) the implementation of intracytoplasmatic sperm injection; and, more recently, (iii) the development of successful protocols of vitrification for cryopreservation of embryos and oocytes. Veterinarians contribution and participation in the field of human assisted procreation could be distinguished between technological development and/or clinical embryology. Despite of the 3 year delay on the birth of the first in vitro fertilization calf in relation to the birth of the first IVF baby, many steps and protocols of these technological advancements relied on the knowledge acquired from animal models or biotechnologies now used for commercial purposes in animal production and breeding. Furthermore, new embryo culture technologies such as microfluidics environments and controlled atmospheres are initially evaluated in animal models. Another example of the role of veterinarians on the development of human assisted reproduction is the emergence of the widespread concept of ovarian dynamics pattern of follicular waves in monovulatory animals from Theriogenology to Reproductive Medicine in the last decade. The translation of this concept to Reproductive Medicine might allow the development of different approaches of controlled ovarian stimulation. In a human assisted reproduction clinic, the clinical embryology laboratory is responsible for selecting oocytes after transvaginal ovum pick-up, fertilizing these oocytes, cultivating the supposed zygotes until the stage of 8 cell embryos or blastocysts, and embryo preparation for transference to the uterine cavity. Several tasks enrolled in these steps of human IVF are identical to those performed by veterinarians in an animal IVF facility; however, a veterinarian must be familiar to the specific guidelines and needs of a human IVF laboratory. Conclusion: Assisted reproduction treatments are performed by groups of professionals with different academic backgrounds. Veterinarians have been playing an important roles in the development and/or adaptation of several biotechnologies utilized for human procreation; additionally, veterinarians working on animal in vitro fertilization facilities have an adequate formation and skills to act on and/or supervise clinical embryology laboratories after specific education, practice and certification.
Keywords: assisted reproduction techniques; in vitro fertilization; embryo culture; controlled ovarian stimulation.
1 Huntington Medicina Reprodutiva, So Paulo, Brazil. *Corresponding authors address: Huntington Medicina Reprodutiva, Av. Repblica do Lbano, 529, So Paulo, SP, Brazil, Postal code: 04501-000, Phone: 5511 30596122, e-mail: arocha@huntington.com.br
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I. INTRODUCTION II. ASSISTED REPRODUCTION EDUCATION IN VETERINARY MEDICINE AND MEDICINE III. THE INTERSECTION BETWEEN ANIMAL AND HUMAN ASSISTED REPRODUCTION IV. CONCLUSIONS
I. INTRODUCTION
Infertility is a disease [1] affecting 10% of the human population in reproductive age and couples experiencing difficulties to achieve pregnancy may benefit from assisted reproduction treatment. Human assisted reproduction comprises a series of methods and techniques to treat infertility from timed intercourse to in vitro fertilization (IVF) by intracytoplasmatic sperm injection (ICSI) [2]. Approximately, 55,500 human embryos were transferred in 21,285 IVF cycles in Latin America according to the 2007s Registro Latinoamericano de Reproduccin Asistida [3]. Centers for treatment of infertility located in Brazil had performed a considerable amount of these IVF cycles (14,428; 42.3%) confirming the great experience of the Brazilian infertility specialists and embryologist in ovulation induction, ovum pick-up, and clinical embryology [3]. Coincidentally, Brazil is the world leader in in vitro production (IVP) of bovine embryos. According to Sociedade Brasileira de Tecnologia de Embries there was a 27% increase in the number of IVP embryos from 2003 to 2007. Additionally, in 2007, 245,257 bovine embryos were produced in the world and the Brazilian yield accounted for 86.6% of this amount [4]. Ninety four percent of these embryos were originated from Zebu cows with technology specifically designed for these breeds [4]; thus Brazilian theriogenologist are also developing new protocols and technologies. Apparently, animal and human assisted reproduction are parallel fields; nevertheless, there are several intersections in the progress of these disciplines, and the interchange of knowledge between veterinarians practicing animal reproduction and those professionals enrolled in human reproduction such as biologists, physicians and nurses might contribute to the development of more efficient protocols and techniques in both areas.
II. ASSISTED REPRODUCTION EDUCATION IN VETERINARY MEDICINE AND MEDICINE

The Ministry of Education of Brazil established guidelines for the instruction of undergraduate students in several traditional careers, among them Veterinary Medicine. These documents determine the minimal knowledge and skills of the professionals of each area; for Veterinary Medicine, the freshly graduated professional is expected to manage and perform assisted reproduction techniques [5]. Veterinary Medicine Schools line up Theriogenology knowledge in several disciplines from physiology to biotechnology of reproduction to accommodate the Veterinary Medicine teaching to Federal guidelines. The specific duration of Theriogenology training varies from 4 to 8% of the educational duration [6-8]; however, principles of reproductive physiology and management are applied in various other subjects as in medicine and breeding of several species of companion and production animals [6-9]. Furthermore, there are several graduate student programs in Animal Reproduction and Reproductive Sciences held by outstanding Universities [10]. Conversely, the guideline for the instruction of future medical doctors determines that the students should acquire several abilities; nevertheless, it does not mention the need to treat infertility or to perform or manage assisted reproduction treatments [11]. Thus, assisted reproduction and infertility treatment comprise a minimal portion of the medical education and these issues would be addressed in the 3rd year of the medical residency in Obstetrics and Gynecology, but they are limited up to 15% of the time spent in the Obstetrics ambulatory [12]. Consequently, few physicians have received education to deal with reproductive issues and assisted reproduction.
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III. THE INTERSECTION BETWEEN ANIMAL AND HUMAN ASSISTED REPRODUCTION

