workshop 5:inter-relações da reprodução humana e veterinária · workshop 5: inter-relações...

Workshop 5: Inter-relações da reprodução humana e veterinária

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 timehas been lowered to 6 month if the female partner is older than 35 years. Infertile couples are offered to low and highcomplexity treatments are available according to their cause of infertility. Low complexity treatments comprise timedintercourse, intrauterine insemination with or without controlled ovarian stimulation. High complexity treatments comprisestandard 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, Huntington’s disease, fragile-X syndrome amongothers. Human assisted reproduction is a dynamic branch of Medicine performed by several medical specialists fromgynecologists to surgeons, and professional as nurses, biologists and veterinarians.

Review: Initially, human infertility was treated by intracervical insemination due to the risk of endometrial inseminationand the occurrence of pelvic inflammation, but the development of better insemination techniques and instrumentsled to the report of a safe intrauterine insemination method in 1974. However, insemination was restricted tooligozoospermia 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 ofthe first IVF baby birth. Since then infertile couples due to tubal factors, ovarian and moderate male infertility factorsstarted to be treated with IVF. Several changes in embryo culture conditions and instruments for IVF were developedand the technology was world wide spread. The first IVF baby was a girl born in 1984 in Brazil. Even though, someovarian conditions and severe male factor infertility couldn’t be treated until 1992. This landmark was the report of thefirst babies originated by in vitro fertilization with ICSI. In parallel to those scientific and medical evolutions, drugs andnew 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 ofcryopreservation techniques for long term storage. Slow rate freezing and vitrification of embryos were reportedapproximately 25 years ago, but just recently vitrification of oocytes and embryos became the first option for fertilitypreservation and/or embryo storage in humans. Preimplantational genetic screening and diagnosis were complementarytechniques developed during the same period. These tests require precise and meticulous micromanipulation techniquesand skills for the obtainment of a single blastomere with intact genetic material for screening of aneuploydies byfluorescent 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 diagnostictests and with the improvement of clinical approaches to infertility and better conditions in embryology laboratoriesand this constant evolution is the result of the synergic interaction of clinical infertility specialists with researchers ofdifferent backgrounds.

Keywords: Assisted reproduction, in vitro fertilization, IVF, intracytoplasmatic sperm injection, ICSI, human reproduction.

1Huntington Medicina Reprodutiva, São Paulo, Brazil; 2Centro de reprodução humana “Governador Mário Covas” do Hospital dasClínicas da Faculdade de Medicina da Universidade de São Paulo*Corresponding author’s address: Huntington Medicina Reprodutiva,Av. República do Líbano, 529, São Paulo, SP, Brazil, Postal code: 04501-000, Phone: 5511 30596122, Fax: 5511 30596100, e-mail:[email protected]

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Workshop 5: Inter-relações da reprodução humana e veterinária.. 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 thistime has been lowered to 6 month if the female partner is older than 35 years [1]. Approximately, 10% of the couplesin reproductive age might require infertile care [2]. The last report on assisted human reproduction in Europe indicatedthat 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. Maleinfertility factors comprise a myriad of malfunctions from retrograde ejaculation to impaired production or release ofsperm such as in oligozoospermia, teratozoosperia, asthenozoospermia, and obstructive and non-obstructiveazoospermia [5]. Conformably to male infertility, several diseases are enrolled in female infertility. Didactically, femaleinfertility 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 andhigh complexity according to the patients needs (Table 1)[8-9].

Low complexity treatments comprise timed intercourse, intrauterine insemination with or without controlledovarian stimulation. High complexity treatments comprise standard in vitro fertilization (IVF) and IVF byintracytoplasmatic sperm injection (ICSI). Additionally to these treatments, infertile patients might be benefited bytechniques 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, byarchaic techniques of artificial insemination. Despite its availability of for more than 200 years, artificial inseminationremained controversial in many countries until the 1970´s [10].

Modern artificial insemination consisted in the intracervical delivery of prepared semen to minimize the riskof occurrence of pelvic inflammation. Nevertheless, since 1974 the use of a of a safe intrauterine inseminationmethodhas replaced intracervical alternative [11].

Intrauterine artificial insemination was restricted to oligozoospermia or cervical factors [12]; however, it wasnot able to overcome ovarian and tubal infertility factors, neither severe male infertility factor. In vitro fertilization is theappropriate treatment for these conditions, and despite of the publication of attempts of in vitro fertilization of animaloocytes in the begging of the 20th Century, the fertilization of human oocytes was first reported in 1969 [13]. Severaldevelopments in embryo culture techniques were developed and in 1978 the birth after of the first IVF baby wasannunciated and it broadened the range of treatable infertility causes; thence, infertile couples due to tubal, ovarianand 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, establishedtreatments with both higher quality standards and have observed an even increasing pregnancy and birth rates. In1992, Palermo and colleagues [15] reported a births of babies originated by in vitro fertilization with intracytoplasmaticsperm 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 inductionincreased 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 ofoocytes and embryos became the first option for fertility preservation for female gametes and embryos storage inhumans [17-18]. In fact, vitrification of oocytes has provided quality oocytes at warming resulting in pregnancy ratessimilar to those obtained with fresh oocytes. This methodology has been chosen in Spain as the main storage methodfor oocytes in an ovodonation program [19-20]

Preimplantation genetic screening and diagnosis were complementary techniques developed during thesame period [21]. These tests require precise and meticulous micromanipulation techniques and skills for the obtainmentof a single blastomere with intact genetic material for screening of aneuploydies using fluorescent in situ hybridizationor the detection of a deleterious allele by single cell PCR [21]. Preimplantation genetic diagnosis is particularlyinteresting for the prevention of allelic disorders such as thalassemia, sickle cell anemia, cystic fibrosis, Duchenne’sMuscular 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 descriptionof follicular waves in women [25] described originally by Baerwald et al. [26-27]. These protocols are currently underclinical 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 asproteomics and genomics come into place. The in vivo environment for embryo development is chemically andphysically dynamic; however, embryo culture systems currently used in Human Assisted Reproduction is static [28-29]. The establishment of physically dynamic conditions through tilting embryo culture media [30] or, in a moresophisticated approach, through a complex series of channels creating microfluidic stream of media [28-29] are stillon progress, but they are promising technology on embryo culture techniques and they may have a great impact onpregnancy rates.

