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TEMPLATE DESIGN © 2008 www.PosterPresentations.com Hena Zaheer [iqbal] MD DNB MRCOG Awatif Al Bahar FACHARTZ Mohammed Elkalyoubi FRCOG MRANZCOG Wael Ismail Madkour MD MRCOG Mays Al Adham MSc. Dubai Gynaecology and Fertility Centre, Dubai,UAE Objectives ResultsConclusions References 1Rinehart W. Sex Preselection - Not Yet Practical. Population Reports 1975 May (2) series 1: periodic Abstinence 1-12 2.CM chia et al Triplod pregnancy after ICSI of frozen testicular spermatozoa into cryopreserved human oocytes Human Reprod 2000 vol15 no 1962-64. 3. Al-Hasani S, Diedrich K, van der Ven H, Reinecke, Hartje, Krebs D. Cryoperservation of human oocytes. Hum Reprod 1987;2:695-700. 4.van Uem JF, Siebzehnrübl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987 Mar 28;1(8535):752-3. 5.Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat-Baroux J, Alvarez S, Tibi C, Cohen J, Debache C, Tesquier L. Cryopreservation of human oocytes and embryos Hum Reprod 1988;3:117-19. 6.Siebzehnruebl ER, Todorow S, van Uem J, Koch R, Wildt L, Lang N. Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propranediol. Hum Reprod 1989;4:312-17. 7.Todorow SJ, Siebzehnrübl ER, Spitzer M, Koch R, Wildt L, Lang N. Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. Hum Reprod 1989;4:812-16. 8.Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986 Apr 19;1(8486):884-6. 9.Kippen R, Evans A, Gray E. Australian attitudes toward sex-selection technology. Fertil Steril 2011;95:1824-26. 10.Puri SMS, Nachtigall RD. The ethics of sex selection: a comparison of the attitudes and experiences of primary care physicians and physician providers of clinical sex selection services. Fertil Steril 2010;93:2107-14. 11.Ehrich K, Williams C, Farsides B, Sandall J, Scott R. Choosing embryos: ethical complexity and relational autonomy in staff accounts of PGD. Sociol Health Illn 2007;7:1091-106 12.Shenfield f. Ethical dilemmas in ART: Current Issues. In Botros R, Gracia-Velasco J, Sallam H, Makrigiannakis A. Infertility and Assisted Reproduction. Cambridge University press 2008, p717- 22..13. Mazur P, Rall WF, Leibo SP. Kinetics of water loss and the likelihood of intracellular freezing in mouse ove: influence of the method of calculating the temperature dependence of water permeability Cell Biophys. 1984:6:197-213. 14. Fahy GM, McFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiliogy 1984;21:407-26. 15koichi kyono et al Birth of a healthy male infant after transfer of vitrified warmed blastocyst derived from ICSI with vitrified warmed oocytes and frozen thawed sperm j of Assissted Reprod genetics2009 26 451-453. 16.Van Der Elst J. oocyte freezing, here to stay? Hum Reprod update 2003 9 463-70. 17.Podsiandly BT, wolcot RJ et al pregnancy resulting from intracytoplasmic injection of cryopreserved spermatozoa recoverd from testicular biopsy Hum reprod 1996:11;1306-8. 18.. Grace Ching et al, Birth of a healthy baby after transfer of blastocsyt derived from cryopreserved human oocyte fertilized with frozen sperm Fertil: Steril 2005 83Ehrich K, Williams C, Farsides B, Sandall J, Scott R. Choosing embryos: ethical complexity and relational autonomy in staff accounts of PGD. Sociol Health Illn 2007;7:1091-106:5 19.Tucker et al previous experience with human cryopreservation,Human Reprod 1996 7 183 20. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod. 1999 Dec;14(12):3077-9. Hum Reprod. 21. Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM, Cha KY. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril. 2003 Jun;79(6):1323-6. Yoon TKKim TJPark SEHong SWKo JJChung HMCha KYFertil Steril. 22. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril. 2003 Jul;80(1):223-4 Katayama KPStehlik JKuwayama MKato OStehlik EFertil Steril. 23. Porcu E, Fabbri R, Ciotti PM, Petracchi S, Seracchioli R, Flamigni C. Ongoing pregnancy after intracytoplasmic sperm injection of epididymal spermatozoa into cryopreserved human oocytes. J Assist Reprod Genet. 1999 May;16(5):283-5.Porcu EFabbri RCiotti PMPetracchi SSeracchioli RFlamigni CJ Assist Reprod Genet. 24Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S, Flamigni C. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol. 2000 Nov 27;169(1-2):33-7.Porcu EFabbri RDamiano GGiunchi SFratto RCiotti PMVenturoli SFlamigni CMol Cell Endocrinol. CASE REPORT OF A BIRTH OF A HEALTHY BABY BOY AFTER FERTILIZATION OF A CRYOPRESERVED OOCYTE WITH A CRYOPRESERVED TESTICULAR SPERMATOZOA FOLLOWED BY PREIMPLANTATION GENETIC SCREENING. Introduction We report the birth of a healthy male infant from a pregnancy resulting from a frozen thawed oocyte on which ICSI was performed using a frozen thawed spermatozoa obtained from testicular aspiraton. PGS[preimplantation genetic screening] was performed on the cleavage stage embryo[day 3] to determine the gender. People expressing a preference for a child of a particular sex have existed for ages. Greek