Despite of the 3 year delay on the birth of the first in vitro fertilization calf in relation to the birth of the first IVF baby [13-14], many steps and protocols of these technological advancements relied on the knowledge acquired from animal models [15]. Veterinarians contribution and participation in the field of human assisted procreation could be distinguished between technological development and/or clinical embryology. New embryo culture technologies such as microfluidics environments and controlled atmospheres are initially evaluated in animal models [16-18], as well as, some quality control programs for IVF laboratories are performed with the evaluation of mouses embryo development [19]. Another example of the role of veterinarians on the development of human assisted reproduction is the emergence of the widespread concept of ovarian dynamics pattern of follicular waves in monovulatory animals from Theriogenology to Reproductive Medicine in the last decade [20-24]. The translation of this concept to Reproductive Medicine might allow the development of different approaches of controlled ovarian stimulation [25]. In human assisted reproduction clinics, the clinical embryology laboratory is responsible for preparing media and dishes, selecting oocytes after transvaginal ovum pick-up, fertilizing these oocytes, cultivating the supposed zygotes until the stage of 8 cell embryos or blastocysts, and embryo preparation for transference to the uterine cavity [26-27]. Several tasks enrolled in these steps of human IVF are identical to those used by veterinarians for commercial purposes in an animal IVF facility for production and breeding; however, a veterinarian must be familiar to the specific guidelines and needs of a human IVF laboratory [26-27]. The mutual interaction between human reproduction professionals and theriogenologists might also bring new protocols and technologies for animal reproduction, such as in oocyte cryopreservation. The successful cryopreservation by slow rate freezing of human oocyte was initially described in 1986 [28]. However, this technique had low efficiency and several improvements were developed in Italy due to legal constrains [29-34]. However, several reports indicate that vitrification of oocytes is superior to slow rate freezing [35] yielding laboratorial and clinical outcomes similar obtained with fresh oocytes [36-37]. Over than 900 normal babies were born after oocyte cryopreservation [38] and the transposition of the experience in oocyte vitrification from human reproduction to animal IVF might be beneficial and meet the commercial requirements for animal production purposes.
IV. CONCLUSIONS
Veterinarians have been playing important roles in the development and/or adaptation of several biotechnologies utilized for human procreation; additionally, veterinarians working on animal in vitro fertilization facilities have an adequate formation and skills to act on and/or supervise clinical embryology laboratories after specific education, practice and certification.
REFERENCES
1. ASRM. 2008. Practice Committee of the American Society for Reproductive Medicine: Definitions of infertility and recurrent pregnancy loss. Fertility and Sterility. 89 (6): 1603. 2. Hrometz S.L. & Gates V.A. 2009. Review of available infertility treatments. Drugs Today (Barc). 45 (4): 275-291. 3. Registro Latinoamericano de Reproduccin Asistida. 2007. 4. SBTE. 2008. Mudanas e tendncias no mercado de embries bovinos no Brasil. O Embrio. 5-7. 5. Maranho E.A., Macedo A.R. & Okida Y. 2002. Diretrizes Curriculares Nacionais do Curso de Graduao em Medicina Veterinria. C.N.D.: Educao. Dirio Oficial da Unio. 11/4. 6. NAEG. Grade Curricular-Medicina Veterinria. http://naeg.prg.usp.br/numeros/curso.phtm.14/07/2010. 7. Medicina Veterinri- Grade Curricular. http://www.fait.edu.br/principal/index.php?option=com_content&task=view&id=149&Itemid=109. 14/07/2010. 8. Curso de Graduao em Medicina Veterinria - Grade Curricular. http://www.fcav.unesp.br/graduacao1/vet/grade_curric.php. 14/07/ 2010. 9. Pfuetzenreiter M.R. & Zylbersztajn A. 2004. O ensino de sade e os currculos dos cursos de medicina veterinria: um estudo de caso. Interface - Comunicao, Sade, Educao. 8 (15): 349-360.
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10. CAPES. Relao de Cursos Recomendados e Reconhecidos. http://conteudoweb.capes.gov.br/conteudoweb/ P r o e jt o R e a lc a o C u r s o s S e r v e lt ? a c a o = p e s q u s ia r I e s & c o d g io A r e a = 5 0 5 0 0 0 0 7 & d e s c r c ia o A r e a = C I % C A N C I A S + A G R % C 1 R I A S + & d e s c r c ia o A r e a C o n h e c m ie n t o = M E D I C I N A + V E T E R I N % C 1 R I A & d e s c r c ia o A r e a A v a a ilc a o = M E D I C I N A + V E T E R I N % C 1 R I A . 11. EDUCAO C.N.D. Diretrizes Curriculares Nacionais do Curso de Graduao em Medicina. C. d. E. Superior. Dirio Oficial da Unio. 09/11/2001. 38. 12. Educao M.D. Contedos do Programa de Residncia Mdica de Obstetrcia e Ginecologia. C. N. D. R. MDICA. Dirio Oficial da Unio. 31-32. 13. Steptoe P.C. & Edwards R.G. 1978. Birth after the reimplantation of a human embryo. Lancet. 2 (8085): 366. 14. Brackett B.G., Bousquet D., Boice M.L., Donawick W.J., Evans J. F. & Dressel M.A. 1982. Normal development following in vitro fertilization in the cow. Biology of Reproduction. 27 (1): 147-58. 15. Varago F.C., Mendona L.F.& Lagares M.A. 2008. Produo in vitro de embries bovinos: estado da arte e perspectiva de uma tcnica em constante evoluo. Revista Brasisleira Reproduo Animal. 32 (2): 100-109. 16. Krisher R.L. & Wheeler M.B. 2010. Towards the use of microfluidics for individual embryo culture. Reproduction, Fertility and Development. 22 (1): 32-9. 17. Heo Y.S., Cabrera L.M., Bormann C.L., Shah C.T., Takayama S. & Smith G.D. 2010. Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Human Reproduction. 25 (3): 613-622. 18. Higdon H.L., Blackhurst D.W. & Boone W.R. 2008. Incubator management in an assisted reproductive technology laboratory. Fertility and Sterility. 89 (3): 703-710. 19. Lane M., Mitchell M., Cashman K.S., Feil D., Wakefield S. & Zander-Fox D.L. 2008. To QC or not to QC: the key to a consistent laboratory? Reproduction, Fertility and Development. 20 (1): 23-32. 20. Ginther O.J., Beg M.A., Gastal E.L., Gastal M.O., Baerwald A.R. & Pierson R.A. 2005. Systemic concentrations of hormones during the development of follicular waves in mares and women: a comparative study. Reproduction. 130 (3): 379-88. 21. Baerwald A. R. & Pierson R.A. 2004. Endometrial development in association with ovarian follicular waves during the menstrual cycle. Ultrasound in Obstetrics and Gynecoogy.. 24 (4): 453-60. 22. Ginther O.J., Gastal E.L., Gastal M.O., Bergfelt D.R., Baerwald A.R. & Pierson R.A. 2004. Comparative study of the dynamics of follicular waves in mares and women. Biology of Reproduction. 71 (4): 1195-1201. 23. Baerwald A.R., Adams G.P. & Pierson R.A. 2003. A new model for ovarian follicular development during the human menstrual cycle. Fertility and Sterility. 80 (1): 116-122. 24. Baerwald A.R., Adams G.P. & Pierson R.A. 2003. Characterization of ovarian follicular wave dynamics in women. Biology of Reproduction. 69 (3): 1023-1031. 25. Bianchi P., Serafini P., da Rocha A. M., Hassun P., da Motta E., Baruselli P. & Baracat E. 2010. Follicular Waves in the Human Ovary: A New Physiological Paradigm for Novel Ovarian Stimulation Protocols. Reproduction Science (in press). 26. 2008. Revised guidelines for human embryology and andrology laboratories. Fertility and Sterility. 90 (5): S45-59. 27. Magli M.C., Van den Abbeel E., Lundin K., Royere D., Van der Elst J.&Gianaroli L. 2008. Revised guidelines for good practice in IVF laboratories. Human Reproduction. 23 (6): 1253-1262. 28. Chen C. 1986. Pregnancy after human oocyte cryopreservation. Lancet. 1 (8486): 884-886. 29. Porcu E. 2001. Oocyte freezing. Seminars in Reproductive Medicine. 19 (3): 221-230. 30. Fabbri R., Porcu E., Marsella T., Rocchetta G., Venturoli S. & Flamigni C. 2001. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Human Reproduction.16 (3): 411-416. 31. Porcu E., Fabbri R., Damiano G., Giunchi S., Fratto R., Ciotti P.M., Venturoli S. & Flamigni C. 2000. Clinical experience and applications of oocyte cryopreservation. Molecular and Cellular Endocrinology. 169 (1-2): 33-37. 32. Fabbri R., Porcu E., Marsella T., Primavera M.R., Rocchetta G., Ciotti P.M., Magrini O., Seracchioli R., Venturoli S. & Flamigni C. 2000. Technical aspects of oocyte cryopreservation. Molecular and Cellular Endocrinology. 169 (1-2): 39-42. 33. Porcu E. 1999. Freezing of oocytes. Current Opinion in Obstetrics and Gynecology. 1 (3): 297-300. 34. Porcu E., Fabbri R., Seracchioli R., Ciotti P.M., Magrini O. & Flamigni C. 1997. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertility and Sterility. 68 (4): 724-726. 35. Smith G.D., Serafini P.C., Fioravanti J., Yadid I., Coslovsky M., Hassun P., Alegretti J. R. & Motta E.L. 2010. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertility and Sterility. (in press). 36. Cobo A., Meseguer M., Remoh J. & Pellicer A. 2010. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Human Reproduction. (in press). 37. Almodin C.G., Minguetti-Camara V.C., Paixao C. L. & Pereira P.C. 2010. Embryo development and gestation using fresh and vitrified oocytes. Human Reproduction. 25 (5): 1192-1198. 38. Noyes N., Porcu E. & Borini A. 2009. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reproductive Biomedicine Online. 18 (6): 769-776.
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Application of the Concept of Follicular Waves in Human Assisted Reproduction

