**3. Results**

As shown in **Figure 1**, 849 oocytes from 120 patients were warmed, 61 cycles were from women <35 years old, 27 cycles were from women 35–37 years old, and 32 cycles were from women >37 years old. The resulting blastocysts were either fresh transfer (74 cycles) at Day 5 or cryopreserved at Day 5–7 (23 cycles) for later FET Twenty-two patients had frozen all blastocysts due to PGT-A, and one patient (45 years old) had plan for fresh blastocyst transfer but did not have blastocyst at Day 5, so blastocyst transfer was canceled. However, one embryo developed to blastocyst at Day 7, so it was frozen and then processed for frozen/warmed blastocyst transfer.

A total of 67 blastocysts from 22 patients were biopsied and 55.2% (37) blastocysts were euploid after PGT-A. Euploid blastocysts in 16 patients and a Day 7 blastocyst without PGT-A in 1 patient were transferred in FET cycles.

Twenty-three cycles did not have embryo transfer due to no blastocyst formation, including no fertilization, no cleavage or arrested embryo development before blastocyst stage.

Women's ages at the time of oocyte cryopreservation on post-warming outcomes were shown in **Table 1**. It was found that average numbers of oocytes warmed

#### **Figure 1.**

*Diagram of patient population and cycle information. Patients were grouped based on ages of <35, 35–37 or > 37 years old at the time of oocyte cryopreservation. Only the first embryo transfer (either fresh or FET) was included in the data analysis.*


*\* No. of patients without blastocysts includes all cases in which oocytes were not fertilized after ICSI, fertilized oocytes did not cleave, and cleaved embryos did not develop to blastocyst stage.*

*abValues are significantly different with different superscripts in the same row, P < 0.05.*

#### **Table 1.**

*Women's ages at the time of oocyte cryopreservation on post-warming laboratory outcomes.*

(7.2 ± 6.0, 7.2 ± 5.5 and 6.7 ± 3.7, respectively), proportions of oocytes survived (93.4, 90.8 and 94.9%, respectively), fertilized (71.2, 72.3 and 73.0%, respectively), cleaved (95.2, 92.2 and 94.0%, respectively) and developed to blastocysts (56.8, 54.2 and 49.3%, respectively) were similar (P > 0.05) among patients <35, 35–37 and > 37 years old. However, cancelation rates, which were determined by no available blastocyst for transfer were significantly (P < 0.05) higher in patients >37 years old (31.2%) than in patients <35 years old (13.1%).

As significant differences in the cancelation rates were present in the three age groups, we further analyzed the detailed reasons of the cancelation. As shown in **Table 2**, it was found that oocyte survival after warming did not cause any cancelation, and all patients had survived oocytes after warming. However, cancelation was observed in 30.4% (7/23) patients without fertilization after ICSI, in 8.7%


#### **Table 2.**

*Detailed reason analysis of patients without blastocysts\* .*


#### **Table 3.**

*Relationship between number of oocytes warmed and no blastocyst formation\* .*

#### *Efficiency of Autologous Egg Cryopreservation: Eight Years' Experiences and Clinical Outcomes DOI: http://dx.doi.org/10.5772/intechopen.98675*

(2/23) patients without embryo cleavage and in 60.9% (14/23) patients without blastocyst development that was the main reason of cancelation. However, no statistical differences were found among three age groups.


*\* P > 0.05 in all comparison groups within the same age group between transfers (only fresh embryo transfers were compared).*

#### **Table 4.**

*Women's ages at the time of oocyte cryopreservation on post-warming clinical outcomes\* .*


*abP < 0.05 at least in the same row with different superscripts.*

*\* Semen samples were not able to collect or samples did not have motile sperm, all oocytes were frozen.*

*\*\*Semen sample had motile sperm but sperm number was not enough to fertilize all of the oocytes or patients wanted to fertilize partial oocytes and to freeze the remaining oocytes.*

*\*\*\*All oocytes were frozen for fertility preservation.*

#### **Table 5.**

*Patient categories for oocyte cryopreservation and clinical outcomes.*

As shown in **Table 3**, when oocyte number (1–3, 4–8 and > 8 oocytes per warming cycle) and cancelation were analyzed, it was found that no blastocyst formation was found in all groups: 34.8% with 1–3 oocytes, 52.1% with 4–8 oocytes and 13.0% with >8 oocytes. There is increased tendency that less cancelation was observed if more than 8 oocytes were warmed. However, no statistical differences were found among three age groups or three oocyte number groups.