Complementary techniques such as PGD are also becoming more sophisticated through the implementationof feasible microarray based comparative genomic hybridization [31]. This technique allows high resolution investigationof the embryo genome and detection of deleterious copy number variation of genes, causes of miscarriage and it stillallows the determination of chromosomal numbers [32-33].

IV. CONCLUSIONS

Human Assisted Reproduction is constantly evolving with the development of more precise diagnostictests and with the improvement of clinical approaches to infertility and better conditions in embryology laboratoriesand this constant evolution is the result of the synergic interaction of clinical infertility specialists with researchers ofdifferent backgrounds.

REFERENCES

1 ASRM. 2008. Practice Committee of the American Society for Reproductive Medicine: Definitions of infertility and recurrent pregnancyloss. 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 AndersenA. 2006. Assisted reproductive technology in Europe: results generated from European registers by ESHRE. Human Reproduction. 1-12.

4 RedLARA. 2009. Registro Latinoamericano de Reproducción Asistida. Red Latino Americana de Reproducción 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 infertilityin 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 YorkAcademy 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. EuropeanJournal 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 husband’s 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 spermatozooninto 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 Prospectiverandomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertility and Sterility (in press).

19 Cobo A., Meseguer M., Remohí 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 bankingand 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 preimplantationgenetic 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 diagnosisfor 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 ofReproduction. 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 mouseembryo 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., LeLorc’h M. & Achour-Frydman N. 2009.Preimplantation genetic diagnosis: state of the art. European Journal of Obstetrics & Gynecology and Reproductive Biology. 145(1): 9-13.

32 Perrin A., Delobel B., Andrieux J., Gosset P., Gueganic N., Petit F., De Braekeleer M. & Morel F. 2010. Molecular cytogeneticanalysis 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

André Monteiro da Rocha1

ABSTRACT

Background: Infertility is a disease affecting 10% of the human population in reproductive age and couples experiencingdifficulties to achieve pregnancy may benefit from assisted reproduction treatment. According to the Red Latinoamericanade Reproducción Asistida, approximately 34,100 in vitro fertilization (IVF) treatments were performed in Latin Americain 2007 and 42.3% (14,428) of these treatments were conducted in Brazil. Standard techniques and treatments forinfertility were developed within the last 4 decades and some of them are still under technological improvements orthey are frequently considered experimental or “state of art” technology. Usually, these treatments and techniques toovercome 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 humanassisted procreation, developing new assisted reproduction technologies and treatments, and finally acting in clinicalembryology laboratories as embryologists or supervisors.

Review: Human assisted reproduction landmarks were: (i) the birth of the first in vitro fertilization (IVF) child; (ii) theimplementation of intracytoplasmatic sperm injection; and, more recently, (iii) the development of successful protocolsof vitrification for cryopreservation of embryos and oocytes. Veterinarians’ contribution and participation in the field ofhuman 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 animalmodels or biotechnologies now used for commercial purposes in animal production and breeding. Furthermore, newembryo culture technologies such as microfluidics environments and controlled atmospheres are initially evaluated inanimal models. Another example of the role of veterinarians on the development of human assisted reproduction isthe emergence of the widespread concept of ovarian dynamics pattern of follicular waves in monovulatory animalsfrom Theriogenology to Reproductive Medicine in the last decade. The translation of this concept to ReproductiveMedicine might allow the development of different approaches of controlled ovarian stimulation. In a human assistedreproduction clinic, the clinical embryology laboratory is responsible for selecting oocytes after transvaginal ovumpick-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 areidentical to those performed by veterinarians in an animal IVF facility; however, a veterinarian must be familiar to thespecific guidelines and needs of a human IVF laboratory.

Conclusion: Assisted reproduction treatments are performed by groups of professionals with different academicbackgrounds. Veterinarians have been playing an important roles in the development and/or adaptation of severalbiotechnologies utilized for human procreation; additionally, veterinarians working on animal in vitro fertilization facilitieshave an adequate formation and skills to act on and/or supervise clinical embryology laboratories after specificeducation, practice and certification.

Keywords: assisted reproduction techniques; in vitro fertilization; embryo culture; controlled ovarian stimulation.

1Huntington Medicina Reprodutiva, São Paulo, Brazil. *Corresponding author’s address: Huntington Medicina Reprodutiva, Av. Repúblicado Líbano, 529, São Paulo, SP, Brazil, Postal code: 04501-000, Phone: 5511 30596122, e-mail: [email protected]

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I. INTRODUCTION

II. ASSISTED REPRODUCTION EDUCATION IN VETERINARY MEDICINE ANDMEDICINE

III. THE INTERSECTION BETWEEN ANIMAL AND HUMAN ASSISTEDREPRODUCTION

IV. CONCLUSIONS

I. INTRODUCTION

Infertility is a disease [1] affecting 10% of the human population in reproductive age and couples experiencingdifficulties to achieve pregnancy may benefit from assisted reproduction treatment.

Human assisted reproduction comprises a series of methods and techniques to treat infertility from timedintercourse to in vitro fertilization (IVF) by intracytoplasmatic sperm injection (ICSI) [2]. Approximately, 55,500 humanembryos were transferred in 21,285 IVF cycles in Latin America according to the 2007’s Registro Latinoamericano deReproducción Asistida [3]. Centers for treatment of infertility located in Brazil had performed a considerable amountof these IVF cycles (14,428; 42.3%) confirming the great experience of the Brazilian infertility specialists andembryologist 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 SociedadeBrasileira de Tecnologia de Embriões 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 specificallydesigned 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 severalintersections in the progress of these disciplines, and the interchange of knowledge between veterinarians practicinganimal reproduction and those professionals enrolled in human reproduction such as biologists, physicians andnurses might contribute to the development of more efficient protocols and techniques in both areas.

II. ASSISTED REPRODUCTION EDUCATION IN VETERINARY MEDICINE ANDMEDICINE

The Ministry of Education of Brazil established guidelines for the instruction of undergraduate students inseveral traditional careers, among them Veterinary Medicine. These documents determine the minimal knowledgeand skills of the professionals of each area; for Veterinary Medicine, the freshly graduated professional is expected tomanage and perform assisted reproduction techniques [5].