Paulo Homem de Mello Bianchi
ABSTRACT
Background: Ovulation induction (OI) is one of the cornerstones in human assisted reproduction treatments. OI is based on the understanding of ovarian follicular physiology. Current knowledge of human follicular dynamics relies on hystologic observations of ovaries in distinct moments during one menstrual cycle (interval between two menses). According to this data follicles in diferent stages of maturation are seen at any time during a menstrual cycle. Initial recruitment and grow of the primordial follicles is still a poorly understood process, but continued development of antral follicles, selection of the dominant follicle and ovulation depends on follicle stimulating hormone (FSH) and luteinizing hormone (LH) serum levels, which are influenced by estradiol and progesterone concentrations. With the degeneration of the corpus luteum at the end of the luteal phase, serum estradiol and progesterone concentrations fall, leading to menstruation and an increase in FSH levels. All this observations have led to the formulation of the current theory of human follicular dynamics by which follicles continuously grow from the primordial pool randomicaly, but only those which happen to be at the antral stage (FSH sensitive) during menses (FSH elevation) may continue to develop until the pre ovulatory stage and ovulate. Accordingly, in current OI protocols for human in vitro fertilization (IVF) stimulation usually beggins 2 to 5 days after the first day of menstruation. Review: Recently a new model of human folliculogenesis, based on the occurence of follicular waves, has been described. Major development of medical ultrasound technology has allowed for the precise detection of small antral follicles in the transvaginal route. Using this technology and shifting the landmark for observations from menses to ovulation, synchronic grow of a group of antral follicles (follicular wave) has been observed two or three times in an inter ovulatory interval. Emergence of a wave is preceeded by an elevation in FSH levels. The first wave emerges around ovulation and the last is the ovulatory follicular wave. This is in accordance with previous observations in other mono ovulatory animals like the cows and mares, but in contrast with the current model of human folliculogenesis, Application of follicular wave knowledge has proved to be very usefull to the development of more effective OI protocols used in Veterinary Medicine. It has been shown that synchronization of OI with wave emergence results in more oocytes and embryos with better quality in cows. Protocols to control wave emergence are currently standart of practice for in vitro production of some animals embryos. The description of the follicular waves in humans gives the opportunity to develop new OI protocols for IVF. Synchronization of OI with wave emergence could be achieved through mechanical (aspiration of the dominant follicle folowed by ovarian stimulation) or chemical interventions (initiate ovulation or using high doses of estradiol and progesterone in any phase of the cycle, followed by ovulation induction). However some differences between in vitro production of animal embryos and Human IVF should be enfasized and could challenge the application of this knowledge. For instance, Human embryos are usually obtained and transfered to the same subject, therefore a synchronization of embryo and endometrial development is a major concern. Conclusions: Description of follicular waves in Human ovaries could have profound implications in OI for IVF, but some challenges should be expected to transpose the animal model to the Human setting.
Keywords: Human follicular waves; ovulation induction; in vitro fertilization.
Centro de Reproduo Humana do Hospital das Clnicas da Faculdade de Medicina da USP, Huntington Centro de Medicina Reprodutiva. CORRESPONDENCE: Avenida Repblica do Lbano 529, Ibirapuera So Paulo SP Brasil, CEP 04501000, Email: paulobianchi35@gmail.com, pbianchi@huntington.com.br
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I. INTRODUCTION II. CURRENT THEORY OF HUMAN FOLLICULOGENESIS AND OVULATION INDUCTION PROTOCOLS III. DESCRIPTION OF THE FOLLICULAR WAVES IN THE HUMAN OVARIES IV. POSSIBLE APLICATIONS OF THE FOLLICULAR WAVE CONCEPT TO HUMAN ASSISTED REPRODUCTION V. DISCUSSION AND CHALLENGES
I. INTRODUCTION
Despite much development in laboratory techniques in the past years, ovulation induction is still one of the cornerstones in assisted reproduction treatments in humans [10]. The proper use of exogenous gonadotropins to increase the number of mature oocytes available at one time relies on the understanding of key events of reproductive physiology. In this article we are going to review the theory of human ovarian follicular dynamics on which current ovulation induction protocols are based, indicate evidence of the occurence of follicular waves in human ovaries and speculate on the impact of this alternative physiologic paradigm to ovulation induction protocols and human assisted reproduction.
II. CURRENT THEORY OF HUMAN FOLLICULOGENESIS AND OVULATION INDUCTION PROTOCOLS

Since menses are a very distinct event during the womens reproductive years, menstuation has been used as a landmark in the studies of the female reproductive system. Cyclic changes in reproductive organs, tissues and sexual hormones between two menses are called the menstrual cycle. Didactically there are two distinct phases during an intermenstrual interval. In the follicular phase, just after menses, follicles grow, produce estradiol and ultimately ovulate. The ovulated follicle becomes the corpus luteum, which produces progesterone and dominates the next phase, the lutheal phase. If pregnancy did not occur, corpus luteum degenerate in fourteen days, hormonal levels fall and a new cycle begins [25]. The current theory of human follicular dynamics is derived from hystologic observations of excised ovaries in different moments during the menstrual cycle [16] together with serum and follicular fluid steroid measurements of women and animal models in several physiologic and pathologic conditions [11]. Germ cells can be identified in ovaries of human embryos since the 16th week of gestation. Through mitotic divisions the number of germ cells in the ovaries reach its maximum at around 20 weeks of gestational age (maximal ovarian reserve) [25]. Germ cells in the fetal ovaries are surrounded by flattened cells from the germinative epithelium (early granulosa cells) constituting the primordial follicle. From the 20th week of gestational age on mitotic activity of germ cells in the fetal ovaries ends and their number decrease progressively [11]. Gradually primordial follicles activate and beggin the process of maturation. Granulosa cells become cuboidal and proliferate to form a multilayer surrunding the oocyte [11,17]. Stromal cells adjacent to the granulosa transform to constitute the theca, early sensitive to the actions of the luteinizing hormone (LH) [9]. Later a cavity is formed in the center of the follicle, the antrum, which separates two groups of granulosa cells, the cumulus oophorus, surrunding the oocyte, and the parietal cells, close to the theca [11]. At this stage granulosa cells acquire follicle stimulating hormone (FSH) receptors [18,9]. A few follicles are abble to progress beyond this point, reach full maturation (with a big antrum) and finally ovulate. The great majority of follicles actually suffer atresia through apoptosis [11]. Primordial follicles that beggin the process of maturation during the fetal stage and childhood are fated to suffer atresia in the early stages of the process [19]. Only during the menacme (reproductive years) a small proprotion of follicles are able to reach full maturation and ovulate.
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Morphometric studies showed that ovarian follicles in diferent stages of development are present throughout the menstrual cycle [16]. The transition from primordial to mature follicles takes several months [19]. The mechanisms responsible for the activation of the primordial follicles and beggining of follicular development are still unknown, but it appears that the initial steps of folliculogenesis are not under the controll of gonadotropins [24]. From this observations it has been postulated that activation of the primordial follicle and development until the early antral stage occurs continuously and randomly [11]. Final follicular development (from the early antral stage on) and ovulation, on the other hand, is clearly dependent on FSH and LH actions [11]. Follicles in the antral stage demand different concentrations of FSH to continue to grow [11,8,26]. FSH concentration increase above the stimulatory threshold in the late lutheal phase [20]; at this time follicles that happen to be in the antral stage grow and produce estradiol. Estradiol has a negative feedback effect on the production and secretion of FSH and ultimately FSH concentration fall below the threshold again. The time period in which FSH concentrations are above the stimulatory threshold is called FSH window [11]; during this period all follicles that happen to be FSH sensitive (antral stage), are able to grow. When FSH concentrations fall below the threshold and the FSH window is closed, usually just one follicle continues to grow, becoming the dominant follicle, and eventualy ovulates. Actually this follicle is sensitive to lower concentrations of FSH [11] and, during the common follicular growh phase, its granulosa cells also acquire LH receptors [27]. Since during the follicular phase small concentrations of LH are present, the dominant follicle is able to further develop also under the stimulation of LH. This is the mechanism of dominance responsible for the selection of just one follicle to complete the development and ovulate each cycle [11]. Based on the observations above it has been postulated that primordial follicles continuously and ramdomly activate and start to grow since the intra uterine life until menopause [16]. Most follicles suffer atresia but those, which happen to be sensitive to FSH (antral stage) during the rise in FSH concentrations, that occurs in the late lutheal and early follicular phases, continue to grow and eventualy ovulate. This model of folliculogenesis has been called the Propitious Moment Theory [2]. Final follicular development until ovulation is expected to occur only during the follicular phase of the cycle [25]; the presence of the corpus luteum after ovulation is thought to inhibit follicle development to a mature follicle. This model of folliculogenesis has been the base for current ovulation induction protocols in human assisted reproduction treatments. Administration of exogenous gonadotropins takes place in the early follicular phase, beggining more precisely between the second and fifth day of the menstral cycle. The objective is to extend the FSH window allowing for more follicles to complete all stages of development and produce a greater number of mature oocytes for fertilization [11].
III. DESCRIPTION OF FOLLICULAR WAVES IN THE HUMAN OVARIES

