The number of prior pregnancy losses does not impact euploidy rates in young patients with idiopathic recurrent pregnancy loss

Our study aimed to determine the possible factors that might impact the probability of obtaining a euploid blastocyst following intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A) procedures in idiopathic recurrent pregnancy loss (RPL) patients. This single-center retrospective cohort analysis included 180 oocyte retrieval cycles of 166 women under 35 years old and those diagnosed with idiopathic RPL according to American Society of Reproductive Medicine (ASRM) guidelines. Trophectoderm biopsy and next-generation sequencing (NGS) were the techniques used. Patients were stratified by the number of previous losses (Group A: 2, Group B: 3, and Group C: > 3). Baseline and embryological characteristics showed no statistically significant differences. The euploidy rate per analyzed blastocyst was comparable within the groups (63.3%, 58.2%, and 58.5%; p = 0.477). Logistic regression analyses confirmed that only the trophectoderm scores of A and B increased the probability of obtaining a euploid embryo [OR: 1.82, 95% CI (1.120–2.956), p: 0.016]. It is concluded that there was no correlation between the number of previous losses and the chance of finding at least one euploid embryo in ICSI cycles of women younger than 35 years.


hCG
Human chorionic gonadotropin ICSI Intracytoplasmic sperm injection ICM Inner cell mass NGS Next-generation sequencing P 4 Progesterone PGT-A Preimplantation genetic testing for aneuploidy POCs Products of conception RPL Recurrent pregnancy loss SART-CORS Society for assisted reproductive technology clinical outcomes reporting system SNP Single nucleotide polymorphism TV-USG Transvaginal ultrasound

Introduction
Recurrent pregnancy loss (RPL) is defined as two or more clinical losses and is reported to have an incidence of 1-5% [1]. The accepted causes of RPL include parental chromosomal abnormalities, uterine anomalies, endocrine abnormalities, and antiphospholipid syndrome [2]. However, in at least 50% of cases, there is no convincing etiology, and they are thus defined as having idiopathic RPL [3]. Although most early pregnancy losses are believed to be genetic in origin, the debate is still ongoing regarding the association between fetal aneuploidy and RPL. Studies designed on products of conception (POCs) to date have reported conflicting results. Some previous studies have reported an increased incidence of embryo aneuploidy in RPL cases [4][5][6][7]. The hypothesis that is put forward to explain this issue is that some couples have a genetic predisposition to an embryo karyotype imbalance even at young ages [8]. For example, the mother's carrier status of a variant of the PLK4 gene has been associated with mitotic nondisjunction events that cause embryo karyotype aberrations in the early embryogenesis period [9]. Contrary to prior findings, more recent studies [10][11][12] conducted on the evaluation of POC have reported that when stratified by maternal age, RPL cases do not have an increased risk of aneuploidy when compared with sporadic losses. In addition, a recent meta-analysis including 55 published studies that investigated the genetic structure of POC found a similar percentage of chromosomal abnormalities in women who had a history of single loss or recurrent pregnancy losses [13].
It is well known that aneuploid embryos are generally unable to attach to the endometrium or do not have the capability of generating a viable gestational sac. In particular, in aneuploidy cases ending with biochemical losses and spontaneous abortions, the evaluation of POC is not possible; hence, aneuploidy rates in POC might be lower than expected. This is where preimplantation genetic testing for aneuploidy (PGT-A) can come into play.
Advanced maternal age is the major cause of increased meiotic nondisjunction abnormalities seen in the embryos. Therefore, it should be assessed in a different category. PGT-A may improve the live birth rates and reduce the abortion risks per embryo transfer in this group [14,15]. Furthermore, in a recently published Society for Assisted Reproductive Technology Clinical Outcomes Reporting System (SART-CORS) study, the benefit of PGT-A was greater in women of advanced maternal age who suffered from RPL [16]. However, there is insufficient evidence on whether PGT-A improves the clinical outcomes in couples with idiopathic RPL [17,18]. In addition, as PGT-A is a costly treatment strategy, a detailed evaluation would determine which subgroups of patients with idiopathic RPL would benefit more from PGT-A.
To this end, we aimed to identify possible clinical and embryological factors affecting the probability of obtaining euploid embryos in women under 35 years of age with idiopathic RPL and tried to determine any relationship between embryo ploidy and the number of previous losses.

