Clinical efficacy analysis of oocyte cryopreservation: A propensity score matched study.

This study was approved by the Ethics Committee of Guangdong Women and Children Hospital and has been performed in accordance with the principles of the Declaration of Helsinki.

Clinical data of 78 patients receiving ICSI‐assisted pregnancy after vitrification and thawing of oocytes were reviewed from January 2013 to May 2020 in the Department of Reproductive Health and Infertility, Guangdong Provincial Maternal and Child Health Care Hospital. All of these patients had their eggs frozen because their husbands were unable to extract sperm for various reasons on the day of oocyte retrieval. The data of oocyte cryopreservation to extend their reproductive lifespan were not included in this study, and all eggs were used within several years. Based on the age at cryopreservation, patients were divided into three groups: Group A, <30 years old; Group B, 30–34 years old; Group C, ≥35 years old. Using PSM nearest neighbor matching, according to age, cycle date, number of mature eggs, infertility type, and infertility factors, the control group was selected from 2640 cycles of fresh oocytes and ICSI in our center (during the same time period) at a ratio of 1:4, and recorded as A1, B1, and C1 groups. Inclusion criteria: (1) number of cycles ≤2, number of eggs obtained ≥2; (2) number of embryos transferred 1–2. Exclusion criteria: (1) untreated serious hydrosalpinx; (2) Stage III/IV endometriosis and serious adenomyosis; (3) untreated endometrial lesions, such as endometrial polyps, tuberculosis, submucosal myomas; (4) serious underlying diseases, such as severe cardiovascular disease, liver dysfunction, diabetes, and cancer. The detailed vitrification and thawing protocols have been reported in our previous study. 2 The embryos were transferred from culture into basal medium (BM) containing HEPES (Quinn's SAGE, Knardrupvej, Denmark) with 20% (v/v) human serum albumin (Vitrolife, Sweden), and then transferred into vitrification solution 1 (BM solution containing 7.5%, v/v DMSO and 7.5% v/v ethylene glycol) for 2–7 min. When the shrinkage of embryos expanded to 80%, the embryos were transferred into vitrification solution 2 (BM solution containing 15%, v/v DMSO, 15%, v/v ethylene glycol, and 10%, v/v sucrose) for 30 s. Within 5–10 s, the embryos were collected in a minimal volume and put into the cryodevice, which were then immediately put into liquid nitrogen to cryopreserve at _196°C. For thawing, the embryos were transferred from cryodevice into warming solution 1 (BM solution containing 1 M sucrose) for 1 min, and then transferred into warming solution 2 (BM solution containing 0.5 M sucrose) for 3 min. Last, the embryos were transferred into the BM and remained in the solution for 5 min. Next the embryos were rinsed in G2 solution several times and cultured at 37°C in 6% CO 2 incubator overnight. Endometrial preparation for the oocyte thawing cycle included a natural cycle plan and artificial cycle plan. Natural cycle FET: patients with regular menstruation and ovulation will undergo FET in a natural cycle. On the 9th or 11th day of their cycle, transvaginal ultrasound was used to monitor ovarian follicles. When the diameter of dominant follicle was ≥14 mm, urinary luteinizing hormone (LH) level was tested until LH surge occurred. If the dominant follicle with diameter more than 18 mm did not show LH surge, 5000–10 000 IU of human chorionic gonadotropin (HCG, Livzon Pharmaceutical Factory, Zhuhai, China) was injected intramuscularly to induce ovulation. Luteal phase support was started on the day of ovulation with oral dydrogesterone (Duphaston, Abbott, OLST, Netherlands) at a dose of 20 mg twice daily. After ovulation, thawing and transferring were performed. The time for transplantation depended on the time when embryo was frozen. Cycles were canceled if no ovulation occurred. Progesterone injection should be continued after transplantation. Artificial cycle FET: On the fourth or fifth day of the menstrual cycle of FET preparing cycle, patients with EMT>7 mm during menstruation accepted shallow endometrial scratching as we reported before (impact of endometrial thickness during menstruation and endometrial scratching on the pregnancy in frozen–thawed embryo transfer). Patients with normal endometrial thickness (EM ≤ 5 mm) during menstruation begin oral oestradiol valerate (Progynova, Bayer‐Schering Pharma AG, Germany; at a dose of 2–3 mg twice daily). The thickness of endometrium was monitored with transvaginal ultrasound. The dosage of estradiol valerate was adjusted based on the condition of endometrium and hormone level, with the maximum being 10 mg/day. When the endometrial thickness reached at least 7 mm, vaginal progesterone gel (Merck Serono, UK) at a dose of 90 mg once daily or intramuscular progesterone (Zhejiang Xianju Pharmaceutical Co., Ltd.) at a dose of 40 mg once daily combined with oral dydrogesterone at a dose of 20 mg twice daily were added to promote the transformation of endometrium to the secretory phase. On the third day after fertilization, 1–2 high‐quality embryos were selected for transfer. 3 The number of embryos transferred is determined by clinicians based on the patient's age, embryo quality, and ASRM guidelines. On the fifth day of progesterone exposure the embryo transfer was performed and luteal phase support was continued until the time of the pregnancy test, and then prolonged until 10 weeks of gestation in the case of a positive test result. Live birth rate was defined as the delivery of a viable infant at 24 weeks of gestation or more. Clinical pregnancy was defined as presence of at least one gestational sac in the uterine cavity at 5 weeks after ET. Biochemical pregnancy with a serum subunit of human chorionic gonadotropin (b‐hCG, level of ≥5 IU/L at 12 days after ET). Early pregnancy loss was defined as loss of clinical pregnancy before 12 weeks of gestation. Cumulative cycle live birth rate = number of live births after fresh or frozen embryo transfer from embryos harvested during oocyte freezing cycles between January 2013 and May 2020/total freezing cycles of oocytes. Live birth rate per egg = number of live births/number of mature eggs. Number of eggs required per live birth = number of mature eggs/number of live babies. SPSS 21.0 software was used for data analysis. Measurement data were expressed as mean ± standard deviation, and a t ‐test was used for comparison between groups. Enumeration data were expressed as a rate (%). The Chi‐square test was used for comparison between two groups and split Chi‐square test was used for the enumeration data of three groups. Analysis of variance was used to compare measurement data, and a nonparametric test was used when the variance was not uniform. R Software Version 4.0.3 software was used for PSM nearest neighbor matching. One‐way analysis of variance (ANOVA) or the Chi‐square test was used for comparison between groups. p  < 0.05 was considered statistically significant. p  < 0.05 was considered statistically significant.

