**6. Is oocyte cryopreservation "experimental?"**

The American Society for Reproductive Medicine (ASRM) published a committee opinion in 2008 which stated that "the experimental nature of oocyte cryopreservation suggests

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 199

appears to reduce zona pellucida hardening and leads to increased fertilization. Embryos obtained from cryopreserved oocytes have a similar incidence of chromosomal abnormalities when compared to control embryos using fluorescence in situ hybridization (FISH) (Cobo et al., 2001). Multiple recent studies have evaluated pregnancy, live birth, and early childhood outcomes in children born after mature oocyte cryopreservation (Borini et al., 2007; Chian et al., 2008a, 2008b; Noyes et al., 2009; Oktay et al., 2006; Wennerholm et al., 2009); these have not documented an increased rate of congenital anomalies among children born after oocyte cryopreservation. These studies are discussed in more detail in the next

In light of these concerns about cryopreservation, the ASRM maintains that oocyte cryopreservation should be considered an experimental procedure. The ASRM specifically states that assisted reproductive technology (ART) procedures should be considered "experimental" until "the published medical evidence regarding their […] overall safety and efficacy is sufficient to regard them as standard medical practice. [This] medical evidence can derive only from appropriately designed, peer-reviewed, published studies performed by multiple independent investigators" (ASRM Practice Committee, 2009). Other authors have supported this statement, by noting that "because the largest demand for oocyte cryopreservation most probably is going to come from women who wish to delay childbearing electively, it is quite likely that several years will be required before sufficient births have occurred to determine the true safety of cryopreserved oocytes" (Jain & Paulson, 2006). Some authors, however, argue against this labeling of oocyte cryopreservation, stating that a variety of commonly used assisted reproductive technologies have never been studied "under the auspices of an IRB" before implementation into standard practice (Noyes

The safety of oocyte cryopreservation has been evaluated through studies of pregnancy, perinatal, and childhood outcomes, in which over 900 infants have been evaluated (Chian et al., 2000b; Noyes et al., 2009). There does not seem to be an increased risk for adverse pregnancy outcomes or congenital anomalies in pregnancies conceived after oocyte cryopreservation, thaw, fertilization and embryo transfer. Additionally, cryopreservation may introduce an extra safety measure with regard to quarantine for infectious disease, similar to protocols in place for cryobanking of donor sperm. By freezing donated oocytes, additional infectious disease testing can be done months after oocyte retrieval to ensure optimal embryo transfer and pregnancy outcome. In an early 2007 paper by Barritt et al., 4 oocyte donors underwent synchronous ovarian stimulation with 4 recipient patients with impaired ovarian reserve, elevated basal FSH, and prior unsuccessful IVF treatments (Barritt et al., 2007). The donors were given a complete medical examination in accordance with ASRM guidelines for oocyte donors, which included a full history, physical exam, BAFC, and cervical cultures. In addition, these women had serological testing for infectious diseases, including HIV, hepatitis B and C, syphilis, gonorrhea, cytomegalovirus, and a urine drug screen. While this initial workup for oocyte donors seems exhaustive, additional checkpoints for infectious disease testing after an extended period of cryopreservation will further prevent the spread of communicable disease and improve pregnancy outcomes by preventing congenital infections. Data continue to emerge supporting the safety and efficacy of oocyte cryopreservation and it is likely that the "experimental" label will soon be

section.

et al., 2010).

removed from this technique.

