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First On-going Pregnancy via Frozen Embryo Transfer (FET) using Frozen Oocytes and Frozen Sperm

Introduction Results Conclusions

References1. Leibo SP. The early history of gamete cryobiology, LRC Press, 2004: 347-370.2. Polge E, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and

dehydration at low temperatures. Nature 1949; 164: 666.3. Chang MC. The effect of low temperature an fertilized rabbit ova in vitro, and the

normal development of ova kept at low temperature for several days. J Gen Physiol 1948; 31: 385-410.

4. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 1983; 305: 707-709.

5. Zeilmaker GH, Alberta AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 1984; 42: 293-296.

6. Friedler S, Giudice LC, Lamd EJ. Cryopreservation of embryos and ova. Fertil Steril 1988; 49: 743-764.

7. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 327: 884-886.

8. Kuleshova L, Gianaoli L, Magli M, Ferrarelti A, Trounson A. Birth following vitrification of a small number of human oocytes. Human Reprod 1999; 14: 3077-3079.

9. Yoon TK, Kim TJ, Park SE, et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer prodram. Fertil Steril 2003; 79: 1323-1326.

10. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. high survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223-224.

11. Kuwayama M, Vajta G, Leda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod BioMed Online 2005; 11: 608-614.

12. Smith GD, Fioravanti J. Oocyte and embryo cryopreservation. In: Gardner DK, ed. IVF: A Clinical Approach. Informa Healthcare USA, 2007.

The history of human gamete cryopreservation can be traced back to the late nineteenth century when Montegazza observed that human spermatozoa became immotile when cooled in snow1. With the development of freezing technologies and numerous empirical studies on animal gamete cryopreservation in the mid 1900s, rapid advancements in human gamete and embryo cryopreservation had been achieved. The discovery that glycerol imparts cryoprotective properties to spermatozoa during freezing and thawing was a major breakthrough2, and had resulted in thousands of children being born through Intra-uterine Insemination (IUI) of cryopreserved semen. Investigations in the late 1940s and the early 1950s by Chang on the low temperature storage of rabbit oocytes and embryos paved the way for studies on the cryopreservation of female gametes and oocytes3. In the early 1980s, application of slow freezing methodologies led to the establishment of the first human pregnancies following freezing and thawing of cleavage stage embryos4,5. In general, it has been demonstrated that oocytes are more sensitive to cryo-damage than later embryonic stages6. Chen first documented live birth from the use of thawed oocytes in 1986 using dimethylsulfoxide (DMSO) slow-freezing method as cryoprotectant7 and Kuleshova et al. recorded live births from vitrified human oocytes using ethylene glycol and sucrose in 19998.  A successful on-going pregnancy is documented here from cryopreserved embryos obtained from the fertilization of freeze-thawed spermatozoa and vitrified oocytes.  

It is important to realize that various fundamental aspects of gamete/ embryo cryopreservation must be followed for successful cell survival, normal cellular function, and subsequent development. However, numerous slight permutations exist between cryopreservation protocols that do not appear to influence clinical outcomes12. Due to difficulties in performing well-controlled, randomized, prospective clinical trials with human oocytes/ embryo, the future of successful gamete/ embryo cryopreservation lies in the strong understanding of cell development biology, theoretical aspects of cryobiology and practical experience. The last 50 years have been exciting times in cryobiology and one could imagine that the next fifty years may provide currently inconceivable advancements that will lead to betterment of health and happiness12. Our experience could possibly be the first reported case of a successful on going pregnancy via a frozen embryo transfer from frozen spermatozoa and vitrified oocytes.

Materials and Methods

The patient underwent Intra-cytoplasmic Sperm Injection (ICSI) program in November 2010 due to 3 years of male factor primary infertility (oligo-astheno-teratozoospermia). A long down regulation protocol with subcutaneous buserelin and follitropin- was implemented and 36 oocytes were collected, αbut her spouse suffered from erectile dysfunction on the day of oocyte retrieval due to anxiety. Despite repeated attempts with oral sildenafil, semen was not obtained and even an emergency Percutaneous Epididymal Sperm Aspiration (PESA) did not obtain any spermatozoa. Of the available oocytes, twenty-four were suitable for vitrification using Medicult Vitrification, and cryopreserved in liquid nitrogen in 6 straws six hours after oocyte retrieval. A fresh embryo transfer cycle was embarked in March 2011 utilizing the frozen oocytes. As a precaution, spermatozoa were frozen in February 2011 preceding the program. The patient was prepared for uterine receptivity with Intra-muscular depot Gonadotropin-Releasing Hormone (GnRHa) to achieve down regulation, then endometrial development was achieved with oral estradiol valerate in escalating doses. Progesterone pessaries were added on day-14 of the estradiol valerate priming, and the oocytes were thawed out on day-15 using Medicult warming method. Because her spouse was again unable to produce an ejaculate, the previously frozen spermatozoa were thawed out 3½ hours later. Nineteen of the 24 oocytes (79.1%) survived the thawing process and ICSI was performed using the thawed spermatozoa. Only six embryos were obtained and 3 were transferred at day-3 cleavage stage, while the remainder three were cryopreserved with slow freezing method using Vitrolife Freeze-kit 1. This resulted in a viable singleton pregnancy but ended with a missed miscarriage at 8 weeks gestation.  A frozen embryo transfer (FET) cycle was underway in August 2011 utilizing the 3 frozen embryos. The patient was again down regulated with Intra-muscular GnRHa depot and endometrial priming achieved with oral estradiol valerate and progesterone pessaries. The embryos were thawed out at day-18 of endometrial priming using Vitrolife Thawing-kit 1 and all 3 survived the process. Embryo transfer for all 3 embryos was done 2 hours after thawing and luteal support was with oral estradiol valerate and progesterone pessaries. A singleton viable pregnancy was achieved and the pregnancy is currently on going. Both mother and fetus are doing well in the antenatal stage and her delivery is due in June 2012.  

Numerous reports on successful human oocyte vitrification, followed by fertilization, embryo development and transfer, and healthy live births exist (Table 1).

The survival rate from vitrified oocytes of 79.1% (19 of 24) from our experience appears comparable to reports worldwide. The fertilization rate of the vitrified oocytes in this case with frozen spermatozoa was 31.5%(6 out of 19). It is important to note that the fertilization rates of vitrified oocytes ranging from 45% to 91%8-11 reported worldwide was with ‘fresh’ spermatozoa. As such, a lower fertilization rate from our observation is not surprising. Implantation rate from the embryo transfer cycle using the 3 embryos obtained from the frozen spermatozoa and vitrified oocytes was 33.3%, but did not result in a live birth. The current on going pregnancy, with frozen embryos from frozen spermatozoa and vitrified oocytes, also yielded a 33.3% implantation rate. 

Leong WY, Lee SS, Low SY - Alpha International Fertility Centre, MalaysiaAlpha Fertility CentreMalaysia