Abstract
Purpose
It is disputable whether chromosomal translocations lead to an inferior embryo development. The purpose of this study was to evaluate whether structural rearrangements (SR) affect blastocyst formation as compared to monogenic disorders in preimplantation genetic testing (PGT) cycles.
Methods
A total of 791 PGT-SR cycles and 757 PGT-M cycles from January 2021 to May 2023 were included.
Results
Lower blastocyst formation (graded 3BB or higher) rate was detected in the PGT-SR group compared with the control PGT-M group. In addition, lower proportion of day 5 blastocysts was found in the PGT-SR group compared with the control PGT-M group. Overall, a comparatively 12.7% lower proportion of eligible blastocysts in PGT-SR cycles. As expected, there were fewer balanced/normal blastocysts for transfer in the PGT-SR group (balanced/normal blastocysts rate, 32.3 vs. 59.9%, P = 0.02). The estimated curve by inverse model showed that yields of transferrable balanced/normal blastocyst per cycle came to a plateau stage followed with a rapid rise once the oocytes retrieved reached to the number of 20.4 in PGT-M cycle and 28.3 in PGT-SR cycle respectively.
Conclusions
Our results demonstrated that patients with SR had a high chance of obtaining lower blastocyst development and significantly fewer usable blastocysts available for transfer compared to PGT-M in their first ovarian stimulation cycle.
Keywords: Structural rearrangements, Blastocyst development, Preimplantation genetic testing, Blastocyst formation
What does this study add to the clinical work
| Structural rearrangements reduce a comparatively 12.7% proportion of embryos classified as suitable ones for biopsy. |
Introduction
Balanced structural chromosomal rearrangements such as Robertsonian translocations (RT), reciprocal translocations (rcp), and inversions (inv) are common in humans, with an incidence rate of 0.2% in newborns [1] and 0.42% in chorionic villus or via amniocentesis [2]. Although most carriers with balanced translocations are phenotypically normal, they have an increased risk of recurrent miscarriages [3], infertility [4, 5] or birth defects (especially neurodevelopmental disorders) in offspring due to abnormal gametes [6].
In reciprocal translocations, the translocated chromosomes and their normal homologs form quadrivalent chromosomes, which may segregate in alternate or non-alternate (adjacent‐1, adjacent‐2, 3:1, or 4:0 segregation) modes [7, 8]. Whereas in Robertsonian translocations, due to the fusion of the entire long arms of two acrocentric chromosomes, trivalent chromosomes result in unbalanced rearrangements (adjacent and 3:0). Although it remains controversial, other chromosomal abnormalities unrelated to translocation-affected ones were purported to be higher in the prevalence of gametes derived from balanced carriers [9, 10], thus resulting in poor overall clinical outcomes.
Preimplantation genetic testing for structural rearrangements (PGT-SR) is an effective method for translocation carriers. By utilizing this method, euploid embryos with balanced or normal karyotype are selected for transfer. Though the cumulative live birth rates after PGT-SR appeared to be similar with that following natural conception, the risk of miscarriage due to aneuploidy did decline as reported. The benefit of PGT-SR is in the premise of sufficient number of blastocysts generated for biopsy. Till now, it is disputable whether translocations lead to an inferior embryo development.
The aim of the present study was to assess embryo development of couples with reciprocal or Robertsonian translocations, as compare to couples with normal karyotype undergoing PGT for monogenic disorders (PGT-M) cycles. The findings were supposed to be critical when guiding consultations on the prediction of blastocysts available for biopsy for patients with known structural rearrangements planning to undergo PGT-SR.
Materials and methods
Ethics approval and consent
Institutional review board approval was obtained for this study by the institutional review board of the First Affiliated Hospital of Sun Yet-Sen University [Application ID: (2018)029; Date of Approval: January 2018]. This research was conducted in accordance with the Helsinki Declaration. Informed consent was waived due to the retrospective analysis of anonymized data.
