Impact of trophoectoderm biopsy for preimplantation genetic testing on serum β-hCG levels, time of delivery and birthweight following frozen embryo transfer cycles

Abstract Aim: This study investigated whether trophoectoderm (TE) biopsy adversely impacts serum β-human chorionic gonadotropin (hCG) level on the 15th day of embryo transfer (ET), delivery week and birthweight, between biopsied and unbiopsied embryo groups, in a cohort of women who delivered a singleton baby, following frozen–thawed ET. Methods: All women having had a live birth after blastocyst ETs following frozen ET cycles with preimplantation genetic testing (PGT) were included. A control group was selected among women who had a live birth following single frozen blastocyst transfer without PGT-A at the same period in our clinic Results: One hundred fifteen and 173 cycles with- and without-PGT, respectively, were included. Serum β-hCG level on the 15th day after ET was comparable between the groups (p = .336). Average birthweight of the babies born following biopsied embryos were significantly lower (3200 vs. 3380; p = .027). Women who received trophectoderm biopsied embryos had a significantly higher probability of having a baby weighing ≤1500 g and 1500–2500 g (p = .022) or ≤2500 g (p = .008). Proportion of preterm delivery was significantly higher in the biopsy group (p = .023). However, after adjusting for potential covariates, trophectoderm biopsy did not seem to increase the risk of preterm birth (OR 1.525; 95% CI, 0,644–3.611; p = .338) Conclusions: TE biopsy does not seem to impact serum β-hCG level on the 15th day after ET. Average birthweight is lower when a biopsied embryo was transferred. After adjusting for potential covariates, trophectoderm biopsy does not seem to increase the risk of preterm birth


Introduction
Human embryos tend to have chromosomal abnormalities, which become more prevalent as the woman ages.The chromosomomal aneuploidy risk is ~20% to 31% in women between 26 and 34 years old, whereas the incidence of aneuploidy gradually increases from 34% to 75% when the age is ≥35 years [1].Chromosomal aneuploidies are a major cause of implantation failure or embryonic development arrest [2]; thus, the probability of successful natural and in vifertilization (IVF) pregnancy is significantly reduced and miscarriage rates are increased in older women.
To improve IVF outcomes, embryos are being investigated for chromosomal abnormalities before implantation through preimplantation genetic testing for aneuploidies (PGT-A).Although still controversial, the most common clinical indications of PGT-A include advanced maternal age and recurrent implantation failure or pregnancy loss.Since the studies reporting a negative effect of cleavage stage embryo biopsy on implantation rates have been published, blastocyst biopsy has become the mainstream technique of PGT-A.From a technical perspective, PGT includes biopsy of blastocyst stage embryos and retrieval of a multicellular fragment from trophoectoderm (TE), which, in turn, eventually gives rise to the placenta.Subsequently, tissue specimens from the blastocysts are evaluated for chromosomal ploidies.Randomized controlled trials and meta-analyses have demonstrated that clinical outcomes of IVF were ameliorated by PGT-A, particularly in woman beyond the age of 35, but not in those below 35 [3][4][5].Moreover, transfer of biopsied blastocyst stage embryos resulted in significantly higher implantation and delivery rates as compared to non-biopsied blastocysts [6].In addition, the risk of miscarriage was reported to decline significantly in IVF cycles with PGT-A [7].
However, TE biopsy procedure is invasive and there are still perturbations regarding the short-and long-term developmental competency of embryo as well as perinatal outcomes when PGT-A is employed.Some studies reported that the number of biopsied TE cells are negatively correlated with the implantation rates [8,9].As part of the routine biopsy application, when 5 to 10 cells from the TE are removed, developmental potential and implantation rates of the embryo seems not to be affected after the biopsy.However, data on the potential long-term impacts, that would possibly be manifest late at pregnancy, such as growth restriction, hypertensive disorders, or preterm birth, which could be attributed to the placental dysfunction, are still sparse.In 2020, Lu et al. [10] reported that TE biopsy reduced the serum β-human chorionic gonadotropin (hCG) levels on the 12th day after embryo transfer (ET) without jeopardizing perinatal outcomes.Contrarily, in their study, Makhijani et al. [11] demonstrated that probability of developing hypertensive disorders of pregnancy was significantly higher in TE biopsy group compared with unbiopsied group; however, the risk of fetal growth restriction and proportion of low or very low birthweight infants were comparable between the groups.
This study aimed to investigate whether TE biopsy adversely impacts implantation potential of embryos, by comparing serum β-hCG level on the 15th day of ET and perinatal outcomes, such as delivery week and birthweight, between biopsied and unbiopsied embryo groups, following transfer of frozen-thawed blastocysts, which eventually resulted in a singleton live birth.