Ultrasound is a valuable tool to investigate folliculogenesis since it allows for noninvasive, in vivo and dynamic imaging of the ovaries, contrasting with the still images provided by hystological evaluations. With the development of high definition ultrasound technology, currently it is possible to see follicles as small as 2 mm in diameter, through the transvaginal route. Intrigued by previous ultrasound observations of follicular activity during the luteal phase, which challenged the prevailing theory of human folliculogenesis, Dr Angela Baerwald and co-authors decided to investigate ovarian phisiology using serial high definition transvaginal ultrasound and serum hormonal determinations in an interovulatory interval, instead of an intermenstrual interval, of fifty healthy, normo ovulatory women [3,4]. Changing the landmark for observations from the menstruation to the ovulation and using serial ultrasound imaging provides a dynamic, ovarian centered view of folliculogenesis. Their data shown that follicles rather develop in coodinated and synchronic groups apearing two (68% of women) or three (32% of women) times during an interovulatory interval [3]. The first group of folicles starts to grow on the day of ovulation; the interovulatory interval is shorter for women with two groups of follicular development comparing to those with three; in the last group a follicle always ovulate [3,4]. The coordinated growth of a coohort of follicles has been previously described in other mono ovulatoy animals, like the equines and bovines, and has been called follicular waves [12,13,14,15, 21].
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Wave emergence is preceeded by a rise in FSH concentrations. Selection of the dominant follicle occured when a follicle reached 10 mm or more in diameter; it only takes a few days from wave emergence to selection of the dominant follicle. Only a small proportion (15%) of women with two wave pattern had a dominan follicle in the first wave. Nineteen percent of women with three waves pattern had a dominant follicle in the second and third wave and another 19% had a dominant follicle in all waves. Dominant follicles of anovulatory waves didnt reach an ovulatory diameter (20 22 mm). The interval between wave 1 and 2 in women with two waves pattern was approximately 1415 days [3,4], meaning that the last follicular wave emerges just before or in the begining of menstruation. In women with three waves pattern the second wave emerges 11-13 days after the first [3,4] (before menstruation) and the last one emerges 6 7 days after the second [3,4] (after menstruation). All this observations are in accordance with studies in other mono ovulatory animal species like the equines and bovines [12-15]. On the other hand it is in sharp contrast with the current theory of random follicular growth; moreover, it shows that follicular development occurs also in the luteal phase, not only in the follicular phase as thought until now.
IV. POSSIBLE APLICATIONS OF THE FOLLICULAR WAVE CONCEPT TO HUMAN ASSISTED REPRODUCTION
The follicular wave concept has long been used in Veterinary Medicine for in vitro production of animal embryos, particularly in cows. It has been shown that when ovarian stimulation with exogenous gonadotropins starts at the beggining of a follicular wave, there are more mature oocytes at the egg retrieval and embryos are of better quality [23,1]. Detection of ovulation and consequently the emergence of the first wave would be the most obvious approach, but since ovulation detection is a time consuming process considering a herd, protocols to synchronize wave emergence with initiation of gonadotropin administration have been developed. There are mechanical and pharmacological strategies to determine wave emergence. The mechanical strategy relies on the ablation of the dominant follicle before ovulation through aspiration. Shortly after aspiration serum concentrations of estradiol and inhibin fall; without the inhibitory effect of estradiol and inhibin on the hypothalamus and pituitary, FSH concentrations rise followed by a wave emergence [5]. However, this strategy is not common in theriogenology because it is an invasive and time consuming procedure [22]. The pharmacological strategy consists in administer a boost of parenteral estradiol and progesterone anytime during the estrous cycle. The consequent rise in serum concentrations of estradiol and progesteone lowers both FSH and LH concentrations, causing atresia of the dominant follicle and, in the lutheal phase, lutheolysis. In either scenario, after metabolization of the exogenous estradiol, endogenous FSH rises initiating a new follicular wave [7]. Therefore ovulation induction could be initiated four days after the hormonal boost [22]. Current ovulation induction protocols for human assisted reproduction, based on the prevailing theory of human folliculogenesis, are not synchronized with follicular wave emergence but rather with menstruation. Starting gonodotropin administration in the second to fifth day of the menstrual cycle probably does not coincide with the begining of a follicular wave, particularlly for women with a three wave pattern. Actually, by that time several women have probably already selected a dominant follicle and the subordinate ones could have initiated the atresia process. Since the follicular wave phenomenon in humans appear to be very similar to the cows, similar protocols to control wave emergence and synchronize it to the begining of ovarian stimulation could theoretically increase the number of mature oocytes, reduce the requirements of exogenous gonadotropins and improve embryo quality, and consequently pregnancy rates. We have recently published a rewiew article where we speculate on possible protocols for synchronize ovulation induction with wave emergence in humans [6]. Briefly, aspiration of the dominant follicle (or induction of ovulation with hCG) followed some days after by gonadotropins administration are probably the easier strategies to do this synchronization, but rely on the identification of a clearly dominant follicle on the follicular phase of the cycle. Conversely, exogenous estradiol and progesterone boost is expected to be a more flexible strategy since it could be used anytime during the cycle, but concerns might rise regarding possible side effects of a hormonal boost and optimal timing and hormonal requirements to induce wave emergence [6].
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V. DISCUSSION AND CHALLENGES