Patients
All PGT-SR cycles from the period between January 2021 and July 2023 was included in this retrospective cohort study. Routine karyotype analysis confirmed either reciprocal or Robertsonian translocation or inversion (except chromosome 9 inversion) in one of the partners. The control group consisted of patients received PGT-M within the same period. These couples were with normal karyotype and with no history of recurrent miscarriage. A total of 791 PGT-SR cycles and 757 PGT-M cycles were included.
The stimulation protocols included the gonadotropin-releasing hormone (GnRH)-agonist long protocols [11] and GnRH-antagonist protocols [12, 13]. Only the first ovarian stimulation cycles were analyzed.
Embryo culture, grading and biopsy
Oocytes retrieved were treated with 100 IU/mL of hyaluronidase (COOK medical, Brisbane, Australia) for less than 30 s to strip off the cumulus cells using micropipettes. After 2 to 4 h, metaphase II oocytes were injected with the prepared sperm using microinjection techniques. All cleavage-stage embryos were cultured in G1 medium (Vitrolife, Goteborg, Sweden) until day 3 under 6% CO2, 5% O2, and 89% N2 in a COOK incubator (Bloomington, IN, USA). If the cleavage-stage embryo had five or more blastomeres and with fragmentations less than 50%, it was further cultured up to at least day 5 and till day 7 according to the development in sequential G2 medium (Vitrolife, Goteborg, Sweden) under the same incubation conditions. The blastocysts graded better than day 5, day 6 or day7 (4BC, 4CB and better) were selected for biopsy. All embryos were graded according to a modification of the Gardner system, as previously described. A zona hole was drilled by laser. 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.[14].
Genetic testing
There were two platforms for genetic testing in our lab, SNP microarray and NGS. Then, the VeriSeq PGS-MiSeq kit (Illumina) was applied to prepare the NGS libraries and the resulting library pools were sequenced by means of the VeriSeq PGS recipe on a MiSeq instrument (Illumina) as reported previously [15].
Clinical outcomes
The outcomes of interest were the number of blastocysts eligible for biopsy on either day 5, day 6 or day 7, the number of day 5 eligible blastocyst, the eligible blastocyst formation rate, as well as the proportion of “usable” embryos generated per cycle. An eligible blastocyst was that graded as 3CB/3BC or higher stage. The blastocyst formation rate (%) was defined as follows: [number of eligible blastocysts / numbers of cleaved embryos at day 3] × 100. A “usable” embryo was defined as a euploid unaffected or a euploid carrier embryo for PGT-M (if PGT-M testing for an autosomal-dominant disorder, only included a euploid unaffected embryos), and a normal or balanced embryo for PGT-SR. Additional outcomes included fertilization rate after ICSI, which was defined as the number of 2PNs of all MII oocytes retrieved, and the number of cleavage-stage embryo for blastocyst culture.
Statistical analysis
All statistical analysis was performed with 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 One-way ANOVA. 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.
To reduce selection bias, confounding factors covering maternal age, AMH, basal hormone levels including basal FSH level, LH and estradiol, the dosage of GnRH-antagonist for down-regulation, the duration, and total consumption of Gn, and the oocytes retrieved 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. Logarithmic and inverse model were utilized for curve estimation. All analysis was two-sided and P < 0.05 was considered statistically significant.
Results
Among 791 PGT-SR cycles and 757 PGT-M cycles, the average female age, BMI, AMH, and the basal hormone levels (FSH, LH, and estradiol) were similar between the two groups. There were also no significant differences detected on the semen parameters (including morphology rate, concentration, and motility of sperm). However, a significantly higher starting dosage and total gonadotropin (Gn) consumptions as well as a significantly higher level of the peak estradiol on HCG-trigger day were detected in the GnRH-SR group as compared to the PGT-M group. No significant differences were found with regards to the mean number of total oocytes retrieved and MII oocytes, the number of cleavage-stage embryos for blastocyst culture, and the number of blastocyst eligible for biopsy. Of interests, there was fewer number of day 5 blastocysts graded as 3BB or better in PGT-SR group compared to the control (1.7 vs 2.4, P = 0.03) (as shown in Table 1).
Table 1.
