Twice vitrication-warming procedures has no effect on frozen-thawed embryo transfer outcomes

Background To evaluate the frozen-thawed embryo transfer (FET) outcomes of repeated cryopreservation by vitrication of blastocysts derived from vitried-warmed day3 embryos in patients who experienced implantation failure previously. Methods single frozen-thawed our to of blastocysts from vitried-warmed day3 once the previous FET. The were between two groups after a propensity (PSM).


Abstract Background
To evaluate the frozen-thawed embryo transfer (FET) outcomes of repeated cryopreservation by vitri cation of blastocysts derived from vitri ed-warmed day3 embryos in patients who experienced implantation failure previously.

Methods
We retrospect the les of patients who underwent single frozen-thawed blastocyst transfer cycles in our reproductive medical center from January 2013 to December 2019. 127 patients transfer of vitri ed-warmed blastocysts derived from vitri ed-warmed day3 embryos were de ned as twice-cryopreserved group. 1567 patients who transfer blastocysts that had experienced once vitri ed-warmed were used as once-cryopreserved group. None of them was pregnant at the previous FET. The outcomes were compared between two groups after a 1:1 propensity score matching (PSM).

Results
The clinical pregnancy rate was 52.76%, live birth rate was 43.31% in twice-cryopreserved group. After PSM,108 pairs of patients were generated for comparison. The clinical pregnancy rate, live birth rate or miscarriage rate was not signi cantly different between two groups. Logistic regression analysis indicated that double vitri cation-warming procedures did not affect FET outcomes in terms of clinical pregnancy rate (OR 0.83, 95%CI 0.47-1.42), live birth rate (OR 0.93, 95%CI 0.54-1.59), miscarriage rate (OR 0.72 95%CI 0.28-1.85). Furthermore, the pregnancy complications rate, gestational age or neonatal abnormalities rate between two groups was also comparable, while twice vitri cation-warming procedures might increase the macrosomia rate (19.6% vs. 6.3%, P = 0.05).

Conclusion
Transfer of double vitri ed-warmed embryo at cleavage stage and subsequent blastocyst stage did not affect live birth rate and neonatal abnormalities rate, but there was a tendency to increase macrosomia rate, which needs further investigation.

Background
It has been decades since the rst IVF baby born, and then assisted reproduction has acquired signi cant advances,the number of available embryos in each IVF cycle has also increased, resulting in a slew of surplus embryo cryopreservation. Cryopreservation of embryos will increase the cumulative pregnancy rate for a single cycle of ovarian stimulation, the clinical pregnancy rate and live birth rate was similar with fresh embryo transfer, while frozen-thawed embryo transfer (FET) decreases the risk of ovarian hyperstimulation syndrome (OHSS), provides optimal endometrial preparations in case of premature progesterone rises, adenomyosis or hydrosalpinx [1][2][3]. Embryo cryopreservation has become a widespread reliable procedure in assisted reproductive technologies [4]. However, cryopreservation procedure may be have negative effects on both mothers and babies when compared with fresh embryo transfer such as higher rate of hypertensive disorders of pregnancy, large for gestational age (LGA) and higher birthweight [5][6][7].
Single embryo transfer was global recommended to reduce multiple pregnancies [8]. In case of multiple embryos were frozen in one cryo-straw previously, these vitri ed embryos will be warmed together, when only one embryo were transferred, the other warmed embryos have to be vitri ed again, so these embryos would undergo twice vitri cation-warming procedures. Some studies have focused on whether repeated cryopreservation process affect the pregnancy and perinatal outcomes. One study showed that twice-frozenthawed embryos have a lower post-thaw survival rate but equivalent pregnancy outcomes to once-frozen embryos [9]. Others found repeated cryopreservation process impairs embryo implantation potential, with lower birth rate and higher miscarriage rate [10,11]. However, the patients underwent repeated cryopreserved embryo transfer may be always experienced previous embryo transfer failure, so the control group should be consistent with this, which may the sample selection bias in these studies. And the most common situation in our center was that two embryos were frozen in one cryo-straw at cleavage stage, after single embryo transfer, the surplus thawed embryos will culture to blastocyst stage and vitri ed again, so these embryos underwent twice vitri cation-warming procedures at cleavage stage and subsequent blastocyst stage, which was different from some other studies when the repeated cryopreservation process was performed at the same stage.
In this retrospective study, we investigated the effectiveness and safety of twice vitri cation-warming procedures at cleavage stage and subsequent blastocyst stage in in patients who experienced previous failure. The pregnancy and perinatal outcomes were compared between the twice-cryopreserved group and the oncecryopreserved group.