Before matching, the data of Groups A (<30 years old), B (30–34 years old), and C (≥ 35 years old), and 2640 cases in the control group had different deviations in age, mature egg number, infertility factors, and infertility types. After matching, the covariates of the three observation groups and control group had no significant differences. The comparison and balance between the groups before and after matching are shown in Figure  1 . The comparison and balance between the groups before and after propensity score matching matching There were no significant differences in general data between the observation groups and the control group ( p  > 0.05). The numbers of high‐quality embryos in the observation groups were significantly lower than that in the control group ( p  < 0.05). The cleavage number of two pronuclei zygotes (2PN) and the fertilization rate in Group A were significantly lower than that in Group A1 ( p  < 0.05). The 2PN cleavage and high‐quality embryo rates in Groups B and C were significantly lower than those in Groups B1 and C1 ( p  < 0.05). There were no significant differences in embryo implantation, clinical pregnancy, biochemical pregnancy, ectopic pregnancy, early abortion, or live birth rates between the three groups and the control group ( p  > 0.05) (Tables  1 , 2 , 3 ). The outcome of comparison between fresh versus vitrified oocytes form patients <30 years old Abbreviation: MII, metaphase II. * p <  0.05; *** p <  0.0005. The outcome of comparison between fresh versus vitrified oocytes form patients 30–34 years old Abbreviation: MII, metaphase II. ** p <  0.005; *** p <  0.0005. The outcome of comparison between fresh versus vitrified oocytes form patients ≥35 years old Abbreviation: MII, metaphase II. * p <  0.05; *** p <  0.0005. The cleavage rate of 2PN in Group C was significantly lower than that in Groups A and B. The cycle cumulative live birth rate in Group C was significantly lower than those in Groups A and B ( p  = 0.0230), and the high‐quality embryo rate in Group A was significantly higher than that in Group B ( p  = 0.0333). There were no significant differences in other indicators. The live birth rates per egg of the three groups were 5.03%, 5.61%, and 3.57%, respectively, and the numbers of eggs per live birth were 13.72, 14.43, and 21.0, respectively (Table  4 ). The outcome comparison between different age patients' oocytes after vitrification Abbreviation: COS, controlled ovarian stimulation. † p  < 0.05 Group A vs. Group B; ‡ p  < 0.05 Group A vs. Group C; § p  < 0.05 Group B vs. Group C.

Written informed consent was obtained from the couples. Patient anonymity was maintained.