Veterinary Medicine Schools line up Theriogenology knowledge in several disciplines from physiology tobiotechnology of reproduction to accommodate the Veterinary Medicine teaching to Federal guidelines. The specificduration of Theriogenology training varies from 4 to 8% of the educational duration [6-8]; however, principles ofreproductive physiology and management are applied in various other subjects as in medicine and breeding ofseveral species of companion and production animals [6-9]. Furthermore, there are several graduate student programsin Animal Reproduction and Reproductive Sciences held by outstanding Universities [10].

Conversely, the guideline for the instruction of future medical doctors determines that the students shouldacquire several abilities; nevertheless, it does not mention the need to treat infertility or to perform or manageassisted reproduction treatments [11]. Thus, assisted reproduction and infertility treatment comprise a minimal portionof the medical education and these issues would be addressed in the 3rd year of the medical residency in Obstetricsand Gynecology, but they are limited up to 15% of the time spent in the Obstetrics ambulatory [12]. Consequently, fewphysicians have received education to deal with reproductive issues and assisted reproduction.

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III. THE INTERSECTION BETWEEN ANIMAL AND HUMAN ASSISTEDREPRODUCTION

Despite of the 3 year delay on the birth of the first in vitro fertilization calf in relation to the birth of the firstIVF baby [13-14], many steps and protocols of these technological advancements relied on the knowledge acquiredfrom animal models [15].

Veterinarians’ contribution and participation in the field of human assisted procreation could be distinguishedbetween technological development and/or clinical embryology.

New embryo culture technologies such as microfluidics environments and controlled atmospheres areinitially evaluated in animal models [16-18], as well as, some quality control programs for IVF laboratories are performedwith the evaluation of mouse’s embryo development [19].

Another example of the role of veterinarians on the development of human assisted reproduction is theemergence of the widespread concept of ovarian dynamics pattern of follicular waves in monovulatory animals fromTheriogenology to Reproductive Medicine in the last decade [20-24]. The translation of this concept to ReproductiveMedicine might allow the development of different approaches of controlled ovarian stimulation [25].

In human assisted reproduction clinics, the clinical embryology laboratory is responsible for preparingmedia and dishes, selecting oocytes after transvaginal ovum pick-up, fertilizing these oocytes, cultivating the supposedzygotes 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 commercialpurposes in an animal IVF facility for production and breeding; however, a veterinarian must be familiar to the specificguidelines and needs of a human IVF laboratory [26-27].

The mutual interaction between human reproduction professionals and theriogenologists might also bringnew 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] yieldinglaboratorial and clinical outcomes similar obtained with fresh oocytes [36-37]. Over than 900 normal babies were bornafter oocyte cryopreservation [38] and the transposition of the experience in oocyte vitrification from human reproductionto 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 severalbiotechnologies utilized for human procreation; additionally, veterinarians working on animal in vitro fertilization facilitieshave an adequate formation and skills to act on and/or supervise clinical embryology laboratories after specificeducation, practice and certification.

REFERENCES

1. ASRM. 2008. Practice Committee of the American Society for Reproductive Medicine: Definitions of infertility and recurrent pregnancyloss. 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 Reproducción Asistida. 2007.

4. SBTE. 2008. Mudanças e tendências no mercado de embriões bovinos no Brasil. O Embrião. 5-7.

5. Maranhão E.A., Macedo A.R. & Okida Y. 2002. Diretrizes Curriculares Nacionais do Curso de Graduação em Medicina Veterinária.C.N.D.: Educação. Diário Oficial da União. 11/4.

6. NAEG. Grade Curricular-Medicina Veterinária. http://naeg.prg.usp.br/numeros/curso.phtm.14/07/2010.

7. Medicina Veterinári- 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 Graduação em Medicina Veterinária - 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 saúde e os currículos dos cursos de medicina veterinária: um estudo de caso.Interface - Comunicação, Saúde, Educação. 8 (15): 349-360.

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10. CAPES. Relação de Cursos Recomendados e Reconhecidos. http://conteudoweb.capes.gov.br/conteudoweb/ProjetoRelacaoCursosServlet?acao=pesquisarIes&codigoArea=50500007&descricaoArea=CI%CANCIAS+AGR%C1RIAS+&descricaoAreaConhecimento=MEDICINA+VETERIN%C1RIA&descricaoAreaAvaliacao=MEDICINA+VETERIN%C1RIA.

11. EDUCAÇÃO C.N.D. Diretrizes Curriculares Nacionais do Curso de Graduação em Medicina. C. d. E. Superior. Diário Oficial da União.09/11/2001. 38.

12. Educação M.D. Conteúdos do Programa de Residência Médica de Obstetrícia e Ginecologia. C. N. D. R. MÉDICA. Diário Oficial daUnião. 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 vitrofertilization in the cow. Biology of Reproduction. 27 (1): 147-58.

15. Varago F.C., Mendonça L.F.& Lagares M.A. 2008. Produção in vitro de embriões bovinos: estado da arte e perspectiva de uma técnicaem constante evolução. Revista Brasisleira Reprodução 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 mouseembryo 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. Fertilityand 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 consistentlaboratory? 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 duringthe 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 offollicular 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 ofReproduction. 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 IVFlaboratories. 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 perspectivesregarding 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 andapplications 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. & FlamigniC. 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 sperminjection 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. Prospectiverandomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertility and Sterility. (in press).

36. Cobo A., Meseguer M., Remohí 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 vitrifiedoocytes. 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 congenitalanomalies. Reproductive Biomedicine Online. 18 (6): 769-776.