As shown in **Table 4**, when fresh blastocyst transfers were compared in terms of clinical pregnancy, mean no. of embryo transferred, embryo implantation, live birth and birth weight in three age groups, there were no statistical difference be observed although the rates were lower in the patients at age of >37. FET cases in each age group were small (7, 3 and 7 cases for age of <35, 35–37 and > 37, respectively), the clinical pregnancies (2, 2 and 2 cases, respectively) and live births (2, 2 and 2 cases, respectively) were not included in the comparisons.

When the data were analyzed based on three categories of patients for oocyte cryopreservation, as shown in **Table 5**, it was found that the number of oocytes warmed and reasons for oocyte cryopreservation had significant impact on clinical outcomes. The patients who had partial oocyte cryopreservation had significantly (P < 0.05) fewer oocytes to be warmed (5.0 ± 3.7) as comparing with patients who had all oocyte cryopreservation, including patients for backup oocyte cryopreservation (9.8 ± 5.8) and women for fertility preservation (9.4 ± 4.4). There was no statistical difference in the oocyte survival (91.3–96.7%) among three categories, however, significantly (P < 0.05) lower rates in fertilization (67.4 vs. 76.6%), clinical pregnancy (36.0 vs. 88.9%), live birth (36.0 vs. 77.8%) and embryo implantation (26.6 vs. 73.3%) were observed in patients with partial oocyte cryopreservation as compared with women for fertility preservation. Other comparisons, including women's age, cleavage, blastocyst formation, cancelation, and mean no. of embryos transferred in all groups did not show statistical differences.

#### **4. Discussion**

It has been demonstrated that oocyte cryopreservation does not compromise in vitro development and pregnancy rates as compared with fresh oocytes [1, 9–12]. Because of its reliability and efficiency, oocyte cryopreservation allows young cancer patients to have their oocytes collected prior to the initiation of chemo- or radiotherapy for the treatment of various malignant diseases, with the expectation of having their oocytes fertilized after recovery [2, 3, 19, 20]. It also would permit healthy women to have their oocytes collected and preserved for use in the future [4, 7, 8, 16–18] and for donor oocyte bank establishment [24, 25, 33].

It has been found that live birth rate was reduced significantly in women >37 years old after fresh oocyte IVF and the reduced live birth rate was mainly caused by embryonic aneuploidies [34–37]. Therefore, women's age at the time of oocyte cryopreservation is the most important factor affecting live birth rates. Present and previous data [3, 9, 16–18] suggest that women should preserve their oocytes before 37 years old if they plan to rely on oocyte cryopreservation to have a live birth. However, for the women who are more than 37 years old, it is still possible to have their oocytes to be cryopreserved for future use, but success mainly relies on oocyte quality and number [3, 9, 13].

In the present study, when we analyzed the efficiency of oocyte cryopreservation in women at different age groups, we found that live birth rates can reach to 51.1, 46.7 and 28.6% in women <35, 35–37, and > 37 years old, respectively, with their first embryo transfer (fresh), which is comparable to live birth rates with embryo transfer from fresh oocytes in our clinic or other published data [2, 3, 9, 23, 25]. A decreased

#### *Efficiency of Autologous Egg Cryopreservation: Eight Years' Experiences and Clinical Outcomes DOI: http://dx.doi.org/10.5772/intechopen.98675*

tendency in live birth rate was observed in women >37 years as compared with women ≤37 years old. This is true because embryo quality (competence to develop to blastocysts and chromosome status) decreases when women reach >37 years old [37, 38]. Even when high quality blastocysts were transferred, embryo implantation rates also dropped, which eventually reduced live birth rates. Furthermore, morphological assessment of embryos does not always choose chromosomally normal embryos, thus high miscarriage rates were found in this population [35, 36]. In the present study, most patients had fresh blastocyst transfer without PGT-A. Actually frozen/warmed euploid blastocyst transfer after PGT-A did not further increase embryo implantation in all age groups in the present study. Similar outcomes have recently been found when embryos (with or without PGT-A) from fresh oocytes were transferred [39, 40], especially the benefits of PGT-A were not found in patients <37 years old. However, large data analysis of pregnancy outcomes in women aged 35–40 years demonstrated a significant improvement in clinical pregnancy rate and live birth rate with the use of PGT-A per embryo transfer [41]. Thus, it is difficult to explain the differences between reports.