philosophers and medical men, offered detailed advice on the means of ensuring the production of a child of a chosen sex. Aristotle gave advice on positions for sexual intercourse, and desirable diet for the mother. Anaxagoras believed that each testicle determined one sex, and advocated tying off one testicle before coitus. Hippocrates deduced that male and female children developed in different parts of the uterus (1). Preimplantation genetic screening for gender selection is an extremely sensitive issue. The application of techniques for sex selection, including the use of pre-implantation genetic diagnosis creates moral and ethical concerns in the opinion of some, while the advantages of sensible use of selected technologies is favoured by others. Since this is uncommon to perform in many IVF centres, no standard treatment protocol has been established.Although a number of births have been recorded, cryopreservation of oocytes combined with ICSI could offer young women wth chemo/radiotherapy a reasonable option to preserve and balance their family. ( 2 ) Although, freezing of embryos is ethically more acceptable that cryopreservation of oocytes, variable oocyte survival and pregnancy rates have precluded the application of oocyte cryopreservation in assisted reproductive technology. Cytotoxicity of the cryoprotectants, poor survival, low fertilization rates and high rates of polyploidy (3-7) has prevented the widespread use of oocytes cryopreservation, albeit of reports of healthy and normal babies (3,4,5,8). Resurgence of interest in human oocyte cryopreservation may be safe in appropriate circumstances, and it’s clinical application has resulted in a number of live births. Development of oocyte cryopreservation is predominantly as a consequence of prohibition of option of embryo cryopreservation in the countries like Italy, U.A.E. More than 1000 babies are born after pre-implantation genetic testing suggesting the accuracy and safety of the procedure and it has gained a place among the choices offered to the couples at risk of transmitting serious and incurable genetic diseases. It is a good alterative. We report the first case of gender selection of an embryo resulting from a frozen oocyte and a frozen sperm obtained from a TESA done earlier and preimplantation genetic screening was performed for non genetic reasons (gender selection)for social reasons. CASE REPORT The couple visited us for the complaint of secondary infertility of 5 years duration. Mrs X is a 31 year old lady,P3+1, all her pregnancies were spontaneous and had three full term normal deliveries and the last pregnancy ended up in a spontaneous miscarriage at six weeks of gestation. In the due course of time, husband had developed non obstructive azospermia and has been a diabetic for the past 9 years,his hormonal profile was normal, testicular aspiration of sperm was performed and thesperm was cryopreserved[TESA] Testicular sperm freezing was carried out with the use of a HEPES-buffered freezing medium (SpermFreeze solution; origio, medicult Denmark) containing 0.4% (vol/vol) human serum albumin. After centrifugation of partly disintegrated testicular tissue (2500 _ g; 10 minutes), the pellet was rinsed with 0.4 mL of IVF culture medium ( origio, medicult Denmark ) mixed (1:1) with Sperm Freeze solution, equilibrated at room temperature for 10 minutes, and loaded into vials. The vials were first placed vertically in liquid nitrogen vapours for 15 minutes and then plunged into liquid nitrogen. In conclusion, this case is one of its kind in the Gulf region,probably in the world to our knowledge, where PGS was performed on an embryo obtained from a cryopreserved oocyte and a cryopreserved sperm obtained from testicular aspiration on which PGS was done for gender selection.This was done as a social indication to balance the family which is allowed under U.A.E law at that time. This case also supports the concept that the develpomental competence of the oocyte is not affected by vitrification of oocytes and its biopsy. Methods. Mrs X had regular periods,has a strong family history of diabetes mellitus.Her hormonal profile was within normal limits and had polycystic ovaries on scan. She had a controlled ovarian hyperstimulation on a long downregulation protocol with rFSH when 35 eggs, 20 [MII] mature oocytes were collected and frozen to prevent Ovarian hyperstimulation. Oocyte cryopreservation was done by vitrification. The oocytes were vitrified according to the Cryotop method (Kuwayama et al., 2005, 2007). Briefly, Basic solution(BS) is a buffer solution.Equilibration solution (ES) was made up of 7.5% (v/v) ethylene glycol (EG; Ketazato BioPharama, Japan) and 7.5% (v/v) dimethylsulphoxide (;Ketazato BioPharama, Japan).Vitrification solution(VS) was prepared with 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/l sucrose. One drop on BS was aligned with another three ES drops in a small petri dish