potential for clinical application […] it might therefore be acceptable […] with appropriate informed consent under the auspices of an IRB" (ASRM Practice Committee, 2008). This "experimental" label was first published by ASRM in 2006. Some studies have looked at provider compliance with this ASRM practice guideline and likelihood of referral for oocyte cryopreservation. In a retrospective study of 530 IVF centers in the United States, 69% of these centers (365/530) were found to offer oocyte cryopreservation. Of these centers, only 62% do so under IRB approval, while 15% reported having an IRB pending and 18% did not use an IRB at all for oocyte cryopreservation (Beck et al., 2009). Compliance with ASRM guidelines was highest in the northeast (71%) and the size of the program was inversely related to the likelihood that oocyte cryopreservation occurred in conjunction with IRB approval. Still, these numbers indicate relatively high compliance with ASRM guidelines. In a different survey of healthcare providers at 5 United States IVF centers, physician preferences and recommendations were analyzed for practice patterns regarding oocyte cryopreservation. More than half of providers considered the ideal age for oocyte cryopreservation to be less than 35 years and 50% found it acceptable for a woman to preserve fertility in this way with a day 3 FSH value of <13 IU/L. A large proportion of providers were less likely to recommend egg freezing to patients with a low BAFC. Additionally, 89% of physicians were more likely to offer oocyte cryopreservation to their patients if there was a medical indication for the procedure, instead of elective reasons for fertility preservation (Luna et al., 2008). Providers recognized the emerging role oocyte cryopreservation will have in the field of fertility preservation. Thus, despite current reservations regarding which patients to refer for oocyte cryopreservation and ASRM guidelines, physicians view oocyte cryopreservation as a technique that will continue to be used with increasing frequency.

Discussion about the safety and efficacy of oocyte cryopreservation has focused on potential concern about meiotic spindle interruption from freezing, hardening of the zona pellucida (which may decrease rates of fertilization), and the potential risk of anomalies and abnormalities that may arise in the setting of a new technique without much outcome data. The meiotic spindle is a dynamic structure that forms during mitosis and meiosis to facilitate chromosomal segregation. Disruption of the meiotic spindle increases the risk of aneuploidy. These concerns were studied by Rienzi et al. by slow freezing oocytes and looking at the meiotic spindle using computer-assisted polarization microscopy (Rienzi et al., 2004). This technique allowed visualization of the spindle in real time by evaluating living oocytes. Previous studies had used electron microscopy or immunocytochemistry, which requires cell fixation and does not permit evaluation of dynamic spindle activity (reviewed in Eichenlaub-Ritter et al., 2002). Though spindles disappeared in oocytes during the thawing process, all surviving post-thaw oocytes were noted to have intact, functional meiotic spindles. Thus, it appears that cryopreserved oocytes are capable of reforming the meiotic spindle apparatus after thawing (Noyes et al., 2010). Hardening of the zona pellucida (ZP, the transparent glycoprotein envelope that surrounds a mature mammalian oocyte) is thought to occur due to premature cortical granule release during cryopreservation (Jain & Paulson, 2006). This release leads to early hardening of the ZP, which impedes penetration and fertilization by sperm. The advent of ICSI in 1992 introduced a solution to ZP hardening, in which the zona is bypassed by direct injection of the sperm into the oocyte. Additionally, vitrification of oocytes in calcium-free media

potential for clinical application […] it might therefore be acceptable […] with appropriate informed consent under the auspices of an IRB" (ASRM Practice Committee, 2008). This "experimental" label was first published by ASRM in 2006. Some studies have looked at provider compliance with this ASRM practice guideline and likelihood of referral for oocyte cryopreservation. In a retrospective study of 530 IVF centers in the United States, 69% of these centers (365/530) were found to offer oocyte cryopreservation. Of these centers, only 62% do so under IRB approval, while 15% reported having an IRB pending and 18% did not use an IRB at all for oocyte cryopreservation (Beck et al., 2009). Compliance with ASRM guidelines was highest in the northeast (71%) and the size of the program was inversely related to the likelihood that oocyte cryopreservation occurred in conjunction with IRB approval. Still, these numbers indicate relatively high compliance with ASRM guidelines. In a different survey of healthcare providers at 5 United States IVF centers, physician preferences and recommendations were analyzed for practice patterns regarding oocyte cryopreservation. More than half of providers considered the ideal age for oocyte cryopreservation to be less than 35 years and 50% found it acceptable for a woman to preserve fertility in this way with a day 3 FSH value of <13 IU/L. A large proportion of providers were less likely to recommend egg freezing to patients with a low BAFC. Additionally, 89% of physicians were more likely to offer oocyte cryopreservation to their patients if there was a medical indication for the procedure, instead of elective reasons for fertility preservation (Luna et al., 2008). Providers recognized the emerging role oocyte cryopreservation will have in the field of fertility preservation. Thus, despite current reservations regarding which patients to refer for oocyte cryopreservation and ASRM guidelines, physicians view oocyte cryopreservation as a technique that will continue to be

