Patients
This retrospective study included data from thalassemia carriers who received PGT and underwent FWBT between June 2014 and December 2015 in the Reproductive Medical Center of the First Affiliated Hospital of Sun-Yat San University, Guangzhou, China. Our center has a history of more than 15 years using fluorescent gap PCR analysis for detection of α-thalassemia with SEA deletion, and PCR-reverse dot blot (RDB) hybridization analysis for β-thalassemia diagnosis. As previously stated, couples were initially divided into two groups according to the mutation types of thalassemias: couples with α-thalassemiaSEA genotype receiving CB (Group CB), and couples with one of the 16 common β-thalassemia mutations receiving BB (Group BB). Embryos were frozen by vitrification following blastocyst culture in Group CB, or just after TE biopsy for Group BB.
There had to be at least one blastocyst diagnosed as a normo-homozygote or heterozygote (unaffected blastocysts jointly), which survived for transfer after warming. Confounders which may have had an adverse impact on implantation were excluded. These included female age ≥35, intrauterine adhesion, uterine malformation/scar, unexplained recurrent miscarriage, immune disorders (systemic lupus erythematosus, antiphospholipid syndrome), and thyroid dysfunction. Finally, 202 cycles in Group CB and 237 cycles in Group BB met our criteria.
All study subjects were required to accept an initial consultation at both the Reproductive Medical Center and its affiliated Genetic Laboratory to acquire information on all aspects of the COS and PGT procedures.
Embryo Culture and Biopsy
In Group CB, oocytes after ICSI were cultured in G1 medium (Vitrolife, Sweden) for 3 days under 6% CO2, 5% O2, and 89% N2 in a COOK incubator (Bloomington, IN, USA). Embryos that had at least five or more cells with <50% of fragmentation (qualified cleavage-stage embryos, QCSEs) early on day 3 after oocyte retrieval were placed into 20 µl Ca2+/Mg2+ free medium under mineral oil (SAGE, BioPharma, US) and subjected to biopsy following zona opening using a non-contact laser (OctaxShotTM laser system). A single blastomere from each embryo was removed by a sampling micropipette. Biopsied blastomeres were placed immediately in RNAse and DNAse-free PCR tubes containing 10-20 µl of PCR-grade water. Biopsied embryos were rinsed thoroughly and transferred gently into G2 medium (Vitrolife, Sweden) until day 5 or 6 after oocyte retrieval for extended culture. Obtained blastocysts graded better than 3BC/3CB on day 5 or 6 were vitrified using the Cryotop (KitazatoBiopharma Co. Ltd., Shizuoka, Japan) method.
In Group BB, embryos were directly cultured to the blastocyst stage in sequential media (G1 and G2, Vitrolife, Goteborg, Sweden) under the same incubation conditions. Blastocysts graded better than 3BC/3CB on day 5 or day 6 were selected for biopsy. A zona hole was drilled by laser on the morning of day 5. TE biopsy was performed when TE cells were herniated from the zona hole. Typically, 5-10 TE cells were aspirated according to two steps described by Kokkali et al.(16). The protocol of cryopreservation in this group was the same as blastocysts from Group CB as previously mentioned.
Genetic testing
Fluorescent gap PCR analysis was utilized for detecting α-thalassemia SEA deletion, while PCR-reverse dot blot (RDB) hybridization analysis was applied for β-thalassemia diagnosis, as shown in our previously published papers. Sixteen β-thalassemia mutations, including CD41-42, IVS-2nt 654, CD17, −28, CD71/72, −29, βE, CD43, −32, −30, Int, CD14/15, CD27/28, CD1/1, CD1/5, and CD31, were covered. The primer sequences and product sizes are shown in Table 1 and all PCRs were performed following the same reaction system and PCR conditions as described previously(18). No chromosome screening was performed in either group.