and another two drops of VS were loaded in the same dish. Then these solutions were allowed to equilibrate to room temperature (25C) for 15 min. Oocytes were then placed briefly in BS droplets; where a channel was opened with next drop of the ES to allow a gradual increase of the cryoprotective concentration and keep them on the edge of the BS drop for 3 minutes in room temperature. Then a new channel was made with next ES and transfer the oocytes to the edge of the first ES droplet and keep them there for another 3 minutes at room temperature. These oocytes were then transferred to the third ES for 9 minutes on room temperature. then oocytes were transferred to first Drop of VS for 1 min and then the second VS for 30 Seonds and subsequently loaded on the flattened tip of the Cryotop with approximately 2 μl of VS. The Cryotop was then immerse direct in liquid nitrogen and stored in the freezing tanks. Two months later Mrs X had a trial of ICSI of frozen oocytes by a sperm obtained from a frozen TESA done earlier, At the time of thawing, the vials were removed from liquid nitrogen and rapidly placed on warm water(30_C).After expulsion from the vials, the thawed samples were washed with IVF culture medium (origio, medicult Denmark) centrifuged 2500-g; 10min twice,where the pellet finally resuspended in a small volume of the same medium, and incubated at 37_C for 30 minutes before being used for ICSI. 9 oocytes were thawed. The vitrified oocytes were warmed by swiftly immersing the Cryotop in the Thawing solution (TS) solution Ketazato BioPharama, Japan) containing 1 mol/l sucrose for 1 min at 37 C temperature. The oocytes were then equilibrated in a Diluent solution(DS) Ketazato BioPharama, Japan) containing 0.5 mol/l sucrose for 3 min and two 5-min consecutive flushes in Washing Solution(WS) Ketazato BioPharama, Japan). These oocytes were then placed in the Fertilization media ( Cook) and incubated at 37C and 6% CO2 in air for 2 h prior to ICSI. 8 oocytes survived thawing and were injected, all were fertilized and cleaved and PGS was performed on all embryos on day three. Biopsy media (cook) was warmed on 37c and supplemented with HAS (sage).numbered droplets of biopsy media were layered in a Petri dish under oil (cook).Embryos were loaded in the numbered droplet. A hole was made in the zona pellucida of 20Mm using Laser assisted hatching. The location of the embryo was made in order to have a nucleated blastomere on the three clock position.The blastomere was slowly aspirated using aspiration pipettes of 42Mm (cook) and then released into the medium The biopsied embryos were then washed thoroughly from the biopsy media and Incubated in culture media (cook) until transfer. The blastomere was then spread on frosted slides till the blastomere burst and the nucleus is spread. Fixation of the blastomere was done by acetic acid, Methanol 1:3. The slides then under went genetic analysis for Sex selection, and a single embryo of XY karyotype was transferred. Endometrial preparation was done on a down regulated cycle Triptorelin 3.75mg was given on a deep intramuscular injection on the second day of a spontaneous period, a scan was performed to confirm down regulation and a serum estradiol value of 14 pg/ml was obtained to confirm the same.Hormone replacement was done by oral estradiol valerate 2mg three times a day for 9 days after which scan was performed to assess endometrial thickness, which was 9.5mm, progesterone suppositories 400mg twice daily were prescribed as a luteal support which was started on the day of thawing the oocytes.the luteal support and HRT was continued till the 12th week of gestation.[unit protocol] She got pregnant, and had a single intaruterine gestational sac with live fetus, and the pregnancy progressed till 35 weeks uneventfully when she had preterm premature rupture of membranes and had a vaginal delivery of an alive healthy baby boy who weighed 1.9 kg without any congenital abnormalities DISCUSSION Reproductive specialists who provide non-medical sex selection service argue that the technology is an expression of reproductive rights, was initiated and pursued by women, and is a sign of female empowerment. Besides family balancing and fulfillment of culture and religious practice (9), non-medical sex selection allows couples to make an informed family planning decisions, prevents unintended pregnancy and abortion, and minimizes intimate partner violence and / or child abuse (10) Opponents question whether women could truly express free choice under family and community pressures (11), incompatibility with unconditional parental acceptance of offspring, potential distortion of sex ratios (9) and sex discrimination (12). In addition, PGD only fro sex selection involves preferential use of embryos and termination of not-chosen embryos that raises the ethical questions of abortion.. In our Center we consider social sex selection only for family balancing