Which factors affect the live birth outcome of the first single euploid frozen-thawed blastocyst transfer in couples with balanced chromosomal translocations?

Objective The objective of this study is to investigate the factors that influence the live birth rate (LBR) of the first single euploid frozen-thawed blastocyst transfer (FBT) cycles after preimplantation genetic testing for structural rearrangements (PGT-SR) in couples with balanced chromosomal translocations (BCT). Design Single center, retrospective and observational study. Methods A total of 336 PGT-SR and the first single euploid FBT cycles between July 2016 and December 2022 were included in this study. The patients were divided into two groups according to the live birth outcomes. The parameters of the study population, controlled ovarian stimulation cycles, and FBT cycles were analyzed. Multivariable binary logistic regression was performed to find the factors that affected the LBR. Results The percentage of blastocysts at developmental stage Day 5 compared to Day 6 (51.8% vs. 30.8%; P<0.001) and with morphology ≥BB compared to <BB (49.7% vs. 32.2%; P=0.001) was significantly different between the group that resulted in live births (n=193) and the group that did not (n=143). The results of the multivariable binary logistic regression analysis indicated that the developmental stage (adjusted OR: 2.068, 95%CI 1.291-3.313; P=0.003) and morphology (adjusted OR: 1.697, 95%CI 1.039-2.773; P=0.035) of the blastocyst were significantly correlated with live birth. Patients with blastocysts that reached the developmental stage at Day 5 and had a morphology of ≥BB had a higher likelihood of having a live birth. Conclusion The developmental stage and morphology of blastocyst affect the live birth outcome of the first single euploid FBT in BCT carriers undergoing PGT-SR.


Introduction
There are two main types of structural chromosomal rearrangements: reciprocal translocations and Robertsonian translocations.Reciprocal translocation have an incidence of 1/ 700 in healthy individuals, while Robertsonian translocations have an incidence of 1/1000 (1).These are also known as balanced chromosomal translocations (BCT) because they do not involve significant loss of chromosomal material.Most carriers of BCT are phenotypically normal and may not even be aware of their carrier status until they experience infertility, miscarriage, or the birth of a child with congenital anomalies due to the chromosomally abnormal embryos (2).Preimplantation genetic testing for structural rearrangements (PGT-SR) which selects euploid blastocysts, can increase the chance of having a healthy live birth, reduce the rate of pregnancy loss, and shorten time to pregnancy (3), although the value of PGT-SR is still under debate (4,5).
The reported live birth rate (LBR) per single euploid frozenthawed blastocyst transfer (FBT) is 50-60% (6,7).There is still room for improvement.Several studies have focused on the factors that influence the LBR of euploid FBT afterpreimplantation genetic testing for aneuploidies (PGT-A), as reviewed by Cimadomo et al. (8).The study found that blastocysts of poor quality, blastocysts that were developed on day 6-7, blastocyst that were frozen twice (though biopsied only once) maternal age of 38 years or older, a body mass index (BMI) of 30kg/m 2 or higher, and a history of repeated implantation failure (RIF) were significantly associated with low LBR (8).PGT-A is recommended for women with advanced maternal age (AMA), recurrent miscarriage (RM) with normal parental karyotypes, and RIF (3).In PGT-A cycles, 40% of biopsied blastocysts were euploid (9), and all aneuploids were de novo sporadic.Apart from the embryo chromosomal abnormalities, the etiologies of RM and RIF are complex, such as thrombophilia, immunological factors, endocrine dysfunction, uterine endometrium, and lifestyle (10,11).In BCT carriers, only 26% of the diagnosed blastocysts were found to be euploid (12,13).This is due to the fact that BCT can result in gametes with a high risk of partial duplications, deletions, and whole chromosome aneuploidies.Additionally, there may be some de novo abnormalities of chromosomes that are not related to the rearrangement (14).Theoretically, embryonic chromosomal factors account for a large proportion of adverse pregnancies in BCT carriers compared to RM and RIF populations.However, carriers of BCT may experience frustration when a certain number of euploid FBT do not result in a live birth, but instead in failure of implantation or miscarriage.Few studies have evaluated the factors that influence LBR after a single euploid FBT in patients with BCT.
The objective of this study was to investigate the factors that affect the LBR of single euploid FBT in BCT carriers.The findings may offer supplementary information for the genetic counseling and clinical strategies for this specific group of couples who experience anxiety and stress.