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Application of the Concept of Follicular Waves in Human AssistedReproduction

Paulo Homem de Mello Bianchi

ABSTRACT

Background: Ovulation induction (OI) is one of the cornerstones in human assisted reproduction treatments. OI isbased on the understanding of ovarian follicular physiology. Current knowledge of human follicular dynamics relies onhystologic 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. Initialrecruitment and grow of the primordial follicles is still a poorly understood process, but continued development ofantral follicles, selection of the dominant follicle and ovulation depends on follicle stimulating hormone (FSH) andluteinizing hormone (LH) serum levels, which are influenced by estradiol and progesterone concentrations. With thedegeneration of the corpus luteum at the end of the luteal phase, serum estradiol and progesterone concentrationsfall, leading to menstruation and an increase in FSH levels. All this observations have led to the formulation of thecurrent 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 continueto 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 beendescribed. Major development of medical ultrasound technology has allowed for the precise detection of small antralfollicles in the transvaginal route. Using this technology and shifting the landmark for observations from menses toovulation, synchronic grow of a group of antral follicles (follicular wave) has been observed two or three times in aninter ovulatory interval. Emergence of a wave is preceeded by an elevation in FSH levels. The first wave emergesaround ovulation and the last is the ovulatory follicular wave. This is in accordance with previous observations in othermono 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 OIprotocols used in Veterinary Medicine. It has been shown that synchronization of OI with wave emergence results inmore oocytes and embryos with better quality in cows. Protocols to control wave emergence are currently standart ofpractice for in vitro production of some animals’ embryos. The description of the follicular waves in humans gives theopportunity to develop new OI protocols for IVF. Synchronization of OI with wave emergence could be achievedthrough 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 ovulationinduction). However some differences between in vitro production of animal embryos and Human IVF should beenfasized and could challenge the application of this knowledge. For instance, Human embryos are usually obtainedand transfered to the same subject, therefore a synchronization of embryo and endometrial development is a majorconcern.

Conclusions: Description of follicular waves in Human ovaries could have profound implications in OI for IVF, butsome challenges should be expected to transpose the animal model to the Human setting.

Keywords: Human follicular waves; ovulation induction; in vitro fertilization.

Centro de Reprodução Humana do Hospital das Clínicas da Faculdade de Medicina da USP, Huntington Centro de Medicina Reprodutiva.CORRESPONDENCE: Avenida República do Líbano 529, Ibirapuera São Paulo SP Brasil, CEP 04501000, Email:[email protected], [email protected]

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I. INTRODUCTION

II. CURRENT THEORY OF HUMAN FOLLICULOGENESIS AND OVULATIONINDUCTION PROTOCOLS

III. DESCRIPTION OF THE FOLLICULAR WAVES IN THE HUMAN OVARIES

IV. POSSIBLE APLICATIONS OF THE FOLLICULAR WAVE CONCEPT TO HUMANASSISTED REPRODUCTION

V. DISCUSSION AND CHALLENGES

I. INTRODUCTION

Despite much development in laboratory techniques in the past years, ovulation induction is still one of thecornerstones in assisted reproduction treatments in humans [10]. The proper use of exogenous gonadotropins toincrease the number of mature oocytes available at one time relies on the understanding of key events of reproductivephysiology. In this article we are going to review the theory of human ovarian follicular dynamics on which currentovulation induction protocols are based, indicate evidence of the occurence of follicular waves in human ovaries andspeculate on the impact of this alternative physiologic paradigm to ovulation induction protocols and human assistedreproduction.

II. CURRENT THEORY OF HUMAN FOLLICULOGENESIS AND OVULATIONINDUCTION PROTOCOLS

Since menses are a very distinct event during the women’s reproductive years, menstuation has beenused as a landmark in the studies of the female reproductive system. Cyclic changes in reproductive organs, tissuesand sexual hormones between two menses are called the menstrual cycle. Didactically there are two distinct phasesduring an intermenstrual interval. In the follicular phase, just after menses, follicles grow, produce estradiol andultimately ovulate. The ovulated follicle becomes the corpus luteum, which produces progesterone and dominates thenext phase, the lutheal phase. If pregnancy did not occur, corpus luteum degenerate in fourteen days, hormonal levelsfall and a new cycle begins [25].

The current theory of human follicular dynamics is derived from hystologic observations of excised ovariesin different moments during the menstrual cycle [16] together with serum and follicular fluid steroid measurements ofwomen 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 mitoticdivisions the number of germ cells in the ovaries reach it’s maximum at around 20 weeks of gestational age (maximalovarian 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 ofgerm cells in the fetal ovaries ends and their number decrease progressively [11]. Gradually primordial folliclesactivate and beggin the process of maturation. Granulosa cells become cuboidal and proliferate to form a multilayersurrunding the oocyte [11,17]. Stromal cells adjacent to the granulosa transform to constitute the theca, early sensitiveto the actions of the luteinizing hormone (LH) [9]. Later a cavity is formed in the center of the follicle, the antrum, whichseparates two groups of granulosa cells, the cumulus oophorus, surrunding the oocyte, and the parietal cells, closeto the theca [11]. At this stage granulosa cells acquire follicle stimulating hormone (FSH) receptors [18,9]. A fewfollicles are abble to progress beyond this point, reach full maturation (with a big antrum) and finally ovulate. The greatmajority of follicles actually suffer atresia through apoptosis [11]. Primordial follicles that beggin the process ofmaturation during the fetal stage and childhood are fated to suffer atresia in the early stages of the process [19]. Onlyduring 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 throughoutthe menstrual cycle [16]. The transition from primordial to mature follicles takes several months [19]. The mechanismsresponsible for the activation of the primordial follicles and beggining of follicular development are still unknown, butit appears that the initial steps of folliculogenesis are not under the controll of gonadotropins [24]. From this observationsit has been postulated that activation of the primordial follicle and development until the early antral stage occurscontinuously and randomly [11].

Final follicular development (from the early antral stage on) and ovulation, on the other hand, is clearlydependent on FSH and LH actions [11]. Follicles in the antral stage demand different concentrations of FSH tocontinue 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 negativefeedback effect on the production and secretion of FSH and ultimately FSH concentration fall below the thresholdagain. 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 folliclecontinues to grow, becoming the dominant follicle, and eventualy ovulates. Actually this follicle is sensitive to lowerconcentrations of FSH [11] and, during the common follicular growh phase, it’s granulosa cells also acquire LHreceptors [27]. Since during the follicular phase small concentrations of LH are present, the dominant follicle is ableto further develop also under the stimulation of LH. This is the mechanism of dominance responsible for the selectionof 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 ramdomlyactivate 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 latelutheal and early follicular phases, continue to grow and eventualy ovulate. This model of folliculogenesis has beencalled the Propitious Moment Theory [2]. Final follicular development until ovulation is expected to occur only duringthe follicular phase of the cycle [25]; the presence of the corpus luteum after ovulation is thought to inhibit follicledevelopment to a mature follicle.