Application of the follicular wave concept in human assisted reproduction is very promising based on the results observed in farm animals. A particular useful application of this concept is for fertility preservation in women with cancer, when the time span between diagnosis and initiation of chemotherapy or radiotherapy often is not sufficient to wait for menstruation and perform a conventional ovulation induction protocol. Howerver some challenges and differences between human treatments and animals should be discussed. Assisted reproduction in animals, particularly in cows, is intended to produce an elevate number of animals with high genetic potencial. Donnor animals are usually young and healthy individuals; embryos produced are transfered to several recipients in order to increase the number of descendants at each time. In humans, on the other hand, assited reproduction is intended to help patients with some degree of subfertility/infertility; embryos are often transfered to the same woman that was submitted to ovarian stimulation. This carateristics have two main consequences: first, results of treatment could be inferior in women comparing to animals; second, concerns should be raised with the endometrial quality of the patient. Ovulation induction synchonized with wave emergence could be assynchronous to the endometrial development with possible impairment of implantation. Vitrification of embryos for posterior endometrial preparation and transference is an option if endometrium assynchrony occurs. Protocols to determine follicular wave emergence based on animal models have to be tested in humans. For instance if aspiration of the dominant follicle occurs before complete dominance has been stablished, ovulation induction can cause the subordinate follicles in the same wave to grow and not a new wave to emerge. The exact size of the dominant follicle when complete dominance is stablished in humans should be investigated to avoid this situation. Moreover, the interval between follicle aspiration and wave emergence in humans should be determined to set the ovulation induction. The dose and duration of administration of exogenous steroids to cause atresia of the dominant follicle and lutheolysis should also be determined, as long as the time interval between steroids administration and wave emergence. Also, side effects of such pharmacological manipulation should also be weighted in cost benefits analisys before it become standard of practice.
REFERENCES
1. Adams G.P. 1994. Control of ovarian follicular wave dynamics in cattle: implications for synchronization and superstimulation. Theriogenology 41: 19-24. 2. Adams G.P. & Jaiswal R. 2008. Follicular dynamics in cattle: Historical overview and research update. Acta Scientiae Veterinariae 36: 387-396. 3. Baerwald A.R., Adams G.P. & Pierson R.A. 2003. A new model dor ovarian follicular development during the human menstrual cycle. Fertility and Sterility. 80 (1): 116-122. 4. Baerwald A.R., Adams G.P. & Pierson R.A. 2003. Characterization of ovarian follicular wave dynamics in women. Biology ofl Reproduction. 69: 1023-1031. 5. Bergfelt D.R., Bo G.A., Mapletoft R.J. & Adams G.P. 1997. Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle. Animal Reproduction Science.. 49(1):1-12. 6. Bianchi P., Serafini P., da Rocha A.M., Hassun P., da Motta E., Baruselli P. & Baracat E. 2010. Follicular Waves in the Human Ovary: A New Physiological Paradigm for Novel Ovarian Stimulation Protocols. Reproduction Science. (in press). 7. Bo G.A., Adams G.P., Pierson R.A. & Mapletoft R.J. 1996. Effect of progestogen plus estradiol-17beta treatment on superovulatory response in beef cattle. Theriogenology 45: 897-910. 8. Brown J.B. 1978. Pituitary control of ovarian function: concepts derived from gonadotrophin therapy. Australian and New Zealand Journal of Obstetrics and Gynaecology. 18: 47-54. 9. Channing C.P. & Kammerman S. 1973. Characteristics of gonadotropin receptors of procrine granulosa cells during follicle maturation. Endocrinology 92: 531-540. 10. Control CfD. 2005 Assisted Reproductive Technology (ART) Report. Atlanta. 98 p. 11. Fauser B.C.J.M. & Van Heusdn A.M. 1997. Manipulation of Human Ovarian Function: Physiological Concepts an Clinical Consequences. Endocrine Reviews. 18 (1): 71-106. 12. Ginther O.J. 1990. Folliculogenesis during the transitional period and early ovulatory season in mares. Journal of Reproduction and Fertility. 90(1):311-320.
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13. Ginther O.J. & Bergfelt D.R. 1993. Growth of small follicles and concentrations of FSH during the equine oestrous cycle. Journal of Reproduction and Fertility. 99(1):105-111. 14. Ginther O.J., Beg M.A., Donadeu F.X. & Bergfelt D.R. 2003. Mechanism of follicle deviation in monovular farm species. Animal Reproduction Science. 78: 239-257. 15. Ginther O.J., Gastal E.L., Gastal M.O., Bergfelt D.R., Baerwald A.R. & Pierson R.A. 2004. Comparative study of the dynamics of follicular waves in mares and women. Biology of Reproduction. 71(4): 1195-1201. 16. Gougeon A. 1986. Dynamics of follicular growth in the human: a model from preliminary results. Human Reproduction. 1: 81-87. 17. Gougeon A. & Chainy G.B. 1987. Morphometric studies of small follicles in ovaries of women at different ages. Journal of Reproduction and Fertility. 81: 433-442. 18. Gougeon A. 1993. Dynamics of human follicle growth. A morphologic perspective. In: Adashi E.., Leung P.C.K. (eds) The ovary. New York. Raven Press, pp 21-39. 19. Gougeon A. 1996. Regulation of ovarian follicular development in primates: facts and hypothesis. Endocrine Reviews. 17: 121-155. 20. Hall J.E., Schoenfeld D.A., Martin K.A. & Crowley W.F. 1992. Hpothalamic gonadotropin-releasing hormone secretion and folliclestimulating hormone dynamics during the lutheal-folicular transition. Journal of Clinical Endocrinology & Metabolism. 74: 600-607. 21. Knopf L., Kastelic J.P., Schallenberger E. & Ginther O.J. 1989. Ovarian follicular dynamics in heifers: test of two-wave hypothesis by ultrasonically monitoring individual follicles. Domestic Animal Endocrinology. 6(2):111-119. 22. Mapletoft R.J., Bo G.A. & Baruselli P.S. 2009. Control of ovarian function for assisted reproductive technologies in cattle Animal Reproduction. 6: 114-124. 23. Nasser L.F., Adams G.P., Bo G.A. & Mapletoft R.J. 1993. Ovarian superstimulatory response relative to follicular wave emergence in heifers. Theriogenology 40: 713-724. 24. Peters H., Himelstein-Braw R & Faber M. 1976. The normal development of the ovar in childhood. Acta Endrocrinol (Copenh) 82: 617630. 25. Speroff L. & Fritz M.A. 2005. Regulation of the menstrual cycle. In: Clinical Gynecologic Endocrinology and Infertility. Philadelphia. Lippincott Williams & Wilkins, pp187-232. 26. Van Weissenbruch M.M., Schoemaker H.C., Drexhage H.A. & Shoemaker. 1993. Pharmaco-dynamics of human menopausal gonadotropin (hMG) and follicle-stimulating hormone (FSH). The importance of the FSH concentration in initiating follicular growth in polycystic ovary-like disease. Human Reproduction. 8: 813-821. 27. Yong E.L., Baird D.T., Yates R., Reichert Jr L.E. & Hillier S.G. 1992. Hormonal regulation of the growth and steroidogenic fuctions of human granulosa cells. Journal of Clinical Endocrinology & Metabolism. 74: 842-849.
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Diagnstico Gentico Pr-Implantacional