Patient population
This retrospective study was performed at Bahceci Fulya IVF Center in Istanbul, Turkey. The data of patients who underwent intracytoplasmic sperm injection (ICSI) followed by PGT-A between January 2016 and July 2019 were reviewed. As recommended by the American Society of Reproductive Medicine (ASRM), women with RPL had a complete RPL workup that included blood work for parental karyotypes and detection of the presence of antiphospholipid antibodies, including anticardiolipin antibody, lupus anticoagulant, and beta-2-glycoprotein, as well as a uterine cavity evaluation. Women were also routinely screened for hypothyroidism and hyperprolactinemia by measuring the levels of serum thyroid-stimulating hormone and prolactin. Patients with a complete RPL workup and unknown etiology for RPL were included in this study. Considering that maternal age and segmental rearrangements have a significant effect on embryo ploidy, females aged ≥ 35 years and couples who were carriers of chromosomal translocation or inversion were excluded. A total of 166 women who underwent 180 oocyte retrieval cycles with PGT-A were included in the study. PGT-A was performed using a trophectoderm biopsy followed by analysis with a next-generation sequencing (NGS) platform. Ovarian stimulation, oocyte retrieval, ICSI embryo culture, and trophectoderm biopsy Controlled ovarian hyperstimulation was performed with the GnRH antagonist protocol. Recombinant FSH (Gonal-F; Merck Serono, Germany) and/or hMG (Merional; IBSA, Switzerland) was initiated on Day 2 of the menstrual period. The dosage of gonadotropins was determined based on the physician's preference regarding the antral follicle count, BMI, and female age. Starting on the fifth or sixth day of stimulation, the ovarian response was monitored by serial transvaginal ultrasound (TV-USG) and by measuring serum estradiol (E 2 ) and progesterone (P 4 ) levels. When the leading follicle exceeded 13 mm in diameter, 0.25 mg of GnRH antagonist (Cetrotide; Merck Serono, Germany) was started daily until the day of the maturation trigger. Patients were administered 250 μg of human chorionic gonadotropin (hCG; Ovitrelle, Serono) or 0.2 mg of triptorelin (Gonapeptyl; Ferring, Spain) for final oocyte maturation when at least two follicles reached 18 mm in diameter, and TV-USGguided follicle aspiration was performed after 35 h.
The oocyte retrieval, denudation, and ICSI procedures were performed as described previously by Serdarogullari et al. [19]. After microinjection, oocytes were cultured individually in a special preequilibrated culture dish. In our study, single-step media, namely Continuous Single Culture Complete with Human Serum Albumin (Irvine Scientific, USA), were used for embryo culture throughout the culture period.

Embryo morphology assessment and trophectoderm biopsy
All embryos were kept in benchtop incubators (MIRI, ESCO Medical, Singapore) and cultured until Day 6 of embryo development. Embryo development was recorded daily. Blastocyst grading was performed at 114 h (Day 5) and 138 h (Day 6) after ICSI using the Gardner and Schoolcraft grading scale [20]. For all developing embryos on Day 3, the zona pellucida was breached by assisted hatching (AH; a hole with a diameter of approximately 20 μm was created) with the help of a laser (OCTAX Navilase, Sweden). Embryo biopsy was performed in mHTF with gentamicin (mHTF, Irvine Scientific, CA, USA) containing 10% SSS (Irvine Scientific, CA, USA) using the pulling method, as previously described by Zhao et al. [21]. All embryos with proper blastocoelic expansion and showing trophoblastic herniation on Day 5 and Day 6 underwent biopsy. The biopsied material consisted of five to eight cells. After the biopsy, each biopsied specimen was individually transferred into a polymerase chain reaction tube and kept frozen at − 20 °C until genetic analysis.
The biopsy samples were sent according to the number and payment requested by the patient.

Preimplantation genetic test for aneuploidy (PGT-A)
The NGS platform performed genetic analysis of biopsied materials (Reproseq PGS Kit, Life Technologies/Thermo Fisher, USA), as previously validated and published [22,23]. NGS steps were then subsequently performed by a PGM sequencing machine using a 318 chip or in an S5 TM XL sequencer using a 530 chip (Thermo Fisher Scientific). Ion Reporter software (version 5.4; Thermo Fisher Scientific) was used for data analysis. For each biopsied sample, a report was generated for each corresponding embryo as being euploid, aneuploid, or chaotic abnormal. Mosaic findings below 30% were reported as 'euploid', and mosaic findings above 30% were reported as 'aneuploid' as per the policy of the genetic laboratory.

