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The development of an effective oocyte cryopreservation system will have a significant impact on the clinical practice of reproductive medicine. However, an important option of emergency oocyte cryopreservation has yet to be well documented. In this report, we review the cases of 15 women with male partners who were diagnosed with nonobstructive azoospermia and for whom testicular sperm extraction on the day of oocyte retrieval failed. Emergency oocyte vitrification was performed and after two months, the vitrified oocytes were thawed and the surviving oocytes inseminated with frozen-thawed donor sperm by intracytoplasmic sperm injection (ICSI). A total of 117 mature oocytes from the 15 women were frozen and thawed. The post-thaw survival rate was 84.6% (99/117), and the fertilization rate following ICSI was 83.8% (83/99). We selected 30 embryos for transfer to 15 patients, 8 of whom became pregnant. The clinical pregnancy rate was 53.3% (8/15) and the implantation rate was 30.0% (9/30). Nine healthy live births resulted from 8 pregnancies. These results indicate that emergency oocyte vitrification is an effective rescue technique that can be applied clinically with acceptable pregnancy and live birth rates when testicular sperm extraction from the male partner failed on the day of oocyte retrieval. These results also highlight another important option for oocyte cryopreservation through the use of vitrification technology.
The development of an effective oocyte cryopreservation system would have a significant impact on the clinical practice of reproductive medicine. Such a program could be offered to cancer patients before gonadotoxic treatment and to infertile couples with moral or religious objections to embryo cryopreservation. In addition to fertility preservation for young women requiring sterilizing medical and surgical treatment, cryobanking of oocytes would benefit a large population of single women who wish to delay motherhood for personal, professional, or financial reasons.
Women suffering from premature ovarian failure who wish to conceive must rely on donor oocytes. Oocyte donation can be complicated and time consuming, requiring hormonal synchronization of the donor and recipient menstrual cycles. A successful oocyte cryopreservation protocol would eliminate the need for synchronization and enable the establishment of egg banks, facilitating the logistics of coordinating egg donors with recipients. Furthermore, egg cryopreservation allows for temporary quarantine of donor eggs to test the donors for transmissible diseases.
Although oocyte cryopreservation provides many benefits, the importance of emergency oocyte cryopreservation has not been emphasized. Many unpredicted situations can occur during infertility treatments; for example, a male partner is unable or refuses to produce semen. Surgical testicular retrieval of sperm for intracytoplasmic sperm injection (ICSI) is a widely practiced technique for treating men with diagnosed with nonobstructive azoospermia (NOA) (Devroey et al., 1995). However, spermatogenesis in NOA is usually limited, with only small foci spread over limited areas, so retrieving sperm can be a challenge. Diagnostic biopsy has significant predictive value, but the chance that no sperm will be found is 25%-30% (Glina et al., 2005). If no sperm can be obtained, cryopreservation of oocytes or use of a sperm donor is the only possible alternative. However, in some countries like China, the use of donor sperm as an emergency measure is illegal and male partners may require more time to consider this option. Therefore, emergency oocyte cryopreservation may be employed to avoid this situation. So far, the information available for emergency oocyte cryopreservation is very limited (Emery et al., 2004; Kyono et al., 2005).
The objective of this study is to report the pregnancy and obstetric outcomes in 15 women through the use of emergency oocyte vitrification and perinatal outcomes from frozen-thawed sperm.
Materials and Methods
From June 2008 to May 2009, 108 patients with NOA were treated by TESE in the first affiliated hospital of Zhengzhou University, China. Of these, we failed to extract sperm from the testicular tissues in 22 male patients (20.4%) on the day of oocyte retrieval. After informed consent, 15 patients accepted emergency oocyte vitrification, due to failure of sperm extraction from the testicular tissues on day of oocyte retrieval. Institutional review board approval was obtained for this retrospective study.