Discussion about the safety and efficacy of oocyte cryopreservation has focused on potential concern about meiotic spindle interruption from freezing, hardening of the zona pellucida (which may decrease rates of fertilization), and the potential risk of anomalies and abnormalities that may arise in the setting of a new technique without much outcome data. The meiotic spindle is a dynamic structure that forms during mitosis and meiosis to facilitate chromosomal segregation. Disruption of the meiotic spindle increases the risk of aneuploidy. These concerns were studied by Rienzi et al. by slow freezing oocytes and looking at the meiotic spindle using computer-assisted polarization microscopy (Rienzi et al., 2004). This technique allowed visualization of the spindle in real time by evaluating living oocytes. Previous studies had used electron microscopy or immunocytochemistry, which requires cell fixation and does not permit evaluation of dynamic spindle activity (reviewed in Eichenlaub-Ritter et al., 2002). Though spindles disappeared in oocytes during the thawing process, all surviving post-thaw oocytes were noted to have intact, functional meiotic spindles. Thus, it appears that cryopreserved oocytes are capable of reforming the meiotic spindle apparatus after thawing (Noyes et al., 2010). Hardening of the zona pellucida (ZP, the transparent glycoprotein envelope that surrounds a mature mammalian oocyte) is thought to occur due to premature cortical granule release during cryopreservation (Jain & Paulson, 2006). This release leads to early hardening of the ZP, which impedes penetration and fertilization by sperm. The advent of ICSI in 1992 introduced a solution to ZP hardening, in which the zona is bypassed by direct injection of the sperm into the oocyte. Additionally, vitrification of oocytes in calcium-free media

used with increasing frequency.

appears to reduce zona pellucida hardening and leads to increased fertilization. Embryos obtained from cryopreserved oocytes have a similar incidence of chromosomal abnormalities when compared to control embryos using fluorescence in situ hybridization (FISH) (Cobo et al., 2001). Multiple recent studies have evaluated pregnancy, live birth, and early childhood outcomes in children born after mature oocyte cryopreservation (Borini et al., 2007; Chian et al., 2008a, 2008b; Noyes et al., 2009; Oktay et al., 2006; Wennerholm et al., 2009); these have not documented an increased rate of congenital anomalies among children born after oocyte cryopreservation. These studies are discussed in more detail in the next section.

In light of these concerns about cryopreservation, the ASRM maintains that oocyte cryopreservation should be considered an experimental procedure. The ASRM specifically states that assisted reproductive technology (ART) procedures should be considered "experimental" until "the published medical evidence regarding their […] overall safety and efficacy is sufficient to regard them as standard medical practice. [This] medical evidence can derive only from appropriately designed, peer-reviewed, published studies performed by multiple independent investigators" (ASRM Practice Committee, 2009). Other authors have supported this statement, by noting that "because the largest demand for oocyte cryopreservation most probably is going to come from women who wish to delay childbearing electively, it is quite likely that several years will be required before sufficient births have occurred to determine the true safety of cryopreserved oocytes" (Jain & Paulson, 2006). Some authors, however, argue against this labeling of oocyte cryopreservation, stating that a variety of commonly used assisted reproductive technologies have never been studied "under the auspices of an IRB" before implementation into standard practice (Noyes et al., 2010).

The safety of oocyte cryopreservation has been evaluated through studies of pregnancy, perinatal, and childhood outcomes, in which over 900 infants have been evaluated (Chian et al., 2000b; Noyes et al., 2009). There does not seem to be an increased risk for adverse pregnancy outcomes or congenital anomalies in pregnancies conceived after oocyte cryopreservation, thaw, fertilization and embryo transfer. Additionally, cryopreservation may introduce an extra safety measure with regard to quarantine for infectious disease, similar to protocols in place for cryobanking of donor sperm. By freezing donated oocytes, additional infectious disease testing can be done months after oocyte retrieval to ensure optimal embryo transfer and pregnancy outcome. In an early 2007 paper by Barritt et al., 4 oocyte donors underwent synchronous ovarian stimulation with 4 recipient patients with impaired ovarian reserve, elevated basal FSH, and prior unsuccessful IVF treatments (Barritt et al., 2007). The donors were given a complete medical examination in accordance with ASRM guidelines for oocyte donors, which included a full history, physical exam, BAFC, and cervical cultures. In addition, these women had serological testing for infectious diseases, including HIV, hepatitis B and C, syphilis, gonorrhea, cytomegalovirus, and a urine drug screen. While this initial workup for oocyte donors seems exhaustive, additional checkpoints for infectious disease testing after an extended period of cryopreservation will further prevent the spread of communicable disease and improve pregnancy outcomes by preventing congenital infections. Data continue to emerge supporting the safety and efficacy of oocyte cryopreservation and it is likely that the "experimental" label will soon be removed from this technique.