This model of folliculogenesis has been the base for current ovulation induction protocols in human assistedreproduction treatments. Administration of exogenous gonadotropins takes place in the early follicular phase, begginingmore precisely between the second and fifth day of the menstral cycle. The objective is to extend the FSH windowallowing for more follicles to complete all stages of development and produce a greater number of mature oocytes forfertilization [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 anddynamic imaging of the ovaries, contrasting with the still images provided by hystological evaluations. With thedevelopment of high definition ultrasound technology, currently it is possible to see follicles as small as 2 mm indiameter, through the transvaginal route.

Intrigued by previous ultrasound observations of follicular activity during the luteal phase, which challengedthe prevailing theory of human folliculogenesis, Dr Angela Baerwald and co-authors decided to investigate ovarianphisiology using serial high definition transvaginal ultrasound and serum hormonal determinations in an interovulatoryinterval, 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 ultrasoundimaging provides a dynamic, ovarian centered view of folliculogenesis. Their data shown that follicles rather developin coodinated and synchronic groups apearing two (68% of women) or three (32% of women) times during an interovulatoryinterval [3]. The first group of folicles starts to grow on the day of ovulation; the interovulatory interval is shorter forwomen with two groups of follicular development comparing to those with three; in the last group a follicle alwaysovulate [3,4]. The coordinated growth of a coohort of follicles has been previously described in other mono ovulatoyanimals, 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 occuredwhen a follicle reached 10 mm or more in diameter; it only takes a few days from wave emergence to selection of thedominant follicle. Only a small proportion (15%) of women with two wave pattern had a dominan follicle in the firstwave. Nineteen percent of women with three waves pattern had a dominant follicle in the second and third wave andanother 19% had a dominant follicle in all waves. Dominant follicles of anovulatory waves didn’t reach an ovulatorydiameter (20 – 22 mm). The interval between wave 1 and 2 in women with two waves pattern was approximately 14-15 days [3,4], meaning that the last follicular wave emerges just before or in the begining of menstruation. In womenwith three waves pattern the second wave emerges 11-13 days after the first [3,4] (before menstruation) and the lastone 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 theequines and bovines [12-15]. On the other hand it is in sharp contrast with the current theory of random folliculargrowth; moreover, it shows that follicular development occurs also in the luteal phase, not only in the follicular phaseas thought until now.

IV. POSSIBLE APLICATIONS OF THE FOLLICULAR WAVE CONCEPT TO HUMANASSISTED REPRODUCTION

The follicular wave concept has long been used in Veterinary Medicine for in vitro production of animalembryos, particularly in cows. It has been shown that when ovarian stimulation with exogenous gonadotropins startsat the beggining of a follicular wave, there are more mature oocytes at the egg retrieval and embryos are of betterquality [23,1].

Detection of ovulation and consequently the emergence of the first wave would be the most obviousapproach, but since ovulation detection is a time consuming process considering a herd, protocols to synchronizewave emergence with initiation of gonadotropin administration have been developed. There are mechanical andpharmacological strategies to determine wave emergence. The mechanical strategy relies on the ablation of thedominant follicle before ovulation through aspiration. Shortly after aspiration serum concentrations of estradiol andinhibin fall; without the inhibitory effect of estradiol and inhibin on the hypothalamus and pituitary, FSH concentrationsrise followed by a wave emergence [5]. However, this strategy is not common in theriogenology because it is aninvasive and time consuming procedure [22].

The pharmacological strategy consists in administer a boost of parenteral estradiol and progesteroneanytime during the estrous cycle. The consequent rise in serum concentrations of estradiol and progesteone lowersboth FSH and LH concentrations, causing atresia of the dominant follicle and, in the lutheal phase, lutheolysis. Ineither 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 ofhuman folliculogenesis, are not synchronized with follicular wave emergence but rather with menstruation. Startinggonodotropin administration in the second to fifth day of the menstrual cycle probably does not coincide with thebegining of a follicular wave, particularlly for women with a three wave pattern. Actually, by that time several womenhave 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 tocontrol wave emergence and synchronize it to the begining of ovarian stimulation could theoretically increase thenumber of mature oocytes, reduce the requirements of exogenous gonadotropins and improve embryo quality, andconsequently pregnancy rates.

We have recently published a rewiew article where we speculate on possible protocols for synchronizeovulation induction with wave emergence in humans [6]. Briefly, aspiration of the dominant follicle (or induction ofovulation with hCG) followed some days after by gonadotropins administration are probably the easier strategies to dothis 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 beused anytime during the cycle, but concerns might rise regarding possible side effects of a hormonal boost andoptimal 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 theresults observed in farm animals. A particular useful application of this concept is for fertility preservation in womenwith cancer, when the time span between diagnosis and initiation of chemotherapy or radiotherapy often is notsufficient 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 animalswith high genetic potencial. Donnor animals are usually young and healthy individuals; embryos produced are transferedto 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 transferedto 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 theendometrial quality of the patient. Ovulation induction synchonized with wave emergence could be assynchronous tothe endometrial development with possible impairment of implantation. Vitrification of embryos for posterior endometrialpreparation 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, ovulationinduction can cause the subordinate follicles in the same wave to grow and not a new wave to emerge. The exact sizeof the dominant follicle when complete dominance is stablished in humans should be investigated to avoid thissituation. Moreover, the interval between follicle aspiration and wave emergence in humans should be determined toset the ovulation induction.

The dose and duration of administration of exogenous steroids to cause atresia of the dominant follicle andlutheolysis 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 itbecome 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 oflReproduction. 69: 1023-1031.

5. Bergfelt D.R., Bo G.A., Mapletoft R.J. & Adams G.P. 1997. Superovulatory response following ablation-induced follicular waveemergence 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 HumanOvary: 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 superovulatoryresponse 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 ZealandJournal 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 andFertility. 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 ofReproduction 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. AnimalReproduction 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 offollicular 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 Reproductionand 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. NewYork. 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 follicle-stimulating 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 hypothesisby 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 AnimalReproduction. 6: 114-124.