Methods
We performed a retrospective cohort study that included frozen embryo cycles following a TE biopsy for PGT-A performed in the period between January 2016 and December 2019 at Bahçeci Fulya IVF Center, Istanbul, Turkey.The study was approved by the Institutional Review Board of Üsküdar University, Faculty of Medicine, on February 25, 2021 (Reference number: 61351342/ ŞUBAT 2021-50), and a revision was submitted afterward, which was approved on May 28, 2021 (Reference number: 61351342/ MAYIS 2021-75).All women having had a live birth after blastocyst ETs following frozen embryo transfer (FET) cycles with PGT were included.A control group was selected among women who had a live birth following a single frozen blastocyst transfer without PGT-A at the same period in our clinic.There are strong data in the available literature that PGT-A embryos are associated with higher pregnancy, ongoing pregnancy and live birth rates per ET [3][4][5].This may represent a selection bias in favor of the PGT-A group as compared to untested blastocyst ETs.Thus, the control group included 'good-prognosis and young women' who had untested blastocyst embryos, in our clinical experimental design.To exclude a selection bias favoring the reproductive outcomes of PGT-A group due to the euploid ET, control group comprised of women below the age of 37 with transferred embryo qualities of 4AA, 4AB, 5 and 6 according to Gardner scoring [12].
Exclusion criteria were the main factors possibly affecting the implantation potential of the embryo, which were uterine/endometrial factor such as congenital abnormalities, uterine fibroids or endometrial synechiae, and known chromosomal or immunological problems.In addition, multiple pregnancies, women receiving more than 1 embryo per transfer, fresh ET cycles, and those with PGT for monogenic diseases (PGT-M) were excluded from the study.

Controlled ovarian stimulation and oocyte retrieval
Patients underwent ovarian stimulation using recombinant follicular stimulating hormone (FSH) (rFSH) (150-300 IU, Gonal-F; Meck Serono) or human menopausal gonadotropin (HMG) (75-150 IU; Merional; IBSA) in an antagonist protocol, starting on the second or the third day of the menstrual cycle.Gonadotropin doses were individually adjusted according to the patient's age, body mass index (BMI), medical history and anti-Müllerian Hormone (AMH) values.Pituitary downregulation was performed with daily administration of a gonadotropin-releasing hormone (GnRH antagonist) (Cetrorelix acetate) (Cetrotide; Merck Serono) starting from day 5 or 6 of stimulation.Together with serum estradiol level, periodic transvaginal ultrasound scans were performed to monitor the development of the growing follicles.rFSH/HMG doses were adjusted according to the ovarian response, when necessary.Final oocyte maturation was triggered with 250 mcg of recombinant hCG and/ or 0.2 mg of triptorelin, according to physician discretion, when at least two follicles reached 18-20 mm in diameter.Transvaginal oocyte retrieval was performed 35 h after trigger.