and we have not noticed a persisted preference of a particular sex, social disagreement or religious conflict. The present study indicates that freezing of either oocytes or spermatozoa can be done efficiently and can avoid repetitive surgical procedures for future attempts. PGS for gender selection in resulting embryos can be an option for desirous couples, if the law permits.Although the first frozen-egg pregnancy was achieved by Chen in 1986, (8) this application was not adopted widely for clinical assistance, because survival and pregnancy rates were comparatively low. This freezing method was based on a slow cooling/rapid thawing procedure using propanediol (PROH) and sucrose as cryoprotectants, in line with the conditions originally developed for embryo freezing Alteration of the ooplasm organelles due to the formation of intracellular ice crystals is one of the main alterations inflicted on the oocytes by the freezing process (13). Vitrification was first introduced in order to avoid crystallization damage. Another advantage of this freezing method is that it is very simple and it is based on direct contact between the vitrification solution containing cryoprotectants and liquid nitrogen (14). Vitirification produces ultrarapid freezing method using high concentration of cryoprotectants (ethylene, glycol and DMSO) and rapid cooling -1500°C/min which helps to solidify without crystal formation. This reduces the thermal stress of oocytes and decreases the injury..Borini et al reported pregnancies and births after oocyte cryopreservation with 68 patients. Since then, several other pregnancies have been reported worldwide.(15). Cryopreservation of human oocytes is still considered an experimental technique inspite of improvements achieved over the past few years (16), However the cryopreservation of the sperm is a widely used and an efficient technique that can avoid repetition of surgical procedures for future ICSI attempts (17). Safety of this cryopreservation has now been established in the human reproductive field. Reports on the survival rates of cryopreserved human oocytes using the PROH–sucrose freezing method are highly variable, ranging from 25% to 95%, depending on the individual studyThe establishment of successful pregnancies from frozen oocytes is highly dependent on the efficacy in preserving the meiotic spindle or microtubule structures within the oocytes after cryopreservation. There has been a general consensus that the microtubules within the mammalian oocyte will depolymerize during freezing. Early insemination of frozen-thawed oocytes before full restoration of meiotic spindle may compromise fertilization outcome and subsequent embryonic development,, an incubation of 4 hours before insemination by ICSI may seem to be an appropriate time to allow for the full recovery of normal spindles in cryopreserved human oocytes.(18). Further more, hardening of zona due to vitrification can be overcome by ICSI19]. Applying ICSI to the thawed human oocytes reasonable fertilization and blastocyst rataes 50% and 43% respectively (15), more studies should be carried out to explore the optimal conditions for enhancing the developmental potential of frozenthawed human oocytes. Also, it has a role to play in young women requiring fertility preservation after chemo or radiotherapy, in cases of IVF when a sperm cannot be produced. Additionally, it may play a role in circumventing ethical and legal issues associated with established practice of cryopreservation of embryos (8). A number of babies born are being reported in the past few years much of which is attributed to the changes in the IVF laws. Such laws exist in countries like Italy and the U.A.E(2010). The first birth of the human oocyte cryopreservation was reported in 1986 (18). Until 2004 approximately 100 children were born. Pregnancy rate was low 1-5% due to low oocyte survival 25-40%, poor fertilization rate after IVF, high incidence of polyloidy and poor developmental capacity. Incidence of chromosomal abnormalities was not different from the fresh oocytes (19) ESHRE PGD consortium was set up in 1997 and has been collecting data on PGD and PGS, since then the consortium has analyzed 32.838 cycles for various indications out of which 786 cycles were for social reasons. Kuleshova et al (20) announced the birth of the first child from oocytes stored by vitrification, which was successfully adopted by other authors with the publication of ten additional pregnancies (21,22). Pregnancies from frozen eggs inseminated with epididymal and testicular (23) spermatozoa were published, as well as the birth of a child conceived with frozen eggs and frozen sperms (24). Larger studies are needed to quote a realistic figure of live birth rate by using frozen eggs. 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Veronica Bianchi declares no conflict of interest.
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