Statistical analysis
One hundred and sixty-six patients with a history of idiopathic RPL were evaluated. Patients were divided into three main groups depending on the number of previous losses. Three groups in the study were computed as having 2, 3, or more than 3 losses. All statistical analyses were performed with SPSS for Windows software package version 20 (SPSS, Chicago, USA). A p value of < 0.05 was considered statistically significant for all statistical tests. Continuous quantitative variables were tested to determine whether such parameters followed a normal distribution. From the results of the Kolmogorov-Smirnov and Shapiro-Wilk test statistics, it was found that the continuous parameters under investigation were not normally distributed. Therefore, median (quartile 1-quartile 3) values are reported. An independent-samples median test was run to determine if there were differences in continuous parameters between the three groups. A Chi-square test was performed to identify statistically significant differences in ratios.
Further analysis was performed on 166 patients with 180 OPU cycles who underwent ICSI treatment to identify the factors affecting the outcome of obtaining a euploid embryo. Due to the clustered nature of the data (cycles and embryos originating from the same patient), data analysis was performed by GLMM to build the model. In this model, the effects of female age, male age, BMI (kg/m 2 ), number of previous losses, number of live births, day of trophectoderm biopsy, and embryo quality on the likelihood of euploid embryos were assessed.

Results
A total of 166 patients with a history of idiopathic RPL with 180 oocyte retrieval cycles, including PGT-A, were enrolled in the study. Patients were further categorized into three subgroups based on the number of previous losses (Group A: 2, Group B: 3, and Group C: > 3). Table 1 presents the baseline characteristics of the three subgroups. There were no significant differences in female age (p = 0.056), body mass index (BMI) (p = 0.6), or percentage of patients with previous live births (p = 0.99). Table 2 demonstrates the comparison of the cycle and embryological characteristics of the three subgroups. There was no statistically significant difference in the blastocyst rate per 2PN (46.2% (338/732), 41.9% (262/625), and 44.5% (236/530), respectively; p = 0.288). Trophectoderm and inner cell mass (ICM) scores and the day of embryo biopsy were also not different among groups. The mean number of blastocysts biopsied was 3 (2-4), 3 (1-4), and 3 (2)(3)(4)(5) in Groups A, B, and C, respectively (p = 0.971)   Table 4). The only independent factor that affected the probability of obtaining a euploid embryo was the trophectoderm score. Trophectoderm scores A or B increased the probability of obtaining a euploid embryo