Oocyte denudation, Intracytoplasmic Sperm Injection (ICSI), embryo culture and biopsy
Oocyte denudation, Intracytoplasmic Sperm Injection (ICSI) and embryo culture procedures were performed as previously described in detail by Boynukalin et al. [13].Briefly, oocytes were denuded and ICSI was performed 4 h after retrieval and cultured in a single-step medium, namely, Continuous Single Culture Complete with Human Serum Albumin (Irvine Scientific, CA, USA).Assisted hatching of viable cleavage stage embryos was performed on day 3 and culture was continued in individual droplets until day 5 or 6, at which time biopsy was performed on blastocysts with a viable grade.Micromanipulation and biopsy were performed with the use of laser pulses to release five to ten TE cells for analysis, which were transferred into microcentrifuge tubes and sent to genetic laboratory.Genetic analysis was performed with next generation sequencing (NGS).Following biopsy, all blastocysts were vitrified.

Embryo vitrification and warming procedures
Vitrification and warming procedures were performed as previously described by Boynukalin et al. [13].In this study, a commercial vitrification kit (Vit Kit-Freeze, 90133-SO, Irvine Scientific) for embryo vitrification and a vitrification warming kit (Vit Kit-Thaw, 90137-SO, Irvine Scientific) for warming procedures were used.During embryo vitrification, an open carrier device was used in all cases.Moreover, after completion of the warming procedure, the embryo was transferred to a pre-equilibrated culture dish until ET, and blastocyst grading was performed 2-3 h after the warming procedure.Viability after warming was quantified and classified according to the percentage of surviving (100%, ≥50%, <50%, 0%) intact morphology comprising a distinguishable inner cell mass and trophoblast in a blastocyst-stage embryo and the blastocoel re-expansion ability.

Endometrial preparation for FET
All embryos were transferred in a programmed artificial protocol.Endometrial lesions such as endometrial polyps, submucous myomas and chronic endometritis were managed by hysteroscopy or antibiotic treatment before ET.All women were screened for endometrial thickness using transvaginal sonography, and blood samples were taken to measure estradiol and progesterone levels before performing the frozen embryo transfer (FET).Patients undergoing artificial FET protocol began taking oral estradiol valerate (Estrofem 2 mg tablet, Novo Nordisk, Denmark) on the second day of the menstrual cycle.The tablets were administered with an incremental dose protocol, starting with 2 mg to 6 mg daily.A vaginal ultrasound examination was conducted 10-15 days later to measure endometrial thickness and ensure that no dominant follicle emerged.When endometrial thickness reached 7 mm, 50-mg daily subcutaneous (Prolutex, IBSA, Switzerland) or intramuscular (Progestan, Kocak Farma, Turkey) progesterone supplementation was administered to provide luteal phase support until 10 weeks of gestation if a pregnancy had occurred.ET was performed on the sixth day of progesterone supplementation.
As part of a routine clinical practice, all women are required to give a blood sample for β-hCG levels on the 15th day after ET to confirm pregnancy.

Outcome measures
The primary outcome measure of this study was the average serum β-hCG levels, which was measured by blood test on day 15 after ET, with values >20.0 mIU/mL considered positive and indicative of start of pregnancy.Presence of a gestational sac observed by transvaginal ultrasound at 3-4 weeks after transfer was considered evidence of implantation.Fetal heartbeat was confirmed by endovaginal ultrasound 6-8 weeks after transfer.A live birth was defined as any birth event in which at least one baby was born alive >24 weeks of gestation.
Secondary outcome measures included perinatal outcomes such as gestational age at delivery, birthweight of the neonate and proportion of low birthweight (≤2500 g) and very low birthweight (≤1500 g) infants.

Statistical analysis
Statistical analysis was performed using Statistical Package for the Social Sciences for Windows (version 21.0;IBM Corp., USA).Normality was tested using the Kolmogorov-Smirnov test for continuous variables.Continuous data are presented as the mean ± standard deviation (SD) and median (25th-75th percentile).Categorical data are described by number of cases, including numerator/denominator, percentages and odds ratios (OR) with confidence intervals (CIs).Categorical data and continuous data were analyzed by Pearson's chi-square test/Fisher's exact test or Mann-Whitney U test as appropriate.Potential covariates that may be associated with preterm birth adjusted for in the model included age and BMI as continuous variables and smoking status and parity as categorical variables.A p value <.05 was considered statistically significant.