23. Nasser L.F., Adams G.P., Bo G.A. & Mapletoft R.J. 1993. Ovarian superstimulatory response relative to follicular wave emergencein 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: 617-630.

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 menopausalgonadotropin (hMG) and follicle-stimulating hormone (FSH). The importance of the FSH concentration in initiating follicular growth inpolycystic 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 ofhuman granulosa cells. Journal of Clinical Endocrinology & Metabolism. 74: 842-849.


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Diagnóstico Genético Pré-Implantacional

Juliana Fabricia Cuzzi1, Tânia Maria Vulcani-Freitas1, Priscila Cristina Rodrigues Motta1 &Péricles Assad Hassun Filho1*

ABSTRACT

Background: The ranks of patients seeking preimplantation genetic diagnosis (PGD) to identify embryos with monogenicdisorders like cystic fibrosis or thalassemia are growing rapidly. Even so, the most common indication for preimplantationembryo testing remains the risk of chromosomal imbalance. In most cases, the PGD strategy employed forchromosomal testing involves biopsying a single cell (blastomere) from each embryo at the 6 to 10-cell stage, 3 daysafter fertilization. The cell is placed on a microscope slide, fixed, and then subjected to cytogenetic analysis. Whilethe biopsied cell is being analyzed, the rest of the embryo is maintained in culture. Most infertility clinics prefer totransfer 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 involvesthe hybridization of chromosome specific DNA probes, labeled with different colors, to nuclei spread on a microscopeslide. The method is rapid, performs equally well whether applied to interphase nuclei, and permits enumeration of upto 10 chromosomes per cell. Initially, the PGD for aneuploidy was envisioned that most of the patients seeking PGDfor aneuploidy would be those who carry a chromosomal rearrangement. Couples in which one of the partners carriesa 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 chromosomallynormal individuals undergoing IVF. Early methods employed five FISH probes for PGD, focusing on the chromosomesmost often found to be abnormal in prenatal samples (13, 18, 21, X, and Y). Aneuploidies for these chromosomes aresometimes compatible with survival to term, leading to aneuploid syndromes. This strategy was successful in reducingthe number of such syndromes, but a statistically significant improvement in embryo implantation could not beshown. Later PGD studies expanded the number of chromosomes assessed to eight. This was achieved by performingtwo 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 inmiscarriages. The eight-chromosome PGD protocol led to a doubling of embryo implantation rates in two separatestudies and reduced the number of spontaneous abortions. Although this problem can be partially overcome byperforming sequential FISH experiments on the same cell, the accuracy of the method declines with each additionalhybridization. The good news is that there is a method related to FISH—comparative genomic hybridization (CGH)—that can detect aneuploidy that affects any chromosome within a sample. In addition to offering comprehensivedetection, 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 severalgroups of infertile patients, including women aged 37 and above, those with recurrent miscarriages, women with aprevious comprehensive screening for aneuploidy will likely further increase the benefit of PGD to these patients andmaybe even a broader range of patients. The widespread availability of CGH for PGD is nearly at hand, but while weawait final refinements and validation, PGD strategies can still be improved by making the best use of currentmethods and reassessing the chromosomes selected for screening.

Keywords: PGD, aneuploidy, FISH, CGH.

1Genesis Genetics Brasil, São Paulo – SP, Brasil. *Autor para correspondência: Péricles Assad Hassun Filho, E-mail:[email protected] ou [email protected], Rua Mato Grosso, 306 conjunto 506, Higienópolis, São Paulo – SP,Brasil, CEP 01239-040

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I. INTRODUÇÃO

a. Diagnóstico Genético Pré-Implantacional (PGD)

b. Aplicações do Diagnóstico Genético Pré- Implantacional

i. Análise do Primeiro Corpúsculo Polar

ii. Análise de Blastômeros

iii. Análise de Blastocistos

c. Screening para Aneuploidias (PGS)

II. TÉCNICAS DE CITOGENÉTICA-MOLECULAR

III. HIBRIDAÇÃO GENÔMICA COMPARATIVA (CGH)

IV. CONCLUSÃO

I. INTRODUÇÃO

a. Diagnóstico Genético Pré-Implantacional (DGP)

Estudos citogenéticos por meio da técnica de FISH mostraram que mais de 50% dos embriões em estágiopré-implantacional possuem células aneuplóides [1]. Considerando que a maioria dessas alterações impede odesenvolvimento embrionário e conseqüente implantação, a identificação de embriões geneticamente normais écrucial para a obtenção de gestações viáveis [2]. A identificação confiável de embriões euplóides (cromossomicamentenormais) só pode ser determinada pelo Diagnóstico Genético Pré-implantacional (PGD). O PGD envolve a interaçãoentre as técnicas de reprodução assistida, a biópsia de blastômero ou do corpúsculo polar e posterior análise dacélula única, possibilitando o diagnóstico de doenças geneticamente herdadas em embriões humanos [3,4].

A aplicação clínica desse método proporciona aos casais com alto risco reprodutivo, maiores chances deterem filhos não afetados, identificando os embriões portadores de alterações cromossômicas e evitando que osmesmos sejam transferidos. Segundo Ludwig e colaboradores (2001), a técnica permite diminuir o risco de transmissãode alterações genéticas em mais de 95% [5]. Além de evitar descendentes afetados pelas principais SíndromesGenéticas, o diagnóstico pré-implantacional é uma alternativa para o diagnóstico pré-natal, principalmente nos paísesem que o aborto terapêutico não é permitido.

Os primeiros casos de Diagnóstico Genético Pré - Implantacional em embriões humanos foram descritosindependentemente por Handyside e colaboradores (1990) e Verlinsky e colaboradores (1990) [3,4].