Patients
The medical les of all frozen-thawed blastocyst cycles from January 2013 to December 2019 at Reproductive Medicine Center of Nanjing Drum Tower Hospital were retrospectively reviewed. We established inclusion and exclusion criteria. Inclusion criteria (1) single blastocyst transfer, (2) the patient had experienced at least one frozen-thawed cycle and had not received a live birth in the precede cycle. Exclusion criteria (1) patients received multiple births, (2) endometriosis, (3) preimplantation genetic testing cycles, (4) donor cycles, (5) history of recurrent pregnancy loss, and (6) uterine pathology. Totally 1694 women patients were included in this study. They were divided into two groups based on times of embryos had been cryopreserved oncecryopreserved group n=1567 and twice-cryopreserved group n=127 . Among twice-cryopreserved group, 127 FET cycles were those patients in whom two embryos were frozen by vitri cation in one cryo-straw at cleavage stage, after single embryo transfer, the surplus embryo will culture to blastocyst stage in vitro, then refrozen by vitri cation, then transferred after rethawing. IVF/ICSI procedure and embryos culture Depending on the patients' age, ovarian reserve function, and ovarian responses in the previous ovulation cycle, gonadotrophin-releasing hormone agonist or antagonist protocols were used. When two to three dominant follicles reached a diameter of 18 mm, human chorionic gonadotropin (HCG) was injected. The oocytes were retrieved under transvaginal ultrasound guidance 36-38 hours after HCG administration. Retrieved oocytes were then fertilized in conventional IVF or intracytoplasmic sperm injection (ICSI). Two pronuclei and a second polar body were examined for normal fertilization at 16-18 h post-insemination. Embryos were cultured in G1/G2 sequential media (Vitrolife, Goteborg, Sweden) at 37°C in a 6% CO2, 5% O2 and 89% N2 high-humidity incubator. Blastocyst morphological evaluation was based on the Gardner scoring system [12].

Vitri cation cryopreservation and thawing procedures
The vitri cation cryopreservation and thawing procedures was performed in embryos vitri cation kit was produced and available at Kitazato Co., Fujinomiya, Japan. Before vitri cation, the blastocyst was arti cially shrunken by laser drilling.
Cleavage embryos or shrunken blastocysts were equilibrated in equilibration solution for 5-8 min at room temperature, then placed into vitri cation solution, 1 minute later the embryos were loaded onto the cryotop strip and plunged into liquid nitrogen immediately. For warming, the cryotop strip was removed from the liquid nitrogen and immediately transferred to thawing solution for 1 minute at 37•C, dilution solution for 3 minutes, and then washed twice in washing solution 1 and washing solution 2 for 5 min each. After warming, embryos were cultured in embryo culture medium at 37°C in 6% CO2 for 2-4 h until transfer.

Outcome Measures
Baseline demographics for each patient included age, BMI, duration of infertility, parity, type of infertility and cause of infertility, insemination methods (IVF or ICSI), endometrial thickness and endometrium preparation protocol for FET.
Serum HCG was measured after embryo transfer 12-14 days. Clinical pregnancy was de ned as the presence of gestational sacs observed on an ultrasound scan at least 5 weeks after the embryo transfer. The fetal loss before 28 weeks was de ned as miscarriage. The embryo was implanted outside the uterine cavity called ectopic pregnancy. Such as hypertensive disorders of pregnancy, gestational diabetes mellitus, intrahepatic cholestasis during pregnancy and placenta previa were included in pregnancy complications. Any singleton birth ≥28 weeks of gestation was considered a live singleton birth. The neonatal outcomes evaluated were gestational age, preterm birth (delivery between 28 and 37 weeks), low birthweight ( birthweight <2500 g) macrosomia ( birthweight≥4000 g) gender and neonatal abnormality.

Statistical analysis
Means ± SD for continuous variables percentages for categorical variables were calculated. The data were analyzed using the chi-squared test or Student's t-test. P-values <0.05 was considered statistically signi cant. 1:1 Propensity score matching was used in data analysis to eliminate the in uence of baseline characteristics on outcomes. The differential variables include female age at retrieval, average embryo transfer cycles, parity, basal FSH, type of infertility and cause of infertility. To analyze the associations between the cryopreserved times and pregnancy outcomes, logistic regression models were conducted for each outcome indicator using the after-matching data, and odds ratio (OR) and its 95% CI before and after adjusting for confounders were calculated. Statistical analysis was performed using SPSS statistical package version 26.0.