Discussion
This study highlights the following topic of interest: the factors that may affect the probability of obtaining euploid embryos in PGT-A-implemented ICSI cycles of patients suffering from idiopathic RPL. The trophectoderm score was found to be the only significant factor among the clinical and embryological parameters assessed. There was no relationship between ploidy status and the number of previous pregnancy losses. Couples with RPL are thought to produce higher rates of aneuploid embryos. However, there are limited data on the aneuploidy rates of embryos, and most of the data are based on blastomere biopsy results and FISH technology [24][25][26][27]. In 2009, Rubio et al. reported a retrospective analysis that evaluated the prognostic factors for PGT-A in RPL [28]. The results revealed that the number of previous losses is an important parameter that impacts the outcome. In the subgroup of patients under age 37, aneuploidy was inversely related to the number of previous losses. In addition, in this group, the implantation and pregnancy rates decreased with an increasing number of previous losses.
In their retrospective analysis, Kort et al. analyzed 18,387 trophectoderm biopsy results, and patients with a history of RPL had a significantly higher aneuploidy rate than patients undergoing PGT-A for sex selection with the use of a single nucleotide polymorphism (SNP) microarray (OR: 1.33) [29]. A recent retrospective study comparing the rates of aneuploidy in blastocysts of unexplained RPL cases with the rates of blastocysts from cases with proven fertility who underwent preimplantation genetic testing for monogenic defects revealed an increased rate of aneuploidy in the RPL group aged ≤ 35 years (48.9% vs. 36.9%, p < 0.001), whereas no significant increase was found in the > 35-year-old group (66.9% vs. 61.4%, p = 0.175) [30]. In this study, the evaluated number of embryos in the group ≤ 35 years of age was 274. Although the study had a sufficient sample size for all populations, it was not sufficient for the group ≤ 35 years of age. The main criticism of all these studies [24][25][26][27][28][29] except one [30] is the use of an inappropriate control group. In addition to their drawbacks, all of the studies reported increased aneuploidy rates in RPL cases.
In our study, the aneuploidy rate in idiopathic RPL was 39.6% (358/593), which seems to be similar to the large data in infertile patients (36.9-40.5%) [31]. Our aim in this study was to analyze which factors might affect the probability of obtaining a euploid embryo in a specific group of patients; therefore, we did not establish a control group. The euploidy rate per analyzed blastocyst was similar among the 3 study groups stratified by the number of previous losses (63.3%, 58.2%, and 58.5%, respectively; p = 0.477). GLMM regression analysis revealed that the number of previous losses was not a significant factor in the probability of obtaining euploid embryos.
Previous studies evaluated whether there was any correlation between standard blastocyst morphology and euploidy [32][33][34][35]. These studies reported an increased aneuploidy rate among blastocysts with poor morphologic scores and a However, all these studies had different methodologies for evaluating the quality of blastocysts. Our study included only hatching blastocysts and evaluated the trophectoderm and ICM score separately and revealed that the trophectoderm score was an independent factor that affected the ploidy status of embryos. This is in line with the findings of the above-mentioned studies. It is clear that overall blastocyst morphology does not equivocally correlate with embryo ploidy status; however, there may be a moderate relationship with trophectoderm morphology.
It is difficult to determine the aneuploidy pattern of embryos in RPL because of the inappropriate control group. Aneuploidies detected in the POC describe abnormalities of pregnancies that can promote implantation but are unable to develop further. The most frequently detected aneuploidies in POCs involve chromosomes 16, 22, 21, and 15. Aneuploidies in chromosomes 1 to 7 are seen less frequently [36]. In the present study, we found that the most frequently affected chromosomes were 16 (12.1%), 21 (10.7%), 20 (9.7%), and 22 (9.2%), whereas the least frequently affected chromosomes were 1 and 8 (Fig. 1). Although the frequently affected chromosomes seem to be similar in embryos and POC, the less frequently affected chromosomes are distinct. These results suggest that implantation failures and very early pregnancy losses might be due to aneuploidies with larger chromosomes.
To date, there has been no consensus in the literature on whether PGT-A improves live birth rates in RPL cases. In time, large, well-designed randomized controlled studies will shed light on the efficacy and safety of this technology. Regarding the increased cost of PGT-A treatment, the prognostic factors of PGT-A in RPL and subgroups of patients who are most likely to benefit should be determined. In particular, this is the first study that utilized a multivariate analysis to investigate any association between patient and embryological parameters and PGT-A outcomes. The main limitation of this study was its retrospective nature, which might have led to bias. In addition, the genetic results presented in this study are not displaying the results of low mosaic embryos accepted as euploid. This may have caused the statistics to be misinterpreted. Finally, by design, we included only the cases that had at least one blastocyst suitable for genetic testing in the analysis. Therefore, the present study does not reveal the total cycle potential of all couples applied with an initial diagnosis of idiopathic RPL. Undoubtedly, the most ideal study designs to guide physicians are randomized control studies and prospective studies. However, the idiopathic RPL population is one of the smallest proportions of patients among infertile couples, hence, it would be not easy to make an accurate power analysis in the former type, and time-consuming for the latter. Even though a retrospective cohort design causes limitations in assessing the exact incidence of potential confounders, we gave an effort to eliminate them by strict exclusion criteria.
In conclusion, this study provides evidence that the number of previous losses does not affect the risk of aneuploidy in patients with idiopathic RPL when the female age is under 35. RPL is a multifactorial disorder. Of these, genetic abnormalities of the conceptus constitute 45% percent of cases as detected by conventional karyotype analysis and Arraybased Comparative Genomic Hybridization (array-CGH) [37]. Some evidence from human and mice studies revealed specific genes leading to embryo lethality and causing pregnancy loss [9,38]. If so, the genetic origins of RPL cases can be elucidated by identifying specific genes with more advanced genetic studies. Moreover, successful implantation necessitates compliance between the blastocyst comprising intrinsic genome instability and endometrium that undergoes cyclic waves of rapid growth, decidualization, and menstrual shedding [39]. Apart from the chromosomal status of the embryo, receptivity and selectivity of the endometrium are also crucial throughout the first trimester of pregnancy. Further research should also focus on optimizing the endometrium before conception.