Before ovarian stimulation, all female patients underwent a physical examination, trans-vaginal basal antral follicle count, and a baseline hormonal pro¬le on day 3 for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2). The mean age of the women was 27.9 ± 2.8 years. The mean duration of infertility was 4.1 ± 2.3 years. The mean levels on day 3 were 6.0 ± 1.3 IU/L for FSH, 6.2 ± 3.4 IU/ L for LH, and 45.1 ± 21.9 pg/L for E2, respectively (Table 1).
Ovarian stimulation was performed using a GnRH agonist (Ferring GmbH, Germany) combined with recombinant FSH (Serono Laboratories, Switzerland) and HMG (Ferring GmbH, Germany), and then 10,000 IU of human chorionic gonadotropins (Serono Laboratories, Switzerland) was administered when at least two follicles reached 18 mm in diameter. Oocyte retrieval was performed 36 hours later.
All male partners were diagnosized as NOA by hormonal assessment (FSH, LH and testosterone), biochemical marker assessment (fructose and -glucosidase), karyotype analysis, and Yq microdeletion assessment. Testicular biopsy was performed and histopathological analysis demonstrated that mature sperm were found in the testicular tissue sample in advance, but the testicular tissue was not frozen.
Within 2 hours of oocyte retrieval, oocyte-cumulus complexes (COCs) were exposed to 60 IU/mL hyaluronidase for partial removal of cumulus cells. Only metaphase II (MII) oocytes were selected for vitrification. A modified vitri¬cation method was adopted for cryopreservation of the mature oocytes (Chian et al., 2005; Antinori et al., 2007). Briefly, the oocytes were placed into equilibration medium, containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO), at room temperature for 5 minutes, and then the oocytes were transferred to vitrification medium, containing 15% (v/v) EG, 15% (v/v) DMSO, and 0.50 mol/L sucrose at room temperature for 45 to 60 seconds. Two or three oocytes were loaded on a McGill Cryoleaf (Medicult Company, Denmark) and plunged immediately in liquid nitrogen for vitrification and then for storage.
Two months later, the couples decided to use frozen donor sperm of the same blood type as the male partner and the oocytes were thawed by inserting the McGill Cryoleaf directly into thawing medium (MediCult Company, Denmark) containing 1.00 mol/L sucrose for 1 minute at 37°C. The thawed oocytes were transferred into diluent medium-I containing 0.50 mol/L sucrose and then into diluent medium-II containing 0.25 mol/L sucrose for 3 minutes each. The oocytes were washed twice in washing medium for 3 minutes each time after which they were incubated in culture medium under 6% CO2 at 37°C for approximately 2 hours. Oocyte survival after thawing was evaluated microscopically based on the morphology of the oocyte membrane integrity.
Prior to ICSI, frozen donor sperm (Sperm Bank, Hunan Province, China) were thawed rapidly at 37°C for 10 minutes in water. Cryoprotectant was removed by centrifugation at 500 x g for 15 minutes and re-suspension in 0.5 mL of fresh medium. A motile spermatozoon was injected into each surviving oocyte, and fertilization was checked approximately 16-18 hours after ICSI. Embryo transfer (ET) was performed under trans-abdominal ultrasound guidance on either day 2 or 3, depending on the number and quality of the embryos. Before ET, assisted hatching was performed with a laser system OCTX (Eyeware TM Company, Germany). Clinical pregnancy was defined as the presence of a fetal sac with heartbeats revealed by ultrasonography.
The endometrial lining was prepared for approximately 2 weeks using E2 transdermal patches until the endometrial thickness was 8 mm before oocyte thawing. Progesterone (100 mg/d) was started when the oocytes were thawed, continuing until the time of β-hCG assay. Both E2 and progesterone were continued for luteal support until the 12th week of pregnancy if positive clinical pregnancy was confirmed.
As shown in Table 2, total of 135 COCs were retrieved, and of these 117 oocytes were mature at the MII stage. Following thawing, the oocyte survival rate was 84.6% (99/117). A total of 83 oocytes were fertilized after ICSI, and the fertilization rate was 83.8% (83/99). Of 83 fertilized oocytes, 75 cleaved resulting in a cleavage rate of 90.4% (75/83). A total of 30 selected embryos were transferred to 15 patients, an average of 2.0 ± 0.8 embryos per patient. Ten days after ET, 9 individuals were confirmed pregnant by beta-hCG followed by ultrasound 4 weeks after ET with a clinical pregnancy rate of 53.3% (9/15) and an implantation rate of 30.0% (9/30).