Results
Clinical characteristics and outcomes of the twice-cryopreserved grou p Patients' overall baseline clinical demographics of the twice-cryopreserved group were displayed in Table 1. The female age at retrieval or embryo transfer were 29.61±3.96 and 34.28±4.12 years. The average BMI and basal FSH of the study population were 22.33±2.89 kg/m 2 and 6.7±1.77 mIU/mL, respectively. In the FET cycles, the hormone-replacement therapy (HRT) predominated in endometrium preparation (104 cycles). In the 127 blastocysts, ninety-one were day 5 blastocysts, while the remaining 36 were day 6 blastocysts.  The propensity score matching of basic characteristics between once-cryopreserved group and twicecryopreserved group Among a total of 1694 embryos, 127 were re-thawed and underwent transfer (twice-cryopreserved group), 1567 were underwent a single thawed embryos transfer (once-cryopreserved group). The comparison of clinical characteristics between the two groups were listed in in once-cryopreserved group (mean cycles: 2.94) (P < 0.001) and the parity was more in the twice-cryopreserved group (0.78±0.47 vs. 0.32±0.49, P < 0.001). The distribution of infertility type, cause of infertility was different between the two groups. Table 3 The propensity score matching of basic characteristics between once-cryopreserved group and twicecryopreserved group. .
In order to eliminate the in uence of baseline characteristics on outcomes, a 1:1 PSM was performed between the two groups. Finally, a total of 108 pairs were matched by their propensity score. Before propensity score matching, two groups had signi cantly different maternal and IVF characteristics but the propensity score matching balanced these characteristics between the cohorts (Table 3), suggesting the matched cohorts had highly similar baseline characteristics.
Pregnancy outcomes between the once-cryopreserved group and twice-cryopreserved group after matching The clinical outcomes were summarized in  We adjusted the female retrieval age, BMI, FSH, embryo transfer cycles, parity, the type of infertility and the quality of embryos. AOR is the adjusted odds ratio in logistic regression analysis, representing the risk ratio of clinical pregnancy/live birth/early miscarriage in the twice-cryopreserved group versus once-cryopreserved group. AOR greater than 1 represents increased risk, and AOR less than 1 represents reduced risk. 95% CI is the 95% con dence interval of AOR.
In addition, we conducted a strati ed analysis based on the day of embryo cryopreservation, embryo quality and previous labor history, the difference in live birth rates between the two groups is shown in Table 6. The results showed that transferring D5 or D6 blastocyst,delivery history or not, the live birth rate had no signi cant difference between the two groups (P>0.05). In both groups, high-quality blastocysts for transferring resulted in a higher live birth rate than good-quality blastocysts (P<0.01). However, there was also no signi cant difference in the live birth rate on transferring the same level embryos between the two groups. Table 6 The live birth rate analyzed by strati cation between once-cryopreserved group and twice-cryopreserved group after propensity score matching.  (Table 7). A singleton baby born in the once-cryopreserved group had angioma defect and the rate of congenital birth defects was 1/48 in the once-cryopreserved group. While no newborn anomalies were occurred in the twice-cryopreserved group. Table 7 Neonatal characteristics of live-born singletons between once-cryopreserved group and twicecryopreserved group after propensity score matching. In order to eliminate the possible effect of infant gender on birthweight, we compared the differences in offspring birth weight and mean gestational weeks between the once-cryopreserved group and the twicecryopreserved group in boys and girls, respectively ( Table 7). The results shown that both boys and girls, there was no signi cant difference in gestational week and the birth weight between the two groups. Whether boys or girls, the macrosomia rate in the twice-cryopreserved group was slightly higher than that in the oncecryopreserved group, but it did not reach statistical difference (P>0.05).