Desde as primeiras tentativas na década de noventa, centenas de PGD têm sido realizados, demonstrandouma crescente aceitação da metodologia por diferentes populações. De acordo com a classificação determinadapela ESHRE PGD Consortium, o diagnóstico genético pré-implantacional deve ser dividido em duas categorias deacordo com as características do casal: (1) PGD de alto risco, que inclui casais com anomalias cromossômicas e/ou doenças monogênicas; (2) PGD de baixo risco, que tem por objetivo aumentar a taxa de implantação em casaiscom idade materna elevada (em torno de 37 anos), perdas fetais de repetição ou com freqüentes falhas de fertilizaçãoin vitro. Atualmente, sua indicação mais comum é o screening para aneuploidias em embriões gerados por mães comidade acima de 37 anos [6].

b. Aplicações do Diagnóstico Genético Pré-Implantacional

i. Análise do Primeiro Corpúsculo Polar

Na gametogênese feminina, durante a primeira divisão meiótica, um dos cromossomos homólogos decada bivalente segrega para o primeiro corpúsculo polar (1CP) e o outro para o núcleo do oócito (MII) que se encontraem metáfase II da meiose. Desta forma, conclui-se que o 1CP tem o conteúdo genético complementar ao núcleo

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MII, o que significa dizer que um desequilíbrio em MII implicará na alteração recíproca no corpúsculo polar.

Considerando que o 1CP por sofrer extrusão não tem função reprodutiva, a sua biópsia e posterior análisepermitem a caracterização indireta da constituição cromossômica do MII e a identificação dos oócitos aneuplóides[7] sem comprometer a viabilidade do gameta.

ii. Análise de Blastômeros

Trata-se da metodologia mais empregada no PGD [3,8]. Esse procedimento envolve a dissolução de umaregião da zona pelúcida e a aspiração de uma ou, no máximo, duas células embrionárias. A remoção de um blastômeroem embrião com o equivalente de 6-8 células parece não afetar o seu desenvolvimento, in vitro, para o estágio deblastocisto sendo considerada uma técnica eficiente.

A vantagem mais evidente é que, nesta metodologia, se analisa tanto a contribuição paterna quanto a materna,pois neste estágio a constituição genética do embrião está completamente formada; tornando o PGD em blastômeros ummétodo comparável ao diagnóstico pré-natal. Por outro lado, a desvantagem desta técnica é a coexistência de mais deuma linhagem celular no embrião (linhagens cromossomicamente normais e linhagens aneuplóides), fenômeno chamadode mosaicismo embrionário, que tem origem pós-zigótica e que tem sido detectado em uma porcentagem considerável deembriões [9]. A presença deste mosaicismo dificulta o diagnóstico citogenético pré-implantacional em blastômeros [10],sobretudo nos casos em que apenas uma célula é analisada. Em 2004, Sermon e colaboradores descreveram a necessidadede um estudo prospectivo e randomizado que comparasse a viabilidade embrionária quando realizada a biópsia de uma ouduas células e a incidência de erros no diagnóstico citogenético dos blastômeros [11]. Atualmente esta questão parece,ainda que de forma preliminar, estar solucionada. Goossens e colaboradores (2008), após analisarem 3377 embriões,concluíram que a remoção de 2 blastômeros interferia de forma negativa no desenvolvimento embrionário até o estágio deblastocisto, enquanto a eficiência do diagnóstico por FISH não se alterava [12].

iii. Análise de Blastocistos

Este tipo de biópsia é a mais tardia que se pode proceder. Consiste em cultivar o embrião in vitro atéalcançar o estágio de blastocisto (aproximadamente 5 dias após a fecundação) e remover de 10 a 30 células. Asvantagens são: proporcionar a análise de diversas células e a seleção dos embriões mais viáveis pela manutençãodo seu cultivo até a fase de blastocisto [13]. A principal desvantagem é que não restam mais do que poucas horasapós a biópsia para a realização do diagnóstico, já que segundo o informe do ESHRE PGD Consortium SteeringCommitee (2004), a transferência embrionária deve ser realizada no máximo no 6º dia pós-fecundação. Além disso,muitos centros de reprodução assistida têm dificuldades para cultivar os embriões in vitro até blastocisto e, por estarazão, são poucos os centros no mundo que praticam esta forma de biópsia [14].

c. Screening para Aneuploidias (PGS)

É a aplicação mais freqüente do PGD. As anomalias cromossômicas numéricas estão relacionadas tantocom falhas de implantação quanto com morte e perda embrionária resultando em abortos espontâneos. A incidênciade anomalias cromossômicas é de 1,4% em embriões de mães com até 34 anos de idade e aumenta para 52,4% emmulheres entre 40-47 anos [15]. Da mesma forma, também foi detectada uma incidência elevada de alteraçõescromossômicas em embriões de casais jovens com mais de três falhas de implantação (>55% de embriões sãoaneuplóides) e em casais com pelo menos dois abortos espontâneos (>68%) [16].

Shahine & Cedars (2006) questionaram se a aplicação do PGS em gametas e embriões poderia ajudar aaumentar as taxas de implantação e gestação em casais com idade materna elevada, em casais com falhas recorrentesde implantação, em mulheres que apresentam abortos espontâneos de repetição e também em casais portadores detranslocação equilibrada para detectar alterações dos cromossomos envolvidos no rearranjo [17]. Baseados em dados de20.000 ciclos de PGD, Kuliev & Verlinsky (2008) relataram que o efeito positivo do PGS é evidente quando comparadas astaxas de sucesso reprodutivo dos mesmos pacientes, antes e depois da realização do diagnóstico [18].

II. TÉCNICAS DE CITOGNÉTICA-MOLECULAR

O uso das técnicas de citogenética molecular tem favorecido os estudos em embriões humanos. Baseadasna utilização de diversos tipos de sondas de DNA (centroméricas, loci, específicas ou pinturas cromossômicas) eassociadas a diferentes fluorocromos, essas metodologias vêm se aprimorando com a evolução dos microscópios

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e dos sistemas de captura. Atualmente, as técnicas de citogenética molecular constituem tanto protocolos depesquisa quanto de diagnóstico, adicionando sensibilidade e especificidade aos resultados obtidos por citogenéticaconvencional.