Study design and population
This was a single-center retrospective study of patients with BCT who underwent PGT-SR and had their first single euploid FBT at the Reproductive Medicine Center of Henan Provincial People's Hospital affiliated with Zhengzhou University between July 2016 and December 2022.
The study's inclusion criteria required that either the female or male in the couple was a carrier of BCT, that there were euploid blastocysts available for transfer after PGT-SR, and that the patient had undergone the first single euploid FBT cycle.To avoid confounding by patient-specific factors, only one cycle per patient was included in the study.
The exclusion criteria for this study were as follows: (1) PGT-SR was conducted for reasons other than parental balanced translocation; (2) embryos were unavailable for biopsy or there was a lack of euploid blastocysts for transfer; (3) it was not the first cycle in which a euploid blastocyst was transferred; and (4) FBT was not performed before December 2022.All data were extracted from the medical database.In the end, the study included 336 cycles, as depicted in Figure 1.
All couples underwent genetic counseling and were informed of the advantages and limitations of PGT-SR procedures.They signed the informed consent for PGT-SR therapy.

Treatment protocols
The procedures for controlled ovarian stimulation, oocyte retrieval, embryo transfer, and luteal-phase support were carried out according to our standard protocols, as previously described (15,16).The clinical team utilized controlled ovarian stimulation protocols and gonadotropin doses tailored to each individual patient's characteristics, such as age, BMI, ovarian reserve function, follicle conditions, hormone levels, differences in drugs, prior response to gonadotropins, financial situation, and schedule.The protocols used included GnRH agonist, flexible GnRH antagonist, and progestin-primed ovarian stimulation (PPOS).Final oocyte maturation was induced using a human chorionic gonadotropin (hCG) trigger (Lizhu Pharmaceutical Trading, China) and/or GnRH agonist (Triptorelin Acetate, Ferring, Switzerland) with the dose determined by the number of dominant follicles and peak estradiol level.Oocyte retrieval was then performed 34-36 hours later under transvaginal ultrasound guidance.
In all cycles, fertilization was achieved using intracytoplasmic sperm injection (ICSI).Embryos were cultured until the blastocyst stage on Day 5-6 and those with a grade ≥3BC according to Gardner criteria (17) were selected for trophectoderm biopsy, which was performed by experienced embryologists.Embryos with a grade of 3-6 AA/AB/BA/BB were considered good quality, while others were classified as poor quality.Whole genome DNA from approximately 5 cells per blastocyst was amplified and tested using next-generation sequencing (NGS) technology.
After the biopsy, the blastocysts were vitrified and stored in liquid nitrogen.During the frozen-thawed cycle, only one euploid blastocyst was transferred.The endometrial preparation protocol was determined based on the patient's condition, either a natural cycle or a hormone replacement therapy (HRT).Luteal-phase support was initiated either on the day of ovulation or when the endometrial thickness was ≥ 8mm after taking oral estrogen (4 mg/ day to 8 mg/day) for more than 11 days or when taking the maximum dose of estrogen pills for up to 21 days.This was achieved by using twice-daily oral dydrogesterone tablets (20mg; Duphaston, Abbott, USA) and daily vaginal progesterone gel (90mg; Crinone, Merck Serono, Switzerland) until 10 weeks of gestation.

Outcome measures
The serum b-hCG level was measured 14 days after embryo transfer.A value greater than 10 mIU/mL was considered positive.A transvaginal ultrasound scan was performed 4-5 weeks after embryo transfer to confirm clinical pregnancy by observing the gestational sac, regardless of its location.The clinical pregnancy rate (CPR) was defined by dividing the number of clinical pregnancies by the total number of transfer cycles, expressed as a percentage.Miscarriage was defined as a clinical intrauterine loss that occurred before 22 weeks of gestation.The miscarriage rate was calculated by dividing the number of miscarriages by the number of clinical pregnancies.Live birth was defined as an infant born alive after 28 weeks of gestation.The live birth rate (LBR) was calculated by dividing the number of live births by the total number of transfer cycles (18).