Results
All women who had a live birth following a frozen embryo cycle with PGT-A process in Bahceci Fulya ART Clinic, in the period from January 2016 to July 2019 were assessed.One hundred seventeen PGT-A cycles from 114 patients were assessed for initial inclusion.Two cycles from women with uterine pathologies were excluded and 115 PGT-A cycles were included in the study (Biopsy Group).A control group was composed from women with a live birth following a frozen ET without PGT-A in the same period; thus, 256 cycles were accepted for the initial assessment.Given that the euploid ET would yield higher chances of reproductive outcomes including live birth, we included only women with transferred embryos of grade 5, 6 or 4AA and 4AB in the control group, to exclude a selection bias.With the same rationale, women beyond the age of 37 were excluded from the control group as well.Uterine malformations, such as submucous myoma, intrauterine adhesions or septum were excluded.Eventually, 173 FET cycles without PGT-A were included in the control group.In the biopsy group, embryos were exempt from embryo quality and women age assessments.
Table 1 summarizes the baseline characteristics of all the cycles included.No differences were observed in the two groups with regards to maternal BMI or its subgroups, and parity status.Patients who transferred biopsied blastocysts were significantly older (p < .001),more likely to have a history of at least one abortion (p < .001)and a longer duration of infertility (p = .006).Secondary infertility diagnosis was significantly more frequent in the biopsy group (p < .001).Moreover, the patients in the biopsy group more often had a diagnosis of decreased ovarian reserve (DOR), which could be attributed to advanced maternal age or recurrent pregnancy loss (RPL) (p < .001).
Stimulation characteristics of the included cycles are summarized in Table 2. Biopsy group had significantly shorter period of stimulation (p = .044),required higher gonadotropin doses (p = .002);whereas yielded lower numbers of retrieved oocytes (p < .001)and MII oocytes (p < .001).Endometrium was significantly thicker on ET day (p = .028)and embryos were more frequently frozen on day 5 than on day 6 (p < .001) in unbiopsied group than in biopsied group.
Average serum β-hCG level on the 15th day after ET in the biopsy group was lower than in the unbiopsied group; however, the difference was not statistically significant (p = .336).Obstetric outcomes of the groups are summarized in Table 3. Cesarean delivery was more frequent in the biopsy group (p = .024).Average birthweight of the babies born following biopsied embryos were significantly lower as compared to unbiopsied embryos (3200 vs. 3380; p = .027).Moreover, women who underwent trophectoderm biopsy had a significantly higher probability of having a baby weighing ≤1500 g and 1500-2500 g (p = .022)or ≤2500 g (p = .008).In addition, babies heavier than 4000 g was significantly more common in the unbiopsied embryo group (p = .008).On the other hand, preterm birth (<37 gestational weeks) was significantly more frequent in the TE biopsy group (p = .023).Also, trophectoderm biopsy was associated with an increased risk of preterm birth when compared with unbiopsied blastocyst transfer (OR 1.918; 95% CI, 1.072-3.383;p = .024)(Table 4).Maternal age also increased the risk of having a preterm birth (OR 1.065; 95% CI, 1.009-1.123;p = .022).However, after adjusting for potential covariates including age, BMI, smoking status and parity, the association between trophectoderm biopsy and preterm birth lost its significance (OR 1.525; 95% CI, 0,644-3.611;p = .338).