A hibridação in situ fluorescente (FISH) se tornou a técnica padrão para a avaliação de aneuploidias, sendopossível analisar de duas a nove sondas cromossomo-específicas divididas em uma, duas ou três etapas dehibridação [19]. Cabe destacar que o número de cromossomos que pode ser analisado em cada etapa está ligado aonúmero limitado de fluorocromos disponíveis e à possibilidade de sobreposição dos sinais [2,20]. Em resumo, emuma situação ideal, é possível analisar somente cinco cromossomos em cada hibridação. Além disso, a quantidadede vezes que o material genético pode ser hibridado também é limitada, sendo observada perda considerável naqualidade da amostra a partir da terceira denaturação [21]. Entretanto, o parâmetro técnico mais desfavorável é quea metodologia empregada requer a fixação dos núcleos em lâminas de vidro, o que aumenta o risco de perdacromossômica por artefato de técnica. Em geral, as técnicas que envolvem fixação celular em lâminas são eficientesem diagnosticar casos de hiperploidia, falhando na detecção de hipoploidias. Com o intuito de superar esta limitaçãotécnica, Wells e colaboradores (1999) e Wells & Delhanty (2000) descreveram e aprimoraram a aplicação da HibridaçãoGenômica Comparativa (CGH) [24] em célula única [22,23].

III. HIBRIDAÇÃO GENÔMICA COMPARATIVA

Esta metodologia permite detectar desequilíbrios no número de cópias de qualquer um dos 23 parescromossômicos em apenas uma hibridação. A primeira aplicação da CGH no diagnóstico pré-implantacional foidescrita em blastômeros [25].

De acordo com a literatura, estudos por meio da CGH revelaram que de 25 a 30% dos embriõesdiagnosticados como normais após análise de FISH para nove cromossomos apresentavam aneuploidias paraoutros cromossomos [26]. Estes dados enfatizaram a importância da análise dos 23 pares cromossômicos naeficiência do diagnóstico pré-implantacional. A busca por um PGD altamente eficaz não apenas evita o nascimentode crianças afetadas por cromossomopatias como também favorece a transferência de um único embrião para oútero materno, contribuindo com a diminuição dos riscos envolvimentos em gestações gemelares.

Atualmente a aplicação da CGH no diagnóstico pré-implantacional tem sido muito discutida e, o queparece ser consenso entre os pesquisadores europeus e norte-americanos é que o screening para aneuploidias, pormeio desta técnica, favorece a implantação embrionária aumentando assim as taxas gestacionais, independentementese o diagnóstico foi realizado no oócito ou no embrião de terceiro dia ou até mesmo blastocisto [27].

V. CONCLUSÃO

A aplicação da FISH no diagnostic pré-implantacional tem favorecido o sucesso dos tratamentos dereprodução assistida para vários grupos de paciente inférteis, incluindo idade maternal avançada e abortos derecorrência. No entanto, a aplicação de um screening completo para aneuploidias permitiria que o PGD fosse realizadode forma mais efetiva e abrangente, beneficiando um número maior de casais.

A uso definitivo da CGH no PGD está muito próximo de acontecer, mas enquanto esta metodologia esperapelas últimas validações, as estratégias adotadas hoje para o PGD ainda podem ser aprimoradas, principalmente noque diz respeito à escolha dos cromossomos selecionados para o screening.

REFERÊNCIAS

1. Munné 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 embryossexed 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 geneticdiagnosis. Human Reproduction. 5: 826-829.

05_SBTE_HUMANOS.P65 4/8/2010, 05:22410


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Supl 1

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. & VivilleS. 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 BiomedicineOnline. 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. ESHREPGD 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 balancedstructural 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 andSterility. 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 forpreimplantation 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 abnormalhuman 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 diagnosisand single embryo transfer at Sydney IVF. Fertility and Sterility. 82: 295-298.

15. Márquez C, Sandalinas M., Bahçe M., Alikani M. & Munné S. 2000. Chromosome abnormalities in 1255 cleavage-stage humanembryos. 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 andCellular 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 foraneuploidy. 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 balancedtranslocation 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-situhybridization 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 cellsby 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 genomeamplification 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 hybridizationfor 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 genomichybridization 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 comparativegenomic hybridization or fluorescence in situ hybridization of embryos from patients with recurrent implantation failure. Fertilility andSterility. 80: 860-868.

27. Wells D., Alfarawati S. & Fragouli E. 2008. Use of comprehensive chromosomal screening for embryo assessment: microarrays andCGH. 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 continuesto have a tremendous impact on people’s lives. Since its beginning, scientists have studied and critically analyzedtechniques in order to find ways to improve outcomes; however, little has changed with the actual laboratory technologiesand 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 applicationto chemistry, molecular biology, and cell biology. Two primary advantages exits in micro-scale cell isolation, cultureand 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 thatmore closely mimic the in vivo developmental environment. Although a young field, many developments have occurredwhich demonstrate the potential of this technology for IVF. In this presentation, we briefly discuss physical principlesof microfluidics and highlight some previous utilizations of this technology, ranging from chemical analysis to cellsorting. We then present designs and outcomes for microfluidic devices utilized thus far for each step in IVF: gameteisolation and processing, fertilization, embryo culture, and embryo analysis. Finally, we discuss and speculate on theultimate 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, andgrowth and directed differentiation of embryonic stem cells are evident and progressing.

Keywords: microfluidics, gametes, embryos.


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Embryo Culture: Concerns and Perspectives

Gábor Vajta1

ABSTRACT

Background: Culture of preimplantation-stage embryos plays a crucial role in both domestic animal and humanembryology. During the past 30 years, a considerable advancement has been achieved in this field. The developmentwas most intensive in the 90’s of the last century in domestic animals, and in the first decade of our millennium inhumans. Due to the latter advancement, extended embryo culture and single blastocyst transfer is now the preferredmethod 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 ofmammalian in vitro embryo culture.

Review: In spite of the scientific challenge and considerable commercial interest continuously stimulating researchworldwide, the understanding of the requirements is insufficient, opinions and theories are controversial and theconsensus is missing even in the basic principles of physical, chemical and biological environment. Composition ofmedia, use of two-phase versus single media, need of medium exchange are constant subject of debates, and somebasic principles including the right temperature of incubation have also been questioned. The purpose of this reviewis to summarize and confront different opinions about these issues, explain pro’s and contras, demonstrate thefragility 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 experiencemay play a considerable part of this process. Issues may include improvements in the culture process itself, i.e. newmedia and supplements, different dishes, and changing mentality during embryo handling, but may also be extendedto 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 alreadyapproached the biological limits, a novel, critical and pragmatic approach may result in substantial improvement andmay 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 · workshop 5: inter-relações...

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