Statistical analysis
Statistical analyses were conducted using SPSS software version 27.0.A significance level of P <0.05 was used.The normality of continuous parameters was evaluated using the Shapiro-Wilk test.All the continuous variables in this study were non-normally distributed and were reported as median (quartile 1-quartile 3) [M (P 25 , P 75 )] using nonparametric tests (Mann-Whitney U test).Categorical variables were presented as percentages (number/total number) and analyzed using the Chi-square test.A multivariate binary logistic regression analysis was performed to investigate the parameters associated with the live birth outcome.
The cycles were categorized based on the live birth outcome.One group consisted of cycles that resulted in a live birth (n=193), Flow chart of cycles' selection.The parameters of FBT cycles are shown in Table 2.The two groups had similar endometrial thickness at the start of progesterone treatment (P=0.167).There was no difference in the endometrial preparation protocol between the two groups (P=0.596).The live birth group had a higher percentage of blastocysts biopsied at day-5 compared to the group without a live birth (51.8% vs. 30.8%;P<0.001).Patients with a live birth had a significantly higher percentage of morphologically good quality blastocysts (≥BB) than those without a live birth (49.7% vs. 32.2%;P=0.001).
To identify factors that might affect the live birth outcome, multivariable binary logistic regression was used.The model included parameters with P<0.2, which were analyzed in univariable analyses between the two groups.These parameters consisted of maternal age, estradiol level on trigger day, number of euploid embryos, endometrial thickness at the start of progesterone treatment during the FBT cycles, and developmental stage and morphology of blastocysts.Additionally, the model included BMI and number of previous miscarriages, which were considered as significant factors in previous studies (19).Table 3 indicates that blastocyst developmental stage and morphology significantly affected the probability of having a live birth.The likelihood of having a live birth increased when the blastocyst developmental stage changed from day 6 to day 5 (adjusted OR: 2.068, 95%CI 1.291-3.313;P=0.003).Meanwhile, transferring good-quality blastocysts increased the likelihood of having a live birth compared to transferring poor-quality blastocysts (adjusted OR: 1.697, 95%CI 1.039-2.773;P=0.035).