Obstetric analysis indicated 8 pregnancies with 9 healthy infants born (7 singletons and 1 set of twins) (Table 3). Of the 9 infants, 5 were male and 4 were female; all had normal karyotypes. For the singletons, the mean gestational age was 39 weeks + 5 days and the mean birth weight was 3,807 ± 397.3 g, while for the twins the gestation age was 38 weeks + 6 days and the mean birth weight was 2,625± 530.3 g.
These results indicate that acceptable clinical pregnancy rates and healthy live-births can be achieved from emergency oocyte vitrification followed by the use of donor sperm for insemination when it is not possible to extract sperm from the testicular tissues of the male partner. This suggests that oocyte vitrification technology is an important option that can be applied to this unexpected situation during the treatment.
As mentioned previously, although diagnostic biopsy has significant predictive value, the chance that sperm cannot be retrieved on the day of oocyte retrieval is 25%-30% (Glina et al., 2005). Some have suggested that the cryopreservation of sperm from TESE before treatment should be implemented to prevent this situation (Wu et al., 2005).However, the loss of motility of testicular sperm from frozen-thawing procedures may result in a significant reduction in the fertilization rate. It has been reported that in testicular biopsy samples from the patients with NOA less than 3% of the sperm observed were motile (Lyrakou et al., 2007). Therefore, finding sufficiently motile sperm for ICSI in frozen-thawed testicular tissues in NOA patients is more difficult, resulting in no embryos being available for transfer.
In addition, it has been reported that immature sperm from testicular tissues are much more sensitive to freezing and thawing, possibly leading to a higher rate of aneuploidy among embryos conceived from these frozen-thawed sperm (Ravizzini et al., 2008). Therefore, in our IVF center, we do not routinely freeze immotile sperm from testicular biopsy samples. It remains to be confirmed whether or not testicular tissues from diagnostic biopsy should be frozen as a back up for NOA patients in case sperm cannot be retrieved on the day of oocyte retrieval (Kyono et al., 2005; Chen et al., 2008).
Over the last five years, the new vitrification techniques have significantly improved the survival of cryopreserved oocytes, indicating that vitrification may be more effective than the slow-freezing method of oocyte cryopreservation (Kuleshova and Lopata, 2002). Vitrification of oocytes has resulted in relatively high survival rates (Kuleshova, et al. 1999; Kuleshova and Lopata 2002; Yoon, et al. 2003; Lucena, et al. 2006; Chian, et al. 2008; 2009; Cao, et al. 2009; Cobo, et al., 2008; 2010; Nagy, et al. 2009; Rienzi et al., 2010). The results from this study showed an 84.6% survival rate, 30.0% implantation rate, and 53.3% clinical pregnancy rate following oocyte vitrification and thawing (Table 2), which are comparable to previously reported results.
In our experience, we found the cleavage rate after fertilization from vitrified and thawed oocytes is lower than that of fresh oocytes (Table 2, 90.4% in this observation). Furthermore, the time of cleavage after fertilization from vitrified and thawed oocytes is also slightly delayed compared to that of fresh oocytes. Therefore, the lower cleavage rate and time delay involved in cleavage need to be studied properly using fresh oocytes as controls.
It has been reported that pregnancies and infants conceived following oocyte vitrification are not associated with increased risk of adverse obstetric and perinatal outcomes (Chian et al., 2008; 2009). Although the number of pregnancies and live-births was small, the results from our observation also support this finding (Table 3).
In conclusion, these results indicate that emergency oocyte vitrification is an effective rescue technique that can be applied clinically with acceptable pregnancy and live birth rates when testicular sperm extraction is unsuccessful in the male partner on the day of oocyte retrieval.