Juliana Fabricia Cuzzi1, Tnia Maria Vulcani-Freitas1, Priscila Cristina Rodrigues Motta1 & Pricles Assad Hassun Filho1*
ABSTRACT
Background: The ranks of patients seeking preimplantation genetic diagnosis (PGD) to identify embryos with monogenic disorders like cystic fibrosis or thalassemia are growing rapidly. Even so, the most common indication for preimplantation embryo testing remains the risk of chromosomal imbalance. In most cases, the PGD strategy employed for chromosomal testing involves biopsying a single cell (blastomere) from each embryo at the 6 to 10-cell stage, 3 days after fertilization. The cell is placed on a microscope slide, fixed, and then subjected to cytogenetic analysis. While the biopsied cell is being analyzed, the rest of the embryo is maintained in culture. Most infertility clinics prefer to transfer the embryos no later than day-5 post fertilization. Consequently, PGD methods need to be extremely rapid, providing a result in less than 48 hours. Review: Most chromosomal PGD protocols employ fluorescence in situ hybridization (FISH). This approach involves the hybridization of chromosome specific DNA probes, labeled with different colors, to nuclei spread on a microscope slide. The method is rapid, performs equally well whether applied to interphase nuclei, and permits enumeration of up to 10 chromosomes per cell. Initially, the PGD for aneuploidy was envisioned that most of the patients seeking PGD for aneuploidy would be those who carry a chromosomal rearrangement. Couples in which one of the partners carries a chromosomal rearrangement frequently experience miscarriage or bear children due to chromosome imbalance. However, in recent years the vast majority of patients requesting PGD for aneuploidy have in fact been chromosomally normal individuals undergoing IVF. Early methods employed five FISH probes for PGD, focusing on the chromosomes most often found to be abnormal in prenatal samples (13, 18, 21, X, and Y). Aneuploidies for these chromosomes are sometimes compatible with survival to term, leading to aneuploid syndromes. This strategy was successful in reducing the number of such syndromes, but a statistically significant improvement in embryo implantation could not be shown. Later PGD studies expanded the number of chromosomes assessed to eight. This was achieved by performing two sequential rounds of FISH analysis, assessing five chromosomes in the first round and three more in the second. The three new chromosomes added to the PGD screen (15, 16, and 22) are frequently found to be aneuploid in miscarriages. The eight-chromosome PGD protocol led to a doubling of embryo implantation rates in two separate studies and reduced the number of spontaneous abortions. Although this problem can be partially overcome by performing sequential FISH experiments on the same cell, the accuracy of the method declines with each additional hybridization. The good news is that there is a method related to FISHcomparative genomic hybridization (CGH) that can detect aneuploidy that affects any chromosome within a sample. In addition to offering comprehensive detection, CGH can be used on interphase cells. Conclusion: In summary, the use of FISH for the purpose of PGD has already improved IVF outcome for several groups of infertile patients, including women aged 37 and above, those with recurrent miscarriages, women with a previous comprehensive screening for aneuploidy will likely further increase the benefit of PGD to these patients and maybe even a broader range of patients. The widespread availability of CGH for PGD is nearly at hand, but while we await final refinements and validation, PGD strategies can still be improved by making the best use of current methods and reassessing the chromosomes selected for screening.
Keywords: PGD, aneuploidy, FISH, CGH.
Genesis Genetics Brasil, So Paulo SP, Brasil. *Autor para correspondncia: Pricles Assad Hassun Filho, E-mail: pericles.hassun@gmail.com ou pericles@genesisgenetics.com.br, Rua Mato Grosso, 306 conjunto 506, Higienpolis, So Paulo SP, Brasil, CEP 01239-040
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I. INTRODUO
a. Diagnstico Gentico Pr-Implantacional (PGD) b. Aplicaes do Diagnstico Gentico Pr- Implantacional
i. Anlise do Primeiro Corpsculo Polar ii. Anlise de Blastmeros iii. Anlise de Blastocistos
c. Screening para Aneuploidias (PGS)
II. TCNICAS DE CITOGENTICA-MOLECULAR III. HIBRIDAO GENMICA COMPARATIVA (CGH) IV. CONCLUSO
I. INTRODUO
a. Diagnstico Gentico Pr-Implantacional (DGP) Estudos citogenticos por meio da tcnica de FISH mostraram que mais de 50% dos embries em estgio pr-implantacional possuem clulas aneuplides [1]. Considerando que a maioria dessas alteraes impede o desenvolvimento embrionrio e conseqente implantao, a identificao de embries geneticamente normais crucial para a obteno de gestaes viveis [2]. A identificao confivel de embries euplides (cromossomicamente normais) s pode ser determinada pelo Diagnstico Gentico Pr-implantacional (PGD). O PGD envolve a interao entre as tcnicas de reproduo assistida, a bipsia de blastmero ou do corpsculo polar e posterior anlise da clula nica, possibilitando o diagnstico de doenas geneticamente herdadas em embries humanos [3,4]. A aplicao clnica desse mtodo proporciona aos casais com alto risco reprodutivo, maiores chances de terem filhos no afetados, identificando os embries portadores de alteraes cromossmicas e evitando que os mesmos sejam transferidos. Segundo Ludwig e colaboradores (2001), a tcnica permite diminuir o risco de transmisso de alteraes genticas em mais de 95% [5]. Alm de evitar descendentes afetados pelas principais Sndromes Genticas, o diagnstico pr-implantacional uma alternativa para o diagnstico pr-natal, principalmente nos pases em que o aborto teraputico no permitido. Os primeiros casos de Diagnstico Gentico Pr - Implantacional em embries humanos foram descritos independentemente por Handyside e colaboradores (1990) e Verlinsky e colaboradores (1990) [3,4]. Desde as primeiras tentativas na dcada de noventa, centenas de PGD tm sido realizados, demonstrando uma crescente aceitao da metodologia por diferentes populaes. De acordo com a classificao determinada pela ESHRE PGD Consortium, o diagnstico gentico pr-implantacional deve ser dividido em duas categorias de acordo com as caractersticas do casal: (1) PGD de alto risco, que inclui casais com anomalias cromossmicas e/ ou doenas monognicas; (2) PGD de baixo risco, que tem por objetivo aumentar a taxa de implantao em casais com idade materna elevada (em torno de 37 anos), perdas fetais de repetio ou com freqentes falhas de fertilizao in vitro. Atualmente, sua indicao mais comum o screening para aneuploidias em embries gerados por mes com idade acima de 37 anos [6]. b. Aplicaes do Diagnstico Gentico Pr-Implantacional
i. Anlise do Primeiro Corpsculo Polar

Na gametognese feminina, durante a primeira diviso meitica, um dos cromossomos homlogos de cada bivalente segrega para o primeiro corpsculo polar (1CP) e o outro para o ncleo do ocito (MII) que se encontra em metfase II da meiose. Desta forma, conclui-se que o 1CP tem o contedo gentico complementar ao ncleo
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MII, o que significa dizer que um desequilbrio em MII implicar na alterao recproca no corpsculo polar. Considerando que o 1CP por sofrer extruso no tem funo reprodutiva, a sua bipsia e posterior anlise permitem a caracterizao indireta da constituio cromossmica do MII e a identificao dos ocitos aneuplides [7] sem comprometer a viabilidade do gameta.
ii. Anlise de Blastmeros

Trata-se da metodologia mais empregada no PGD [3,8]. Esse procedimento envolve a dissoluo de uma regio da zona pelcida e a aspirao de uma ou, no mximo, duas clulas embrionrias. A remoo de um blastmero em embrio com o equivalente de 6-8 clulas parece no afetar o seu desenvolvimento, in vitro, para o estgio de blastocisto sendo considerada uma tcnica eficiente. A vantagem mais evidente que, nesta metodologia, se analisa tanto a contribuio paterna quanto a materna, pois neste estgio a constituio gentica do embrio est completamente formada; tornando o PGD em blastmeros um mtodo comparvel ao diagnstico pr-natal. Por outro lado, a desvantagem desta tcnica a coexistncia de mais de uma linhagem celular no embrio (linhagens cromossomicamente normais e linhagens aneuplides), fenmeno chamado de mosaicismo embrionrio, que tem origem ps-zigtica e que tem sido detectado em uma porcentagem considervel de embries [9]. A presena deste mosaicismo dificulta o diagnstico citogentico pr-implantacional em blastmeros [10], sobretudo nos casos em que apenas uma clula analisada. Em 2004, Sermon e colaboradores descreveram a necessidade de um estudo prospectivo e randomizado que comparasse a viabilidade embrionria quando realizada a bipsia de uma ou duas clulas e a incidncia de erros no diagnstico citogentico dos blastmeros [11]. Atualmente esta questo parece, ainda que de forma preliminar, estar solucionada. Goossens e colaboradores (2008), aps analisarem 3377 embries, concluram que a remoo de 2 blastmeros interferia de forma negativa no desenvolvimento embrionrio at o estgio de blastocisto, enquanto a eficincia do diagnstico por FISH no se alterava [12].
iii. Anlise de Blastocistos