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 201

**7. Long-term pregnancy and health outcomes after oocyte cryopreservation**  Given the evolving nature of the technology and the heterogeneity of patient-population and cryopreservation techniques, the actual "success rate" of egg freezing is unknown. Review of the literature, however, suggests that the efficiency of cryopreservation appears to be improving. One analysis of slow-freezing demonstrated improvement in live birth rates from 21.6% per transfer from 1996 to 2004 to 32.4% from 2002 to 2004 (Oktay et al., 2006). Vitrification data shows a similar trend of improvement: 29.4% live birth rate before 2005 versus 39% after 2005. In a study out of McGill University Health Center, 38 women underwent ovarian stimulation and vitrification of retrieved oocytes. After cryopreservation for one full menstrual cycle, there was an 81% thaw survival rate, 75.6% of oocytes were successfully fertilized, and a 50% pregnancy rate per cycle started was achieved (Chian et al., 2008a). Ultimately, 39.5% of women who initiated ovarian stimulation and cryopreservation cycles gave birth to live infants. Nine of these births were singleton, while the remaining six deliveries were multiples (five twins and one triplet). While initial high rates of spontaneous abortion were documented after oocyte cryopreservation (Borini et al.,

2004), these rates have declined with a corresponding increase in live birth rates.

2009).

Since the early 2000s, studies have begun reporting pregnancy and neonatal outcomes following oocyte cryopreservation. Borini et al. reported 13 children born after slow freeze cryopreservation in 2004. All babies born were found to have a normal karyotype and no malformations were seen in their study group. They did note, however, a 20% spontaneous abortion rate in their cohort of patients who had undergone oocyte cryopreservation cycles (Borini et al., 2004). In a later study out of Italy, 149 pregnancies occurred after using a slow freeze protocol for oocyte cryopreservation. This group, again, had a relatively high spontaneous abortion rate of 23.5% (Borini et al., 2007). Reports of live births following oocyte cryopreservation have also emerged from groups in China. Chian et al. found that neonates born after ovarian stimulation and oocyte vitrification were all appropriate birthweights, none weighed <2500g. Additionally, all singletons in their cohort were born at term, with a mean gestational age at delivery of 39 1/7 weeks (Chian et al., 2008a). The same group analyzed 165 pregnancies resulting in 200 babies born after vitrification of oocytes at three centers. In their study, multiple gestations were more likely to deliver in the late preterm period (between 34-37 weeks' gestation) – 57% vs. 22% of singleton pregnancies. This is consistent with current expectations for multiple gestations in the general population. Additionally, 74% of multiples in their study were low birth weight (LBW, <2500g), with 5% of the cohort being very low birth weight (VLBW, <1500g). This was in comparison to singleton neonates born after vitrification, only 17% of which were LBW and 0.7% were VLBW. These birth weights are not significantly different when compared to women who spontaneously conceived or had fresh IVF (Chian et al., 2008b). Birthweight was also analyzed in a systematic review of pregnancy outcome data after oocyte cryopreservation and found to be consistently within normal limits (Wennerholm et al.,

Some concerns have been raised about the rate of malformation or congenital anomalies seen in babies born after any assisted reproductive technology. Epigenetic syndromes (such as Beckwith-Weidemann Syndrome and Angelman Syndrome) have been reported as more common, specifically after ICSI (Noyes et al., 2009). With regard to egg freezing, of 105 babies studied by Borini et al. in 2007, only 2 malformations were seen; one infant was born