Discussion
This study investigated whether TE biopsy impacts serum β-hCG level, as a marker of embryo implantation, as well as perinatal outcomes, such as delivery week and birthweight, following transfer of frozen-thawed blastocysts, in a patient cohort who delivered a live singleton baby.Our results indicate that pregnancies following transfer of biopsied embryos had lower serum β-hCG levels on 15th day after ET as compared to those of unbiopsied embryos, however, this difference did not reach to the statistical significance border.Moreover, birthweight of babies were significantly lower when a TE biopsied blastocyst was transferred, compared to blastocysts without biopsy.In addition, the risk of preterm birth did not seem to increase in TE biopsy group, after adjusting for potential covariates including age, BMI, smoking status and parity.
Human chorionic gonadotropin is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy [14].Maternal hCG concentration in very early pregnancy varies considerably among women, but is a substantial marker of embryo implantation and pregnancy outcomes.There are studies assessing the association between serum β-hCG levels and the number of cells of the transferred embryo [15] or embryo morphology [16].The birth registry trial from Norway also argued that number of cells in the transferred embryo may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations [15].After the introduction of PGT in the current ART practice, there have been concerns as to whether trophectoderm biopsy for PGT has any impact on serum β-hCG in early pregnancies.However, the data regarding the impact of TE biopsy on serum β-hCG levels and perinatal outcomes is sparse.The first study exploring the answer to this question was published in 2020 by Lu et al. [10] and they suggested that the serum β-hCG levels on day 12 after transfer of the biopsied blastocysts were statistically significantly lower than the levels of the control group.In contrary to our study, they only included women with clinical pregnancy.They hypothesized that removal of the TE cells could decrease the maternal β-hCG level to some extent.Another study from China reported comparable serum β-hCG levels between PGT and non-PGT patients and concluded that TE biopsy of blastocysts for PGT did not affect the serum β-hCG level 14 days after transfer [17].Our results are compatible with this report, suggesting that serum β-hCG levels 15 day after ET are statistically comparable between biopsied and unbiopsied groups in a patient cohort who eventually had a live birth.In a similar manner to our study, they reported that serum β-hCG levels from cycles resulting in clinical pregnancy and ongoing pregnancy were comparable between the PGT and non-PGT groups Placenta has a key role in normal fetal growth; thus, it might be, theoretically, plausible to consider that the fetal growth could be hampered due to TE biopsy, manifested by low birthweight or fetal growth restriction (FGR).Thus, we demonstrated a statistically significant decrease in the average birthweight in the biopsy group.Moreover, babies born after a biopsied ET were more likely to be ≤2500 g.Extremely low birthweight babies (≤1500 g) were significantly more common in the biopsy group, as well.Large for gestational age (LGA) (>4000 g) babies were more frequent in the unbiopsied embryo group.There is a limited number of studies exploring the question of whether trophectoderm biopsy negatively impacts perinatal outcomes.A small sample size study explored the effect of blastocyts biopsy by comparing the outcomes of IVF cycles with and without PGT and reported that TE biopsy was associated with an increased risk of preeclampsia but not gestational hypertension, preterm birth and low birth rate [18].However, fresh and frozen cycles were not separated in this study, and, thus, increased adverse outcomes might have resulted from the higher proportion of frozen ET cycles in PGT group.In their study, Makhijani et al. [11] found no significant differences in FGR, mean birthweight, or proportion of LBW or VLBW babies.Similarly, Lu et al. [10] reported, in their study, that there were no statistically significant differences in birth weight, LBW, VLBW, macrosomia, small for gestational age (SGA), LGA, preterm birth or very preterm birth rates between biopsy and control groups, despite a lower serum β-hCG levels in the biopsy group.They attributed this result to the fact that trophectoderm cells of the blastocysts develop into the placenta and fetal membrane and do not participate in the formation of the fetus.However, given the vital role of the placenta in fetal growth, any jeopardize to normal placentation, shown by a lower serum β-hCG levels, should have an influence on the fetal birthweight.We consider that the inconsistency between the literature data and our results in the mean birthweight and LBW/ VLBW newborn rates may have arisen from our control group selection; who are young and healthy women with very high quality embryos, who delivered heavier babies as compared to study group.Indeed, study group women were significantly older than the controls, as the most important PGT-A indication was the advanced maternal age, as reflected by the higher frequency of decreased ovarian reserve, in the biopsy group (Table 1).