Discussion
The aim of this study was to investigate the parameters that may influence the live birth outcomes after the first single euploid FBT in patients with BCT.The results indicate that blastocyst morphology and developmental stage significantly affect the live birth outcome.Blastocysts available for biopsy at day 5 and have morphology≥BB have a higher likelihood of a live birth outcome.Maternal age, BMI, and previous miscarriages were similar between the two groups.
The LBR per single euploid FBT after PGT-SR cycles was found to be affected by the blastocyst developmental stage.However, this finding contradicted some previous studies.For instance, Wu et al. (20)reported no significant difference in the LBR between transfers of single day 5 and day 6 euploid good quality blastocysts (morphology≥4BB).Similarly, Liu et al. (21) found that the LBR of euploid blastocysts on day 5 was comparable to that of blastocysts on day 6 in young women (age < 35 years).Our findings are consistent with Li et al.'s study (9), which also found that day 5 euploid blastocysts had higher LBRs than day 6 blastocysts.Another study with a large sample size (day 5 blastocysts n=2321; day 6-7 blastocysts n=1497) reported similar results (22).Furthermore, in a recent meta-analysis reviewed 18 relative articles published between 2014 and 2021 and found that day 6-7 blastocysts (n=4627) had a significantly lower LBR per single euploid FBT than day 5 blastocysts (n=6716) (OR 0.56, 95%CI 0.48-0.63)(8).It is important to note that the euploid blastocysts analyzed in these studies were from PGT-A cycles, not PGT-SR cycles.The stage of the embryo development may reflect the metabolic health condition of the developing embryo (19).Delayed blastocyst formation may result from consequence of multiple minor embryonic functional defects that prevent an effective implantation and/or subsequent viable pregnancy.
Embryo morphology assessment is a valuable tool for embryo selection and is also suitable for selecting euploid blastocysts.However, it is still controversial whether the morphology of the blastocysts affects the LBR per single euploid FBT.Li et al. (9) found no significant association between the morphology of blastocysts and LBRs of euploid FBT.A multicenter retrospective study conducted by Capalbo et al. (23) found that the euploid embryos of different morphologies and developmental stage yielded a similar ongoing implantation rate.This result was consistent with the findings of the Viñals Gonzalez's study in advanced maternal age (24).However, Cimadomo et al. (25) found that poor-quality blastocysts had a worse prognosis compared to good-quality blastocysts in advanced maternal age.In addition, a meta-analysis revealed that ICM and TE with a score of C, whether considered together or separately, were linked to a decreased LBR per euploid FBT (8).Regarding overall blastocyst quality, blastocysts of poor quality (n=722) had a significantly lower LBR per single euploid FBT compared to blastocysts of good quality (n=4384) (OR 0.20,  95%CI 0.24-0.67).The euploid blastocysts analyzed in these studies were from PGT-A cycles.Due to the subjective nature of morphological grading of embryos, there is a lack of reproducibility between different IVF laboratories (26).This may be one of the reasons for the inconsistent results.Improving interand intra-observer concordance would be helpful.In the future, time-lapse technology combined with artificial intelligence (AI) may offer promising prospects (27).
It is well known that increasing maternal age (especially ≥ 35 years) is associated with decreased reproductive capacity due to decreased ovarian reserve and increased aneuploidy.Additionally, Qiao et al. (28) suggested that the age-related reproductive decline was not only due to increasing aneuploid rates but also to metabolic and epigenetic changes in the embryos.The effect of age on endometrial function is also a concern (29).A meta-analysis conducted recently found that increasing maternal age was linked to a decrease in LBR after euploid FBT following PGT-A (30).However, Idowu et al. (12) and Ogur et al. (31) found no significant difference in the LBR between mothers aged ≥ 35 years and those aged < 35 years (n=18 vs 36, and n=49 vs 164, respectively) after euploid FBT in couples with structural chromosomal rearrangements after PGT-SR.Mateu-Brull et al. (32) found that clinical outcome according to the age group (< 38 years vs 38-42 years) showed no statistical differences for each type of BCT.The present study also found that maternal age had no significant effect on the LBR, which was presented as a continuous variable.Of the 336 patients, 41 were 35 years or older, representing 12.2% of the total.The variations in the results can be attributed to the different chromosomal conditions of the study populations.On the other hand, carriers of BCT tend to be younger.The maternal age of the study population was 29 (26-32) years.
The impact of increased maternal BMI on the LBR after euploid FBT remains uncertain.Boynukalin et al. (19) and Li et al. (9) reported a significant association between BMI and LBR after euploid FBT.Cozzolino et al. (33) and Meng et al. (34) found that obese women (BMI ≥ 30 kg/m 2 ) had a significantly lower LBR after euploid FBT compared to non-obese women.Another study showed that the LBR after euploid FBT was significantly reduced in overweight women (BMI ≥ 25 kg/m 2 ), especially in obese women (BMI ≥ 30 kg/m 2 ) (35).However, some studies have not found a significant association between BMI and LBR after euploid FBT.For instance, In Zhou et al.'s study (36), BMI was comparable among live birth, miscarriage and not-implanted groups.Similarly, in Kim et al.'s study (37), the LBR was similar across all weight categories, including underweight, normal, overweight, and obese groups.And the euploid blastocysts in these studies were from PGT-A cycles.Increased BMI reflects maternal nutritional imbalances and may also be the result of endocrine dysfunction and immunologic factors, and may impair uterine receptivity, alter folliculogenesis, and compromise oocyte quality (38), which could be the cause of RM and RIF.Therefore, there may be confounding factors to consider.The present study found no significant association between BMI and LBR after euploid FBT in BCT carriers.The population in this study had a lower BMI of 22.97(20.70-25.60),which may partially explain the lack of association.Of the 336 patients, 77(21.2%)patients were overweight, with LBR (51.9%) and 21 (5.8%) patients were obese, with LBR (66.7%).Most of the previous studies reporting lower pregnancy rates in obese patients were based on fresh embryo transfers (39,40).The studies by Prost et al. (41) and Insogna et al. (42) which focused on FBT cycles, found that female obesity did not affect LBR.With improved embryo-endometrial synchrony, frozen-thawed embryo transfer may be beneficial for overweight and obese patients.In addition, corpus luteum function may be impaired in overweight or obese patients (35), and increased progestin supplementation may reduce the high risk of miscarriage (37).This finding does not negate the fact that overweight and obesity have adverse effects on maternal and fetal health, such as pregnancy-induced hypertension, gestational diabetes, and fetal growth restriction, so weight intervention is recommended prior to euploid FBT.
Women who have experienced a miscarriage in the past are at a higher risk of experiencing a subsequent miscarriage if they conceive naturally (43).It is uncertain whether previous miscarriages influence LBR after euploid FBT.LBR decreased as the number of miscarriages increased, according to Boynukalin et al (19).Liu et al's study (44) showed that the group with idiopathic recurrent pregnancy loss (iPRL) had a significantly lower LBR compared to the control group.However, Cimadomo et al. (45) found no difference in LBR after euploid FBT (n=1580) among patients with 0, 1, or more than 1 previous miscarriages after PGT-A cycles, which was consistent with the study of Wang et al. (46).Ni et al. (47) investigated the association between previous pregnancy failures and pregnancy outcomes after PGT-A.They found that a history of ≥4 early miscarriages was significantly associated with a higher early miscarriage rate, but was not directly associated with lower LBR after logistic regression.They did not find a clear association between a history of 2 previous early miscarriages and pregnancy outcomes.In the present study, the number of previous miscarriages did not affect LBR after euploid FBT, which is a reassuring.This means that a history of recurrent pregnancy loss has no prognostic value in predicting live birth outcome in euploid FBT after PGT-SR.However, it should be noted that the sample size of high-order miscarriages in our study was relatively limited.Among the 336 patients, 27 (8.0%)patients had 3 previous miscarriages, with LBR (59.3%), and 10 (3.0%) patients had ≥4 previous miscarriages, with LBR (50.0%).Another study, not focused on the PGT cycles, reported by Qiu et al. (48) found that the history of recurrent pregnancy loss was not significantly associated with miscarriage and LBR of the first in vitro fertilization/ICSI frozen embryo transfer or intrauterine insemination cycles, suggesting that fertility treatment itself may mitigate the effect of miscarriage history on subsequent pregnancy.These findings are encouraging.
Whether the parental origin of BCT affects the outcomes of PGT-SR is an interesting question.Tong et al. (49) found that the parental origin of BCT had no significant difference on the laboratory results and ploidy results.In addition, Insogna et al. (50) also found that the type and parental origin of BCT had no significant difference on pregnancy outcomes.Mayeur et al. (51) reported that LBR were not significantly different between female and male BCT carriers.Mateu-Brull et al. (32) observed that ongoing CPR per transfer was comparable between BCT types, and male reciprocal translocation carriers had higher CPR per transfer after day 3 biopsy, but there was no sex effect for CPR per transfer after day 5/6 biopsy.The present study did not find an association between the parental origin of the BCT and the LBR of single euploid FBT.Overall, this means that clinical decisions can be made without regardless of the type and parental origin of the BCT.