Multiple studies from different investigators and institutions have compared the efficacy of oocyte cryopreservation to fresh oocyte cycles. An early IRB-approved prospective study of four donor-recipient oocyte cycles by Barritt et al. demonstrated high pregnancy and implantation rates following slow-freezing and overnight storage before thawing. After ICSI, the authors demonstrated an 89.7% fertilization rate and 91.8% of these fertilized oocytes cleaved normally. Of 23 transferred embryos, 26.1% implanted and 75% of implanted embryos led to clinical pregnancy (Barritt et al., 2007). Cobo et al. performed a study in which fresh oocytes from the same donor were either inseminated directly or vitrified for at least 1 hour before thaw and insemination (Cobo et al., 2008). In comparing embryo quality and clinical outcomes, they found that vitrified/thawed oocytes produced embryos capable of a 47.8% ongoing pregnancy rate, which was similar to fresh oocytes. In addition, Grifo and Noyes performed an age-matched control study of 23 oocyte cryopreservation cycles and fresh oocyte control cycles. Fertilization rates, blastocyst formation, and pregnancy rates were not significantly different between these two matched groups (Grifo & Noyes, 2010). This indicates that frozen/thawed oocytes perform as well as fresh oocytes in ART procedures. Finally, Nagy et al. demonstrated high efficiency of egg cryobanking, with a 55% implantation rate and delivery of 26 live infants. Furthermore, their study showed that twice-frozen gametes (i.e. oocyte cryopreservation followed by fertilization and supernumerary embryo vitrification) can lead to pregnancy after embryo thawing (Nagy et al., 2009). The efficacy of oocyte cryopreservation has thus been established by multiple independent groups in the literature, strengthening the argument to remove its experimental status.

For female cancer patients, treatment regimens of intensive chemotherapy, ionizing radiation, and bone marrow transplantation can lead to premature ovarian failure, with direct impact on the number and viability of remaining oocytes. Gonadotropin-releasing hormone (GnRH) analogues have been studied as a method for fertility preservation before cytotoxic treatments. By suppressing ovarian function and, essentially, rendering the ovary quiescent, it is thought that chemotherapeutics and radiation would not be able to affect post-treatment ovarian function. Unfortunately, this strategy does not have welldocumented efficacy in the literature (Maltaris et al., 2009). Additionally, studies are lacking that have documented resultant oocyte and embryo quality following a course of chemotherapy (ASRM Practice Committee, 2008). Consequently, oocyte or ovarian tissue cryopreservation may be more reliable methods of fertility preservation for female cancer patients.

Finally, it is important to consider the ethical dilemmas of embryo cryopreservation that are bypassed by using oocyte cryopreservation. These issues, while not directly related to safety and efficacy of oocyte cryopreservation, provide additional support for arguments about the importance of this method to avoid the moral impasses generated by embryo cryopreservation and storage. Embryo cryopreservation has legal implications worldwide. Ovarian stimulation cycles and IVF procedures frequently lead to supernumerary cryopreserved embryos. Over 400,000 embryos are currently stored in the United States alone (Hoffman et al., 2003), leading to high rates of embryo abandonment in IVF clinics. The issue of embryo disposal versus continued cryopreservation is one which IVF clinics deal with daily.

Multiple studies from different investigators and institutions have compared the efficacy of oocyte cryopreservation to fresh oocyte cycles. An early IRB-approved prospective study of four donor-recipient oocyte cycles by Barritt et al. demonstrated high pregnancy and implantation rates following slow-freezing and overnight storage before thawing. After ICSI, the authors demonstrated an 89.7% fertilization rate and 91.8% of these fertilized oocytes cleaved normally. Of 23 transferred embryos, 26.1% implanted and 75% of implanted embryos led to clinical pregnancy (Barritt et al., 2007). Cobo et al. performed a study in which fresh oocytes from the same donor were either inseminated directly or vitrified for at least 1 hour before thaw and insemination (Cobo et al., 2008). In comparing embryo quality and clinical outcomes, they found that vitrified/thawed oocytes produced embryos capable of a 47.8% ongoing pregnancy rate, which was similar to fresh oocytes. In addition, Grifo and Noyes performed an age-matched control study of 23 oocyte cryopreservation cycles and fresh oocyte control cycles. Fertilization rates, blastocyst formation, and pregnancy rates were not significantly different between these two matched groups (Grifo & Noyes, 2010). This indicates that frozen/thawed oocytes perform as well as fresh oocytes in ART procedures. Finally, Nagy et al. demonstrated high efficiency of egg cryobanking, with a 55% implantation rate and delivery of 26 live infants. Furthermore, their study showed that twice-frozen gametes (i.e. oocyte cryopreservation followed by fertilization and supernumerary embryo vitrification) can lead to pregnancy after embryo thawing (Nagy et al., 2009). The efficacy of oocyte cryopreservation has thus been established by multiple independent groups in the literature, strengthening the argument to