Consistent with the previous studies [10,11], our results indicate that the risk of preterm birth does not increase after transferring a biopsied embryo.Although, crude analysis revealed an increased frequency of preterm birth, logistic regression analysis revealed no significant increase in the risk of preterm birth in the biopsied ET group (OR 1.525; 95% CI, 0,644-3.611;p = .338).Preterm birth is a leading cause of neonatal mortality and life-long disability [19].Advanced maternal age, ethnic origin, increased BMI, increased parity, previous history of preterm birth and smoking are considered as risk factors of preterm birth [20].Thus, we adjusted our results for potential covariates including age, BMI, smoking status and parity.After adjustment, there was no significant difference in the frequency of preterm birth between biopsied and unbiopsied embryo groups (OR 1.525; 95% CI, 0.644-3.611;p = .338);thus, we may infer that TE biopsy does not increase the risk of preterm birth.This result is compatible with the previous study [11] reporting that embryo biopsy does not increase the risk of preterm delivery.
Mean maternal age discrepancy between the groups (Table 1) is derived from the predetermined selection criterion for the control group.Current data supports the concept that PGT-A increases the pregnancy, ongoing pregnancy and live birth rates per ET [3][4][5].This may have represented a selection bias in favor of the PGT-A group, if we had selected untested blastocyst embryos as the control group.Thus, in our clinical design, the control group contained only 'good-prognosis women' who had untested blastocyst embryos.Those were the women below the age of 37, in order to alleviate the possible negative impact of chromosomal abnormalities of the control group embryos, which are known to increase by age.In other words, to exclude a selection bias favoring the reproductive outcomes of PGT-A group due to the overt euploid embriyo transfer, control group comprised of women below the age of 37 with transferred embryo qualities of 4AA, 4AB, 5 and 6 according to Gardner scoring [12].
Despite its retrospective design, this study has several strengths.First, it included only TE biopsies, not polar body or blastomere biopsies.Second, its control group includes only young women below the age of 37 with transferred embryo qualities of 4AA, 4AB, 5 and 6 according to Gardner scoring [12], to eliminate the positive impact of PGT-A on the clinical outcomes of IVF.Also, only singleton pregnancies were included in the study.Finally, only hormone replacement endometrial preparation protocols were included in the study, to eliminate the effect of endometrial preparation protocols on the outcomes.
The limitations of this study include lack of other perinatal outcomes, such as hypertensive disorders and abnormal placentation, which would reflect the function of placenta.On the other hand, this study included only those IVF cycles, which eventually resulted in a singleton live birth, in the both groups.Pregnancies that ended up with an abortion or stillbirth are excluded from the study, thus, it is not possible to conclude on the impact of TE biopsy on abortion or stillbirths.Moreover, some data in each group are lacking, however, the rate of lacking data is less than 10%.
In conclusion, TE biopsy for PGT-A had no impact on the serum β-hCG levels on 15th day after ET.Average birthweight was lower, and the risk of <2500 g or <1500 g birthweight was significantly higher in the biopsied ET group than in unbiopsied ET group.Moreover, trophectoderm biopsy did not seem to increase the risk of preterm birth, after adjusting for potential covariates including age, BMI, smoking status and parity.was submitted afterward, which was approved on May 28, 2021 (Reference number: 61351342/MAYIS 2021-75).

Table 1 .
Baseline characteristics for the groups with and without Te biopsy.
BMi, body mass index; dor, diminished ovarian reserve; PcoS, polycystic ovary syndrome; rPl, recurrent pregnancy loss; Uei, unexplained infertility.Variables are presented as mean ± standard deviation or median (quartile range).Student's t test was used for continuous variables and chi square test was used for categorical values.a p-value <0.05 was considered statistically significant.

Table 2 .
Stimulation characteristics for the groups with and without Te biopsy.

Table 3 .
obstetric outcomes for the FeT groups with and without Te biopsy.

Table 4 .
Unadjusted and adjusted odds ratios the groups with and without Te biopsy for pretem birth.
Binary logistic regression was used to calculate the adjusted or and controlled for the following covariates: age, BMi, smoking status, parity.a p-value <0.05 was considered statistically significant.