Blastocyst warming and transfer
The blastocyst warming procedure in both groups was conducted as previously described (19). Only those blastocysts that survived warming were considered for transfer. According to the Gardner-Scoring system based on the degree of expansion and the quality of the inner cell mass (ICM) and of the TE cells, blastocysts were morphologically categorized into 3 grades: excellent (expansion degree of 5-6, with both ICM and TE quality of Grade B or better), good (expansion degree of 5-6, with either ICM or TE quality of Grade C; 4BB or better), and average (graded 3BC/3BC or better but not qualified in the foregoing groups)(20). No more than two, usually single unaffected blastocysts, were transferred using a COOK catheter (GuardiaTM) 3-4 hours after warming in both groups (frozen-warmed blastocyst transfer, FWBT).
Tailored natural ovulation cycle (NC) or hormone replacement treatment protocol (HRT) was formulated for endometrial preparation. In HRT cycles, oral estrogen (Progynova, BAYER) was prescribed with an initiation dose of 2 mg, twice daily. Doses of estrogen were increased gradually according to the development of endometrium. When endometrium thickness reached 8 mm, a 60 mg dose of intramuscular progesterone (Zhejiang Xianju Pharmaceutical Co., Ltd.) was given. Alternatively, a dose of 90 mg micronized vaginal progesterone bio-adhesive gel preparation (Crinone 8%, Merck Serono) combined with 10 mg oral progesterone tablets, twice daily (Dupbastone, Abbott) was administered. Blastocysts were warmed and transferred on the sixth day of progesterone administration, or 5 days after ovulation.
Clinical outcomes
We compared baseline characteristics of the two groups in COS cycles, including maternal age, BMI, and levels of basal FSH, LH, and E2, embryo morphology on Day 3 and Day 5/6, and diagnostic results. Pivotal parameters affecting embryo implantation were analyzed. These specifically covered the endometrium preparation protocols, interval time from the OPU day of stimulation cycle to the embryo transfer (ET) day of subsequent first FWBT cycle, blastocysts grades after warming, and average number of embryo transferred, amongst other factors. Outcomes in the successive 1st, 2nd and 3rd FWBT cycles were followed-up.
A preclinical pregnancy was defined as two consecutive positive rising (10 IU/ml) serum β-HCG levels, measured 12 days after transfer day and with an interval of 3 days. A clinical pregnancy was defined as fetal viable heartbeats detected by a transvaginal ultrasound scan at least 6 weeks after embryo transfer. A clinical miscarriage was defined as a spontaneous pregnancy loss (cycles of biochemical pregnancy and multiple pregnancy reduction were excluded) before 28 weeks of gestation. Follow-up data on pregnancies and deliveries were collected via phone contacts (records in delivery file used for birth certificate or discharge records). The implantation rate (detected gestational sacs divided by the total embryos transferred) was taken as a primary outcome measure, whereas the cumulative live birth rate (CLBR, number of patients with first live birth that had been achieved from the first to the current cycle divided by the initial number of patients, irrespective of the duration of gestation and additional live births) was the end-point of the study.
Statistical Analysis
The statistical analysis was performed using Statistical Package for Social Science, version 23.0 (SPSS Inc., IBM corp). Continuous variables with normal distribution were given as mean ± standard deviation (SD) and tested using the Student’s t-test. Otherwise the Mann-Whitney U test was utilized, as demonstrated by the Kolmogorov-Smirnov test. Statistical analysis of contingency tables was performed with the use of Pearson’s chi-square analysis, or, if any expected frequency was ≤4, by a two-tailed Fisher exact test.
To reduce selection bias, confounding factors covering maternal age at OPU, basal hormone levels, history of miscarriage, stimulation protocols, numbers of oocytes retrieved, parameters of the first FWBT as the endometrium preparation protocols, blastocyst grades after warming, number of transferred embryos, endometrial thickness, and levels of E2 and P on the day of transfer, were set as the PSM independents. The PSM module was utilized to calculate propensity score by using the nearest neighbor matching as logistic regression, and 1:1 patient matching proportion was performed. The Hofemer-lemeshow test was used to assess the goodness of categorical variables fit. The calipers value was set as 0.2. The Fine and Gray’s test was applied to perform a competing risk analysis between CB and BB considering comparison of CLBR, where early pregnancy loss was considered a competing risk to the outcome live birth in our study. This analysis was conducted using Stata (version 15.0, StataCorp LLC). All analysis was two-sided and a P value of <0.05 was considered to be statistically significant.