For female cancer patients, treatment regimens of intensive chemotherapy, ionizing radiation, and bone marrow transplantation can lead to premature ovarian failure, with direct impact on the number and viability of remaining oocytes. Gonadotropin-releasing hormone (GnRH) analogues have been studied as a method for fertility preservation before cytotoxic treatments. By suppressing ovarian function and, essentially, rendering the ovary quiescent, it is thought that chemotherapeutics and radiation would not be able to affect post-treatment ovarian function. Unfortunately, this strategy does not have welldocumented efficacy in the literature (Maltaris et al., 2009). Additionally, studies are lacking that have documented resultant oocyte and embryo quality following a course of chemotherapy (ASRM Practice Committee, 2008). Consequently, oocyte or ovarian tissue cryopreservation may be more reliable methods of fertility preservation for female cancer

Finally, it is important to consider the ethical dilemmas of embryo cryopreservation that are bypassed by using oocyte cryopreservation. These issues, while not directly related to safety and efficacy of oocyte cryopreservation, provide additional support for arguments about the importance of this method to avoid the moral impasses generated by embryo cryopreservation and storage. Embryo cryopreservation has legal implications worldwide. Ovarian stimulation cycles and IVF procedures frequently lead to supernumerary cryopreserved embryos. Over 400,000 embryos are currently stored in the United States alone (Hoffman et al., 2003), leading to high rates of embryo abandonment in IVF clinics. The issue of embryo disposal versus continued cryopreservation is one which IVF clinics

remove its experimental status.

patients.

deal with daily.

#### **7. Long-term pregnancy and health outcomes after oocyte cryopreservation**

Given the evolving nature of the technology and the heterogeneity of patient-population and cryopreservation techniques, the actual "success rate" of egg freezing is unknown. Review of the literature, however, suggests that the efficiency of cryopreservation appears to be improving. One analysis of slow-freezing demonstrated improvement in live birth rates from 21.6% per transfer from 1996 to 2004 to 32.4% from 2002 to 2004 (Oktay et al., 2006). Vitrification data shows a similar trend of improvement: 29.4% live birth rate before 2005 versus 39% after 2005. In a study out of McGill University Health Center, 38 women underwent ovarian stimulation and vitrification of retrieved oocytes. After cryopreservation for one full menstrual cycle, there was an 81% thaw survival rate, 75.6% of oocytes were successfully fertilized, and a 50% pregnancy rate per cycle started was achieved (Chian et al., 2008a). Ultimately, 39.5% of women who initiated ovarian stimulation and cryopreservation cycles gave birth to live infants. Nine of these births were singleton, while the remaining six deliveries were multiples (five twins and one triplet). While initial high rates of spontaneous abortion were documented after oocyte cryopreservation (Borini et al., 2004), these rates have declined with a corresponding increase in live birth rates.