Discussion
Embryo cryopreservation is a crucial part of assisted reproductive technology, and the proportion of FET had grown remarkably. In order to reduce the risk of multiple pregnancies and pregnancy complications, single embryo transfer is preferred in the case of suitable patient conditions and embryo quality. Studies have found that compared with embryos in cleavage stage, single blastocyst transfer signi cantly improves clinical pregnancy rate and implantation rate [13][14][15][16]. As multiple cleavage stage embryos might be frozen in one cryostraw previously, after single embryo transfer, the surplus thawed embryos will be cultured to blastocyst stage and vitri ed again, if this time of embryo transfer failed, these patients might choose to transfer these embryos which underwent twice vitri cation-warming procedures. In this study, we found that transfer of twice vitri edwarmed embryo at cleavage stage and subsequent blastocyst stage did not affect pregnancy and perinatal outcomes.
There were several case reports demonstrating healthy live birth after a frozen embryo transfer with embryo that were frozen and thawed twice [17][18][19][20][21]. And some early retrospective studies have also shown that the embryos re-cryopreservation does not affect clinical pregnancy rates [9,[22][23][24]. However, other recent studies have shown that transfer of twice frozen-thawed embryos increase the rate of miscarriage and decreases the clinical pregnancy rate and live birth rate [10,11,25]. The discrepancies in the results of these studies may be due to the different methods used to freeze embryos,embryos frozen at different stages and mismatched baseline of clinical characteristics. Furthermore, these studies may exist the sample selection biases, as the patients underwent repeated cryopresered embryo transfer may always experience previous embryo transfer failure, so the control group should be consistent with this. So, the control group in our study were also experienced previous embryo transfer failure. Furthermore, in order to eliminate the in uence of clinical baseline characteristics on results, a propensity score and logistic regression was used to control for potential confounding differences in our study.
After PSM, the clinical pregnancy rate, miscarriage rate, live birth rate or the incidence of pregnancy complications had no difference between the once-cryopreserved group and the twice-cryopreserved group.
There was a difference in clinical pregnancy and live birth rates between blastocysts developing on Day 5 (D5) and Day 6 (D6),the clinical pregnancy rate and live birth rate were signi cantly higher following D5 compared to D6 blastocysts [26,27]. In addition the quality of embryos plays a crucial role in embryo implantation and live birth rate [28,29]. In the present study, we strati ed the live birth rates based on the day of embryo cryopreservation, embryo quality between once-cryopreserved group and twice-cryopreserved group and found no signi cant difference. Logistic regression after adjusting for relevant confounding factors showed that the number of vitri cation-warming procedure was not related to the clinical pregnancy rate, live birth rate or early miscarriage rate, which was not consist with other studies. Zheng et al. (2017) reported the miscarriage rate of the twice-cryopreserved group was higher and the live birth rate was lower than control group, which may due to two cryopreservation methods were used in their study, slow freezing of cleavage embryos followed by vitri cation of blastocysts [10]. In our study, we only used one cryopreservation method, vitri cation. Compared with slow-freezing method, embryo vitri cation is an ultra-rapid cryopreservation method which prevent ice formation, reduce embryo cryoinjury, have higher embryo survival rate and live birth rate [30,31]. One other study as well indicated that the embryo implantation rate, clinical pregnancy rate and live birth rate in the recryopreservation group were signi cantly lower, and the miscarriage rate also slightly increased [11]. In their study, embryos underwent twice cryopreservation in the blastocyst stage, in addition embryo selection was restrictive compared with once cryopreservation group. Moreover, the blastocyst underwent twice frozen-thawed, and the times of laser drilling and shrinkage increased, which may affect the implantation ability of embryos. Although current studies have con rmed the safety of laser manipulation in human embryo cryopreservation, however the cumulative effect is unclear [32,33]. In our study, we selected embryos underwent twice-cryopreservation, once at the cleavage stage and the second time at the blastocyst stage. Blastocyst culture itself was the selection of embryos, only an embryo with developmental potential can form a blastocyst. And the blastocysts underwent the same number of laser drilling as in the one-freeze group.
More and more children are born depending on ART globally, so the safety of the offspring is more concerned. When compared with fresh embryo transfer,FET is associated with higher risk of LGA and macrosomia in singletons [5,7,34]. The maternal and fetal health that experiences repeated freezing should be paid more attention to. Our results showed that no signi cant differences were observed in pregnancy complication rate, gestational age, fetal birthweight, gender, congenital birth defects rate in the singleton delivery between oncecryopreserved and twice-cryopreserved group. However, the macrosomia rate was 19.56% in the twicecryopreserved group, when it was 6.35% in once-cryopreserved group, whether the time of vitri cation-warming procedure is positively associated with the fetal birthweight need further study. Birthweight may be related to gender, so the incidence of macrosomia, gestational age, birthweight was further analyzed for boys or girls separately, and no signi cant differences was observed may due to few samples.
There are several limitations in our study. First, it was a retrospective study conducted in a single center, a larger prospective cohort study was needed to validate our ndings. Second, although we used propensity score matching to eliminate the in uence of baseline characteristics on FET outcomes as much as possible, it could not eliminate all the bias caused by confounders between the two groups.
In summary, our results presented the pregnancy outcomes and neonatal safety of human refrozen-thawed embryos. Twice vitri cation of human embryos at different developmental stages do not affect clinical outcomes and perinatal outcomes. The re-cryopreservation procedure would be a valuable option to increase the cumulative pregnancy rate while preventing embryo waste under full informed consent. However, the maternal and child safety of re-cryopreservation procedure still needs to be con rmed by a long-term multicenter prospective cohort study.