Cancelation is very common in human ART, especially in poorly responding and/ or older patients. In the present study, we cultured all embryos to Days 5–7 to allow embryos to develop to blastocyst stage and found that more patients >37 years old had to cancel embryo transfer due to lack of blastocyst development than patients <35 years old, and this indicates that oocyte quality in older patients are poorer than that in young patients. Although number of oocytes is also a reason for cancelation of a cycle, oocyte quality may be the main reason. Other factors should also be considered as the reasons for cancelation. For example, some canceled cycles had previous failed IVF cycles with the same cohort of fresh oocytes or had failed cycles due to severe male factor infertility. From laboratory results, it was found that no fertilization (mainly due to male factor infertility) and no embryo cleavage also caused cycle cancelation. Therefore, the reason(s) for cycle cancelation is complicated and multiple factors should be considered to explain the cause of failed blastocyst development.

Blastocyst transfer has been one of the most practical embryo selection strategies in human ART [42, 43], which could reduce number of embryos to be transferred and multiple pregnancy [43–45]. Our clinic has adopted blastocyst transfer for all patients, even in patients with a limited number of oocytes. However, we still do not know if pregnancy can be improved by early-stage embryo transfer for oocyte warming cycles. As some fertilized oocytes (as high as 10%) did not cleave during the culture, it is unknown whether embryo development arrest can be overcome by transferring early stage of embryos to uterus. It would also be possible that embryo arrest is caused by damages of some intra-oocyte structures during oocyte cryopreservation and warming, as embryo arrest is less than 1% in human IVF with fresh oocytes in our laboratory. Thus, current oocyte cryopreservation and warming technology needs further improving.

In the present study, we also found that average birth weight and proportions of babies with low birth weight after fresh embryo transfer were comparative to average weight of babies from fresh oocytes [42, 45, 46]. However, low birth weight was observed in babies from frozen/warmed blastocyst transfer in the present study although there are no statistical differences as compared with babies from fresh blastocyst transfer. This is certainly different from those with fresh oocyte IVF in which birth weights were higher in babies from FET than babies from fresh embryo transfer [47]. Because this is the first time that we noticed the difference in birth weight between fresh blastocyst transfer and frozen/warmed blastocyst transfer from frozen/warmed oocytes and the case number is also very limited, further data collection is necessary to reveal whether low birth weight after transfer of frozen/warmed blastocysts resulting from frozen/warmed oocytes is a common phenomenon.

Oocyte cryopreservation has been widely provided to women for various purposes. In the present study, we found that the efficiency was different among three different patient categories. Women used oocyte cryopreservation as fertility preservation had higher live birth rate as compared with patients who initially underwent infertility treatment. For infertility patients, live birth rates between partial oocyte cryopreservation/warming and all oocyte cryopreservation/warming were not significantly different although fewer oocytes had less opportunity to have a live birth, which was similar as that predicted by other researchers [3], especially when cumulated live birth rates were calculated [9].

However, for patient own oocytes, there are many factors, such as age and ovarian and hormone status. Some patients with oocyte cryopreservation may be due to lack of (enough) sperm for insemination, thus the time for oocyte cryopreservation may be delayed (after insemination of partial oocytes or after waiting for attempt of sperm collection). Occyte cryopreservation is usually performed 3–5 h after egg retrieval [2, 9, 11], thus it is still unknown whether delayed cryopreservation affects oocyte survival, fertilization, and embryo development. We did not examine these factors in the present study because the case numbers are very small in each category. For these patients, male factor infertility may also affect oocyte fertilization and embryo development and implantation.

We found that embryo development is slower with frozen oocytes as compared with fresh oocytes. Recently, Cobo et al. used time-lapse scope to track oocyte fertilization and embryo development, they found that pronuclear formation is about 1 h delayed in frozen oocytes as compared with fresh oocytes [48]. We also found that Day 5 blastocyst rates were lower but overall blastocyst rates (Days 5–7) were same between fresh and frozen donor oocytes in a previous study [11, 49].

A comprehensive analysis should be done whether an oocyte warming cycle can eventually result in a live birth. Many factors, such as oocyte quality, numbers of oocytes warmed, previous IVF outcomes, male factor infertility and others, should be carefully evaluated because women will rely on cryopreserved oocyte IVF to have a live birth in the future. It should be realized that if a fresh oocyte IVF cycle fails (no embryo available for transfer after IVF or no live birth after embryo transfer), the patients can attempt the second or more cycles to achieve a live birth. However, if an oocyte warming cycle fails to have a live birth, it may be too late for the patients to attempt the second or more oocyte retrieval cycles, especially when women use oocyte cryopreservation as their fertility preservation.