Since the early 2000s, studies have begun reporting pregnancy and neonatal outcomes following oocyte cryopreservation. Borini et al. reported 13 children born after slow freeze cryopreservation in 2004. All babies born were found to have a normal karyotype and no malformations were seen in their study group. They did note, however, a 20% spontaneous abortion rate in their cohort of patients who had undergone oocyte cryopreservation cycles (Borini et al., 2004). In a later study out of Italy, 149 pregnancies occurred after using a slow freeze protocol for oocyte cryopreservation. This group, again, had a relatively high spontaneous abortion rate of 23.5% (Borini et al., 2007). Reports of live births following oocyte cryopreservation have also emerged from groups in China. Chian et al. found that neonates born after ovarian stimulation and oocyte vitrification were all appropriate birthweights, none weighed <2500g. Additionally, all singletons in their cohort were born at term, with a mean gestational age at delivery of 39 1/7 weeks (Chian et al., 2008a). The same group analyzed 165 pregnancies resulting in 200 babies born after vitrification of oocytes at three centers. In their study, multiple gestations were more likely to deliver in the late preterm period (between 34-37 weeks' gestation) – 57% vs. 22% of singleton pregnancies. This is consistent with current expectations for multiple gestations in the general population. Additionally, 74% of multiples in their study were low birth weight (LBW, <2500g), with 5% of the cohort being very low birth weight (VLBW, <1500g). This was in comparison to singleton neonates born after vitrification, only 17% of which were LBW and 0.7% were VLBW. These birth weights are not significantly different when compared to women who spontaneously conceived or had fresh IVF (Chian et al., 2008b). Birthweight was also analyzed in a systematic review of pregnancy outcome data after oocyte cryopreservation and found to be consistently within normal limits (Wennerholm et al., 2009).

Some concerns have been raised about the rate of malformation or congenital anomalies seen in babies born after any assisted reproductive technology. Epigenetic syndromes (such as Beckwith-Weidemann Syndrome and Angelman Syndrome) have been reported as more common, specifically after ICSI (Noyes et al., 2009). With regard to egg freezing, of 105 babies studied by Borini et al. in 2007, only 2 malformations were seen; one infant was born

Oocyte Cryopreservation for the Elective Preservation of Reproductive Potential 203

Preservation Experience (HOPE) is a phase IV, multicenter, observational registry in the United States that has been created to prospectively collect data on oocyte cryopreservation and subsequent outcomes (Ezcurra et al., 2009). The goals of this project are twofold: first, to evaluate the safety and efficacy of different oocyte cryopreservation techniques, and second, to assess the safety of these methods in relation to the babies resulting from cryopreserved oocytes. This initiative will follow 400 women over three years who are undergoing oocyte cryopreservation, thawing, and subsequent embryo transfer. Standardized data will be collected for all subjects, including demographic information, laboratory studies, and pregnancy outcomes. Additionally, all babies will be followed for the first year of life to evaluate perinatal and infant outcomes after oocyte cryopreservation. Studies of this nature are crucial for the validation of oocyte cryopreservation as a valuable method for fertility preservation in the United States and removal of its "experimental" categorization by

A variety of ART strategies have been introduced over the past few decades without being deemed "experimental" or requiring IRB approval. Moreover, new procedures in ART have not historically been required to demonstrate improved efficacy over established protocols before being introduced into clinical practice (Noyes et al., 2010). One example is the introduction of ICSI in the 1990s (Palermo et al., 1995). Though ICSI is more invasive than conventional IVF, it was quickly embraced in the field and used widely for couples with severe male factor infertility after extensive informed consent. Other ART techniques, such as frozen embryo storage, prenatal genetic diagnosis (PGD), laser assisted hatching, and even human chorionic gonadotropin (hCG) agonist triggering of ovulation have not required implementation under the "auspices of an IRB." Instead, informed consent documents highlight risks and benefits of these procedures and infertility centers are expected to honestly present data regarding success rates and outcomes. In light of these inconsistencies, it seems incongruous to require such stringent, IRB-approved regulations for oocyte cryopreservation, which has been shown to produce high survival rates. Clinics should be transparent about their experience, their site-specific pregnancy rates, and the

Though oocyte cryopreservation was first introduced more than three decades ago, the past several years have yielded significant enhancement of techniques and documentation of efficacy. Current and future advancements have the potential to preserve reproductive potential for young women with cancer prior to gonadotoxic treatments as well as for those seeking elective preservation of their fertility. As stimulation techniques are simplified, costs are contained, safety and efficacy are documented, and more wide-spread awareness of the reproductive aging process is achieved, it is likely that the number of women who are able

Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos.

to benefit from this new technology will continue to increase.

*Journal of Reproduction and Fertility* 1993; 99(2):471-7.

ASRM.

**8. Conclusion** 

associated perinatal outcomes.