Although oocyte cryopreservation has been gradually improved, the clinical effects of oocyte thawing and recovery are still quite different in distinct studies. In 2013, the American Society for Reproductive Medicine reviewed 80 studies related to the effectiveness of oocyte cryopreservation, 4 and found no significant difference in normal fertilization, embryo implantation or pregnancy success rates between vitrified thawed and fresh oocytes. 5 , 6 , 7 , 8 However, multicenter prospective cohort studies from Italy 9 and large sample retrospective studies 10 have reported that embryo implantation and clinical pregnancy rates of oocyte thawing cycles may be lower than that of fresh oocytes. In China, only four studies have compared the clinical outcomes of oocyte thawing cycles and fresh oocyte cycles, but sample sizes were only 10–37 cases, and the studies reported different conclusions. In this study, the main confounding factors such as age, number of MII oocytes, infertility types was adjusted by propensity score matching. No difference found in number of embryos transferred, proportion of blastocysts transferred, and endometrial thickness. The baseline of the observation and control groups were even. The total survival rate of oocyte thawing cycle was 78.1%, which was consistent with literature reports at home and abroad, 11 , 12 indicating the operation of the laboratory has reached the international standard and ensure the credibility of the outcomes. The current results show that the clinical outcome of the thawing cycle were similar to that of fresh eggs, suggesting that vitrification of oocytes provides a better method for fertility preservation, but the cleavage and embryo quality were affected, to a certain extent, by vitrification and freezing–thawing of oocytes from women of different ages. In addition, the normal fertilization rate of group A was significantly lower than that of the control group, suggesting that the utilization rate of young patients' oocytes after vitrification and freezing was significantly reduced compared with controls. The oocyte is the largest cell in humans, with a large volume and low surface area/volume ratio. Oocyte membrane fluidity is poor because of the high water content of oocytes. The permeability of oocytes to water and cryoprotectants is poor, making them vulnerable to damage during freezing and thawing. The metaphase II (MII) stage is very sensitive to physical and chemical factors. During freezing, the oocyte cytoskeleton may be damaged, such as abnormal distribution of microfilaments, morphological changes to the spindle, loss of mitochondria and ultimately apoptosis, 13 , 14 , 15 affecting the division of embryo cells and reducing their developmental potential. We further compared the clinical data between the three observation groups with patients of different ages. The current results showed that the cumulative live birth rates of oocytes were 66.67%, 65.38%, and 31.82% in Groups A, B, and C, respectively. Thus, the cumulative live birth rate obtained using oocyte freezing before the age of 35 years was still relatively high, then markedly declined after 35 years of age, consistent with the results of Mesen et al. 16 In 2010, one study found that the survival rate of thawed oocytes and embryo development did not seem to be affected by maternal age, however, maternal age was the only factor affecting the cumulative pregnancy rate. 17 In the present study, the live birth rates per oocytes in Groups A, B, and C were 5.03%, 5.61%, and 3.57%, respectively, and the number of oocytes required per live birth was 13.72, 14.43, and 21.0, respectively. These findings suggest that with increasing age, especially after the age of 35 years, the utilization efficiency of thawed oocytes decreases significantly, and the number of eggs required per live birth increases sharply. The current study was consistent with previous results, 1 , 12 , 18 , 19 suggesting that the developmental potential of thawed oocytes decreased with increasing age at the time of oocyte cryopreservation. But because of the low embryo utilization rate of young patients in Groups A and B, the calculated live birth rate per egg was lower than that reported in the literature. The embryo utilization rate of patients in Group C was higher, so the calculated live birth rate per egg and the number of eggs required per live birth in Group C were relatively accurate and consistent with the literature. Based on our findings, we make the following recommendations. First, to maximize the likelihood of a good perinatal outcome after oocyte thawing, women who plan to freeze their eggs should do it before the age of 35. Second, we suggesting the number of frozen eggs should be around 15 in women younger than 35 years. Third, women over 35 age should have a lower expected value of oocyte cryopreservation, the number of frozen eggs should be more than 21.

Cryopreservation of oocytes is a useful complement to assisted reproduction techniques and is currently the most promising approach for preserving female fertility. The average childbearing age of women is increasingly delayed, and more women hope to delay childbearing via oocyte cryopreservation technology. 1 However, oocyte cryopreservation may be assumed to be low risk, making patients ignore other aspects of age‐related declining fertility, leading to adverse pregnancy outcomes. Clinicians need to more accurately evaluate the clinical efficacy of oocyte thawing cycles. Limited numbers of oocytes can be collected, so clinical expectations for successful freezing and recovery are high. Vitrification is considered the most suitable technique for oocyte freezing. However, at present, the recovery rate of eggs undergoing vitrification in China is about 76%–80%, and the subsequent clinical pregnancy rate is about 34%–54%, still lower than expectations of clinicians and patients. Hunan becomes the first province to open up oocyte cryopreservation for single women in China, in August, 2021, but China lacks guidelines on oocyte cryopreservation for single women. Because of national policies on oocyte cryopreservation, there are few high‐quality studies on the subject, the small sizes of cases have limited statistical analysis, and conclusions are not same in different studies. Therefore, there has been no reliable evaluation of the clinical efficacy of oocyte thawing cycles in China. We were unable to conduct a randomized control study (RCT) in oocyte cryopreservation in China, only can eliminate the selection bias in retrospective studies as far as possible. Propensity score matching (PSM) can balance variables identified in a retrospective analysis, minimize the influences of potential confounding factors, and ensuring the reliability of the outcome. In order to accurately evaluate the clinical efficacy of oocyte freezing in women of all ages, in this study, 78 cases of frozen egg cycle data were stratified by age, after PSM processing, the baseline of the observation and control groups were even. The clinical outcomes of intracytoplasmic sperm injection (ICSI)‐assisted pregnancy after thawing of oocytes, and ICSI‐assisted pregnancy using fresh eggs were investigated, the live birth rate per egg, number of eggs required per live birth in each subgroup were calculated, and the utilization rate and clinical efficacy of oocytes after thawing were analyzed, providing reliable information reference for doctors and women expecting to delay fertility through oocyte freezing.

The authors have no conflict of interest to declare.