Este tipo de bipsia a mais tardia que se pode proceder. Consiste em cultivar o embrio in vitro at alcanar o estgio de blastocisto (aproximadamente 5 dias aps a fecundao) e remover de 10 a 30 clulas. As vantagens so: proporcionar a anlise de diversas clulas e a seleo dos embries mais viveis pela manuteno do seu cultivo at a fase de blastocisto [13]. A principal desvantagem que no restam mais do que poucas horas aps a bipsia para a realizao do diagnstico, j que segundo o informe do ESHRE PGD Consortium Steering Commitee (2004), a transferncia embrionria deve ser realizada no mximo no 6 dia ps-fecundao. Alm disso, muitos centros de reproduo assistida tm dificuldades para cultivar os embries in vitro at blastocisto e, por esta razo, so poucos os centros no mundo que praticam esta forma de bipsia [14]. c. Screening para Aneuploidias (PGS) a aplicao mais freqente do PGD. As anomalias cromossmicas numricas esto relacionadas tanto com falhas de implantao quanto com morte e perda embrionria resultando em abortos espontneos. A incidncia de anomalias cromossmicas de 1,4% em embries de mes com at 34 anos de idade e aumenta para 52,4% em mulheres entre 40-47 anos [15]. Da mesma forma, tambm foi detectada uma incidncia elevada de alteraes cromossmicas em embries de casais jovens com mais de trs falhas de implantao (>55% de embries so aneuplides) e em casais com pelo menos dois abortos espontneos (>68%) [16]. Shahine & Cedars (2006) questionaram se a aplicao do PGS em gametas e embries poderia ajudar a aumentar as taxas de implantao e gestao em casais com idade materna elevada, em casais com falhas recorrentes de implantao, em mulheres que apresentam abortos espontneos de repetio e tambm em casais portadores de translocao equilibrada para detectar alteraes dos cromossomos envolvidos no rearranjo [17]. Baseados em dados de 20.000 ciclos de PGD, Kuliev & Verlinsky (2008) relataram que o efeito positivo do PGS evidente quando comparadas as taxas de sucesso reprodutivo dos mesmos pacientes, antes e depois da realizao do diagnstico [18].
II. TCNICAS DE CITOGNTICA-MOLECULAR

O uso das tcnicas de citogentica molecular tem favorecido os estudos em embries humanos. Baseadas na utilizao de diversos tipos de sondas de DNA (centromricas, loci, especficas ou pinturas cromossmicas) e associadas a diferentes fluorocromos, essas metodologias vm se aprimorando com a evoluo dos microscpios
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e dos sistemas de captura. Atualmente, as tcnicas de citogentica molecular constituem tanto protocolos de pesquisa quanto de diagnstico, adicionando sensibilidade e especificidade aos resultados obtidos por citogentica convencional. A hibridao in situ fluorescente (FISH) se tornou a tcnica padro para a avaliao de aneuploidias, sendo possvel analisar de duas a nove sondas cromossomo-especficas divididas em uma, duas ou trs etapas de hibridao [19]. Cabe destacar que o nmero de cromossomos que pode ser analisado em cada etapa est ligado ao nmero limitado de fluorocromos disponveis e possibilidade de sobreposio dos sinais [2,20]. Em resumo, em uma situao ideal, possvel analisar somente cinco cromossomos em cada hibridao. Alm disso, a quantidade de vezes que o material gentico pode ser hibridado tambm limitada, sendo observada perda considervel na qualidade da amostra a partir da terceira denaturao [21]. Entretanto, o parmetro tcnico mais desfavorvel que a metodologia empregada requer a fixao dos ncleos em lminas de vidro, o que aumenta o risco de perda cromossmica por artefato de tcnica. Em geral, as tcnicas que envolvem fixao celular em lminas so eficientes em diagnosticar casos de hiperploidia, falhando na deteco de hipoploidias. Com o intuito de superar esta limitao tcnica, Wells e colaboradores (1999) e Wells & Delhanty (2000) descreveram e aprimoraram a aplicao da Hibridao Genmica Comparativa (CGH) [24] em clula nica [22,23].
III. HIBRIDAO GENMICA COMPARATIVA

Esta metodologia permite detectar desequilbrios no nmero de cpias de qualquer um dos 23 pares cromossmicos em apenas uma hibridao. A primeira aplicao da CGH no diagnstico pr-implantacional foi descrita em blastmeros [25]. De acordo com a literatura, estudos por meio da CGH revelaram que de 25 a 30% dos embries diagnosticados como normais aps anlise de FISH para nove cromossomos apresentavam aneuploidias para outros cromossomos [26]. Estes dados enfatizaram a importncia da anlise dos 23 pares cromossmicos na eficincia do diagnstico pr-implantacional. A busca por um PGD altamente eficaz no apenas evita o nascimento de crianas afetadas por cromossomopatias como tambm favorece a transferncia de um nico embrio para o tero materno, contribuindo com a diminuio dos riscos envolvimentos em gestaes gemelares. Atualmente a aplicao da CGH no diagnstico pr-implantacional tem sido muito discutida e, o que parece ser consenso entre os pesquisadores europeus e norte-americanos que o screening para aneuploidias, por meio desta tcnica, favorece a implantao embrionria aumentando assim as taxas gestacionais, independentemente se o diagnstico foi realizado no ocito ou no embrio de terceiro dia ou at mesmo blastocisto [27].
V. CONCLUSO
A aplicao da FISH no diagnostic pr-implantacional tem favorecido o sucesso dos tratamentos de reproduo assistida para vrios grupos de paciente infrteis, incluindo idade maternal avanada e abortos de recorrncia. No entanto, a aplicao de um screening completo para aneuploidias permitiria que o PGD fosse realizado de forma mais efetiva e abrangente, beneficiando um nmero maior de casais. A uso definitivo da CGH no PGD est muito prximo de acontecer, mas enquanto esta metodologia espera pelas ltimas validaes, as estratgias adotadas hoje para o PGD ainda podem ser aprimoradas, principalmente no que diz respeito escolha dos cromossomos selecionados para o screening.
REFERNCIAS
1. Munn S, Scott R, Sable D & Cohen J. 1998. First pregnancies after preconception diagnosis of translocations of maternal origin. Fertility and Sterility. 69: 675-681. 2. Wells D. & Levy B. 2003. Cytogenetics in reproductive medicine: the contribution of comparative genomic hybridization (CGH). Bioessays. 25: 289-300 3. Handyside A.H., Kontogianni E.H., Hardy K. & Winston R.M. 1990. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 344: 768-770. 4. Verlinsky Y., Ginsberg N., Lifchez A., Valle J., Moise J. & Strom C.M. 1990. Analysis of the first polar body: preconception genetic diagnosis. Human Reproduction. 5: 826-829.
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5. Ludwig M., Diedrich K. & Schwinger E. 2001. Preimplantation genetic diagnosis: the German situation. Trends in Genetic. 17: 473-474. 6. Sermon K.D., Michiels A., Harton G., Moutou C., Repping S., Scriven P.N., SenGupta S., Traeger-Synodinos J., Vesela K. & Viville S. 2007. ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004. Human Reproduction. 22: 323-336. 7. Gitlin S.A., Gibbons W.E. & Gosden R.G. 2003. Oocyte biology and genetics revelations from polar bodies. Reproduction Biomedicine Online. 6: 403-409. 8. Sermon K., Moutou C., Harper J., Geraedts J., Scriven P., Wilton L., Magli M.C., Michiels A., Viville S. & De Die C. 2005. ESHRE PGD Consortium data collection IV: May-December 2001. Human Reproduction. 20:19-34. 9. Malmgren H., Sahlen S., Inzunza J., Aho M., Rosenlund B., Fridstrom M., Hovatta O., Ahrlund-Richter L., Nordenskjold M. & Blennow E. 2002. Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations. Molecular Human Reproduction. 8: 502-510 10. Munne S., Cohen J. & Sable D. 2002. Preimplantation genetic diagnosis for advanced maternal age and other indications. Fertility and Sterility. 78: 234-236. 11. Sermon K., Van Steirteghem A. & Liebaers I. 2004. Preimplantation genetic diagnosis. Lance. 363: 1633-1641. 12. Goossens V., De Rycke M., De Vos A., Staessen C., Michiels A., Verpoest W., Van Steirteghem A., Bertrand C., Liebaers I. & Devroey P. 2008. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Human Reproduction. 23: 481-492 13. Sandalinas M., Sadowy S., Alikani M., Calderon G., Cohen J. & Munne S. 2001. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Human Reproduction. 16: 1954-1958. 14. de Boer K.A., Catt J.W., Jansen R.P., Leigh D. & McArthur S. 2004. Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertility and Sterility. 82: 295-298. 15. Mrquez C, Sandalinas M., Bahe M., Alikani M. & Munn S. 2000. Chromosome abnormalities in 1255 cleavage-stage human embryos. Reproduction Biomedicine Online 1(1):17-26. 16. Gianaroli L., Magli M.C. & Ferraretti A.P. 2001. The in vivo and in vitro efficiency and efficacy of PGD for aneuploidy. Molecular and Cellular Endocrinology. 183 (1): S13-18. 17. Shahine L.K. & Cedars M.I. 2006. Preimplantation genetic diagnosis does not increase pregnancy rates in patients at risk for aneuploidy. Fertililty and Sterility. 85: 51-56. 18. Kuliev A. & Verlinsky Y. 2008. Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome. Reproduction Biomedicine Online 16: 9-10. 19. Pujol A., Durban M., Benet J., Boiso I., Calafell J.M., Egozcue J. & Navarro J. 2003. Multiple aneuploidies in the oocytes of balanced translocation carriers: a preimplantation genetic diagnosis study using first polar body Reproduction, 701-711. 20. Wilton L. 2005. Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization. Human Reproduction Update. 11: 33-41. 21. Liu J., Tsai Y.L., Zheng X.Z., Yazigi R.A., Baramki T.A., Compton G. & Katz E. 1998. Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis. Molecular Human Reproduction. 4: 972-977. 22. Wells D., Sherlock J.K., Handyside A.H. & Delhanty J.D. 1999. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation. Nucleic Acids Research. 27: 1214-1218. 23. Wells D. & Delhanty J.D. 2000. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Molecular Human Reproduction. 6: 1055-1062. 24. Kallioniemi A., Kallioniemi O.P., Sudar D., Rutovitz D., Gray J.W., Waldman F. & Pinkel D. 1992. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science. 258: 818-821. 25. Voullaire L., Wilton L., McBain J., Callaghan T. & Williamson R. 2002. Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure. Molecular Human Reproduction. 8: 1035-1041. 26. Wilton L., Voullaire L., Sargeant P., Williamson R. & McBain J. 2003. Preimplantation aneuploidy screening using comparative genomic hybridization or fluorescence in situ hybridization of embryos from patients with recurrent implantation failure. Fertilility and Sterility. 80: 860-868. 27. Wells D., Alfarawati S. & Fragouli E. 2008. Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH. Molecular Human Reproduction. 14(12):703-710.
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Microenvironment, Microfluidics, and Gametes and Embryos