**9. References** 

with choanal atresia and the other with Rubenstein-Taybi syndrome (Borini et al., 2007). Chian et al. analyzed rates of malformations in their 2008 cohort of 200 babies born after vitrification. Overall, only 5 birth defects were noted, for a malformation rate of 2.5% (Chian et al., 2008b). This rate is consistent with that seen in spontaneously conceived pregnancies and those following fresh IVF (Tan et al., 1992). In the Chian study, 2 ventricular septal defects (VSD), 1 case of biliary atresia, 1 club foot and 1 skin hemangioma were described in neonates. In their systematic review of the literature, Wennerholm et al. found that children who underwent karyotype analysis after oocyte cryopreservation were all within normal limits (Wennerholm et al., 2009).

The largest study to date of congenital anomalies following oocyte cryopreservation was published in 2009 by Noyes, Porcu and Borini. In this literature review, the authors identified 936 infants born after oocyte cryopreservation. In this worldwide population of infants, only 12 of 936 had either a major or minor congenital anomaly, for a malformation rate of 1.3% (Noyes et al., 2009). Defects seen included 3 VSD, 3 clubfoot, 1 choanal atresia, 1 biliary atresia, 1 Rubenstein-Taybi syndrome, 1 Arnold-Chiari syndrome, 1 cleft palate, and 1 skin hemangioma; some of these defects have already been discussed from earlier studies (Borini et al., 2007; Chian et al., 2008b). No difference in rates of major or minor congenital anomalies was found when compared to the United States birth outcome data from the Centers for Disease Control and Prevention (CDC). The CDC reports major structural or genetic birth defects occurring in 3% of live births (CDC, 2011); the number of malformations seen after oocyte cryopreservation is, in fact, lower than this national average. Importantly, the birth defects amassed in this group mirror those seen most commonly in the general population. Additionally, the authors stratified the infants between those born after slow freeze versus vitrification protocols. There was no major difference in the rate of anomalies found after these methods of oocyte cryopreservation (1.1% versus 1.5%, respectively). No epigenetic syndromes were found in this international group of infants born after oocyte cryopreservation, though these have been reported for other types of ART.

Ovarian tissue cryopreservation, which has been less studied and is not as widely used as oocyte cryopreservation, has also resulted in successful pregnancies. The first birth after ovarian tissue cryopreservation and autotransplantation was documented in 2004 (Donnez et al., 2004). To date, there have been 13 infants born to 10 women after ovarian tissue cryopreservation (Donnez et al., 2011). Two of these women conceived and delivered two healthy infants in subsequent pregnancies from thawed, transplanted ovarian tissue. These 10 case-reports suggest that ovarian function may be restored anywhere from 2 to 5 years post-transplant of cryopreserved tissue. Women who received chemotherapy before taking measures to preserve ovarian tissue all had significantly decreased length of graft function, compared to those who cryopreserved ovarian tissue before initiating a chemotherapy regimen. All singleton gestations delivered at term, after 37 weeks' gestational age. Additionally, all of the infants born after this method of fertility preservation are alive and healthy, without any known congenital anomalies or perinatal morbidity (Donnez et al., 2011).

Studies of pregnancy outcome and neonatal well-being are extremely important with any new reproductive technology. Perhaps more crucial, however, is the ability to track and register pregnancies that arise out of oocyte cryopreservation cycles. The Human Oocyte Preservation Experience (HOPE) is a phase IV, multicenter, observational registry in the United States that has been created to prospectively collect data on oocyte cryopreservation and subsequent outcomes (Ezcurra et al., 2009). The goals of this project are twofold: first, to evaluate the safety and efficacy of different oocyte cryopreservation techniques, and second, to assess the safety of these methods in relation to the babies resulting from cryopreserved oocytes. This initiative will follow 400 women over three years who are undergoing oocyte cryopreservation, thawing, and subsequent embryo transfer. Standardized data will be collected for all subjects, including demographic information, laboratory studies, and pregnancy outcomes. Additionally, all babies will be followed for the first year of life to evaluate perinatal and infant outcomes after oocyte cryopreservation. Studies of this nature are crucial for the validation of oocyte cryopreservation as a valuable method for fertility preservation in the United States and removal of its "experimental" categorization by ASRM.