Strengths and limitations
This study investigated the factors affecting the LBR after euploid FBT in the population of BCT carriers who are at high risk of adverse pregnancy outcomes undergoing PGT-SR.Previous studies have mainly focused on the patients with AMA, RIF, or RM undergoing PGT-A.The present study focused only on the population of BCT carriers with euploid blastocysts after PGT-SR.It does not represent the entire population of BCT carriers, who face several uncontrollable factors throughout the PGT-SR process, such as the chromosome involved in the structural rearrangement, ovarian reserve, ovarian response, and embryo development before obtaining euploid blastocysts.After euploid blastocysts are obtained, patients may experience anxiety while waiting for the outcome of FBT.This study provides additional information to help alleviate their anxiety and set appropriate expectations.We only included the first single euploid FBT cycles to eliminate confounding factors.However, it has some limitations.This study solely focused on the live birth outcomes, and did not evaluate other clinical outcomes, such as implantation rate and miscarriage rate.Additionally, the retrospective study design carries a certain risk of bias.Furthermore, the sample size is not large enough and has limited power.Lastly, this is a single-center study, and the conclusions can only reflect the results of the center's clinical and laboratory practices.Large-scale multicenter studies will be necessary to confirm our current findings in the future.
In conclusion, blastocyst morphology and developmental stage are the factors that influence the live birth outcome after single euploid FBT in BCT carriers.This information is valuable for clinical counseling and for electing the embryo to transfer.
Zhang et al. 10.3389/fendo.2024.1378635Frontiers in Endocrinology frontiersin.orgwhiletheothergroup consisted of cycles that did not result in a live birth result(n=143).The parameters of the study population and COS cycles are presented in Table1.No significant differences were observed between the two groups in terms of the type of BCT

TABLE 1
Comparison of basic clinical data and parameters of controlled ovarian stimulation cycles between patients with and without a live birth.

TABLE 3
Parameters related to live birth using binary logistic regression model.

TABLE 2
Comparison of parameters of frozen-thawed embryo transfer cycles between patients with and without a live birth.