Characteristics of cycles induced with GnRH-antagonist protocols
| PGT-M + PGT-A (n = 266) | PGT-SR (n = 328) | P | |
|---|---|---|---|
| Female age, year | 30.8 ± 4.4 | 30.8 ± 3.9 | 0.95 |
| Female BMI, kg/m2 | 22.6 ± 4.3 | 21.5 ± 3.5 | 0.83 |
| Male age, year | 33.4 ± 3.8 | 32.8 ± 4.3 | 0.73 |
| Basal hormone levels of female | |||
| AMH, ng/ml | 5.8 ± 4.6 | 5.5 ± 4.3 | 0.67 |
| Basal FSH, IU/L | 5.6 ± 2.0 | 6.0 ± 2.4 | 0.11 |
| Basal LH, IU/L | 4.3 ± 2.5 | 4.1 ± 2.9 | 0.72 |
| Basal estradiol, pg/ml | 38.0 ± 34.9 | 35.4 ± 29.4 | 0.48 |
| Semen parameters | |||
| Sperm morphology rate | 2.4 ± 0.7 | 3.1 ± 1.2 | 0.82 |
| Sperm concentration, × 106/ml | 32.7 ± 24.5 | 28.1 ± 20.3 | 0.65 |
| Sperm motility, % | 43.2 ± 28.1 | 27.8 ± 19.6 | 0.22 |
| Total GnRH-A days, n | 4.7 ± 1.3 | 4.8 ± 1.4 | 0.78 |
| Total Gn days, n | 9.3 ± 1.7 | 9.6 ± 1.8 | 0.17 |
| Total Gn dosage, iu | 2051.3 ± 720.4 | 2245.6 ± 802.1 | 0.02 |
| The starting Gn dose, iu | 220.1 ± 56.7 | 235.0 ± 57.6 | 0.02 |
| FSH level on HCG day, IU/L | 14.4 ± 5.8 | 15.9 ± 6.2 | 0.06 |
| LH level on HCG day, IU/L | 2.2 ± 2.0 | 2.3 ± 2.9 | 0.63 |
| Peak estradiol, pg/ml | 2816.1 ± 1258.3 | 2353.5 ± 1165.8 | < 0.01 |
| Progesterone level on HCG day, ng/ml | 1.1 ± 0.5 | 1.2 ± 0.8 | 0.13 |
| Oocyte retrieved, n | 19.1 ± 11.3 | 17.6 ± 11.3 | 0.28 |
| MII, n | 15.3 ± 8.1 | 14.2 ± 8.7 | 0.14 |
| Fertilization rate, % | 88.9 ± 6.2 | 91.3 ± 8.2 | 0.84 |
| Cleavage-stage embryos for extended culture, n | 11.8 ± 7.1 | 10.9 ± 7.6 | 0.31 |
| Blastocysts for biopsy, n | 7.2 ± 5.2 | 6.4 ± 5.1 | 0.17 |
| Blastocysts eligible for biopsy/cleavage-stage embryos for extended culture, % | 68.3 | 59.7 | 0.04 |
| D5 biopsied blastocysts, n | 2.4 ± 2.8 | 1.7 ± 2.2 | 0.03 |
Data in a normal distribution were presented as mean with standard deviation. Differences were statistically significant as p < 0.05
As for GnRH-agonist induced cycles (Table 2), despite the similar average age of female within the groups, there was a significantly higher level of mean AMH and correspondingly a lower level of mean FSH with significant differences in the PGT-M group compared to the PGT-SR group. Coincidentally, a milder dosage of GnRH-agonist for pituitary down-regulation and a less dosage of the starting as well as the total Gn for stimulation were demonstrated in the PGT-M group. Aiming to reduce confounding bias from these factors, PSM analysis was conducted. Finally, 238 cycles in each group were matched out and shown in Table 3. A significantly fewer number of day 5 blastocysts eligible for biopsy were detected in the PGT-SR group as compared to the PGT-M group (1.1 ± 2.1 vs. 2.1 ± 2.2 respectively, P = 0.03) while the number of MII, cleavage-stage embryos for blastocyst culture as well as the eligible blastocysts for biopsy were similar between the two groups. Overall, a comparatively 12.7% lower proportion of embryos classified as suitable ones for biopsy in balanced carriers was detected in comparison with the non-carriers for PGT-M (Fig. 1). Patients with structural rearrangements had lower chance of blastocysts eligible for transfer in PGT cycles (Table 4).