Gary D. Smith1
ABSTRACT
Background: In vitro fertilization (IVF) remains one of the most exciting modern scientific developments and continues to have a tremendous impact on peoples lives. Since its beginning, scientists have studied and critically analyzed techniques in order to find ways to improve outcomes; however, little has changed with the actual laboratory technologies and equipment of IVF. Semen is still processed in test tubes and fertilization and culture still occurs in culture dishes. This somatic cell culture approach has also infiltrated the embryonic stem cell (ESC) field. Review: New technologic possibilities exist with the burgeoning advancement of nano- and microfluidic technologies. Microfluidics encompasses the study of physical principles of fluid behavior in a micro-environment and its application to chemistry, molecular biology, and cell biology. Two primary advantages exits in micro-scale cell isolation, culture and analysis. Firstly, microfluidics provides size and mechanical advantages not realized at the macro-scale. Secondly, microfluidics provides a micro-environment that is inherently more physiological and permissive to modifications that more closely mimic the in vivo developmental environment. Although a young field, many developments have occurred which demonstrate the potential of this technology for IVF. In this presentation, we briefly discuss physical principles of microfluidics and highlight some previous utilizations of this technology, ranging from chemical analysis to cell sorting. We then present designs and outcomes for microfluidic devices utilized thus far for each step in IVF: gamete isolation and processing, fertilization, embryo culture, and embryo analysis. Finally, we discuss and speculate on the ultimate goal of this technology development of a single, integrated unit for in vitro assisted reproduction techniques. Conclusion: Benefits of microfluidics for gamete isolation, gamete maturation, embryo culture and analysis, and growth and directed differentiation of embryonic stem cells are evident and progressing.
Keywords: microfluidics, gametes, embryos.
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Embryo Culture: Concerns and Perspectives

Gbor Vajta1
ABSTRACT
Background: Culture of preimplantation-stage embryos plays a crucial role in both domestic animal and human embryology. During the past 30 years, a considerable advancement has been achieved in this field. The development was most intensive in the 90s of the last century in domestic animals, and in the first decade of our millennium in humans. Due to the latter advancement, extended embryo culture and single blastocyst transfer is now the preferred method to decrease the chances of multiple pregnancies while preserving the overall efficiency of the treatment. However, there are still a lot of questions to answer and a lot of possibilities to improve the overall efficiency of mammalian in vitro embryo culture. Review: In spite of the scientific challenge and considerable commercial interest continuously stimulating research worldwide, the understanding of the requirements is insufficient, opinions and theories are controversial and the consensus is missing even in the basic principles of physical, chemical and biological environment. Composition of media, use of two-phase versus single media, need of medium exchange are constant subject of debates, and some basic principles including the right temperature of incubation have also been questioned. The purpose of this review is to summarize and confront different opinions about these issues, explain pros and contras, demonstrate the fragility of some commonly accepted arguments, and stimulate a fertile debate towards improvements and optimization. An open-minded approach expanding, sometimes breaking the traditional frames of embryo culture is suggested. Domestic animal embryology with its unlimited quantitative possibilities, legal freedom and widespread experience may play a considerable part of this process. Issues may include improvements in the culture process itself, i.e. new media and supplements, different dishes, and changing mentality during embryo handling, but may also be extended to related techniques as non-invasive embryo selection methods and active treatments to improve embryo quality. Conclusion: Although some researchers suppose that the currently applied in vitro culture systems have already approached the biological limits, a novel, critical and pragmatic approach may result in substantial improvement and may expand considerably the possibilities of future assisted reproduction in both domestic animals and humans.
Keywords: embryo, blastocyst, mammalian, culture, in vitro, evaluation.
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Workshop 5: Inter-Relações Da Reprodução Humana e Veterinária

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Workshop 5:

Inter-relaes da reproduo humana e veterinria

The interface between human and animal reproduction

Acta Scientiae Veterinariae . 38(Supl 2): s393-s413, 2010.

Human Assisted Reproduction

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

II. HISTORICAL OVERVIEW OF HUMAN ASSISTED REPRODUCTION

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

III. PERSPECTIVES FOR HUMAN ASSISTED REPRODUCTION

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

The Role of the Veterinarian on Human Assisted Reproduction

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II. ASSISTED REPRODUCTION EDUCATION IN VETERINARY MEDICINE AND MEDICINE

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III. THE INTERSECTION BETWEEN ANIMAL AND HUMAN ASSISTED REPRODUCTION

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Application of the Concept of Follicular Waves in Human Assisted Reproduction

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II. CURRENT THEORY OF HUMAN FOLLICULOGENESIS AND OVULATION INDUCTION PROTOCOLS

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III. DESCRIPTION OF FOLLICULAR WAVES IN THE HUMAN OVARIES

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Workshop 5: Inter-relaes da reproduo humana e veterinria. .

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V. DISCUSSION AND CHALLENGES

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Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

Diagnstico Gentico Pr-Implantacional

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i. Anlise do Primeiro Corpsculo Polar

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ii. Anlise de Blastmeros

iii. Anlise de Blastocistos

II. TCNICAS DE CITOGNTICA-MOLECULAR

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

III. HIBRIDAO GENMICA COMPARATIVA

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

Microenvironment, Microfluidics, and Gametes and Embryos

Workshop 5: Inter-relaes da reproduo humana e veterinria. .

Acta Scientiae Veterinariae. 38 (Supl 2): s393-s413

Embryo Culture: Concerns and Perspectives