Table 2.
Characteristics of cycles induced with long GnRH-agonist protocols
| PGT-M + PGT-A (n = 418) | PGT-SR + PGT-A (n = 429) | P | |
|---|---|---|---|
| Female age | 31.5 ± 3.9 | 31.4 ± 3.5 | 0.92 |
| AMH, ng/ml | 4.7 ± 3.6 | 3.4 ± 2.1 | 0.04 |
| Basal FSH, IU/L | 5.5 ± 1.9 | 6.2 ± 2.3 | 0.04 |
| Basal LH, IU/L | 3.7 ± 2.1 | 3.8 ± 1.9 | 0.69 |
| Basal estradiol, pg/ml | 36.2 ± 27.1 | 34.4 ± 21.0 | 0.15 |
| The dosage of GnRH-agonist down-regulation, mg | 1.0 ± 0.7 | 0.7 ± 0.6 | 0.02 |
| Total Gn days, n | 11.4 ± 2.8 | 11.4 ± 1.5 | 0.89 |
| Total Gn dosage, iu | 2886.3 ± 828.4 | 3007.2 ± 706.9 | < 0.01 |
| The starting Gn dose, iu | 245.2 ± 50.9 | 270.1 ± 45.7 | < 0.01 |
| FSH level on HCG day, IU/L | 16.7 ± 5.3 | 18.3 ± 5.1 | < 0.01 |
| LH level on HCG day, IU/L | 0.8 ± 0.4 | 0.7 ± 0.5 | 0.85 |
| Peak estradiol, pg/ml | 2627.4 ± 1166.6 | 2638.1 ± 1099.4 | 0.76 |
| Progesterone level on HCG day, ng/ml | 1.3 ± 4.4 | 1.0 ± 0.5 | 0.14 |
| Oocyte retrieved, n | 15.3 ± 6.7 | 15.7 ± 7.3 | 0.81 |
| MII, n | 12.5 ± 5.8 | 12.6 ± 6.4 | 0.66 |
| Fertilization rate, % | 83.4 | 81.2 | 0.08 |
| Cleavage-stage embryos for extended culture, n | 9.6 ± 4.6 | 9.6 ± 5.4 | 0.95 |
| Blastocysts for biopsy, n | 6.0 ± 3.5 | 5.1 ± 3.6 | 0.04 |
| Blastocysts eligible for biopsy/cleavage-stage embryos for extended culture, % | 69.4 | 55.3 | 0.03 |
| D5 biopsied blastocysts, n | 1.9 ± 1.5 | 0.9 ± 1.5 | 0.02 |
Table 3.
Selected matching variables of study patients and embryonic characteristics after PSM analysis in GnRH-antagonist cycles
| PGT-M + PGT-A | PGT-SR + PGT-A | P | PGT-M + PGT-A | PGT-SR + PGT-A | P | |
|---|---|---|---|---|---|---|
| Before-PSM | After-PSM | |||||
| Cycles | 418 | 429 | 233 | 233 | ||
| Female age | 31.5 ± 3.9 | 31.4 ± 3.5 | 0.92 | 31.7 ± 4.0 | 31.2 ± 3.9 | 0.11 |
| AMH, ng/ml | 4.7 ± 3.6 | 3.4 ± 2.1 | 0.04 | 3.7 ± 2.5 | 3.5 ± 2.4 | 0.33 |
| Basal FSH, IU/L | 5.5 ± 1.9 | 6.2 ± 2.3 | 0.04 | 5.8 ± 1.4 | 6.0 ± 1.9 | 0.31 |
| Basal LH, IU/L | 3.7 ± 2.1 | 3.8 ± 1.9 | 0.69 | 3.5 ± 2.0 | 3.5 ± 1.6 | 0.88 |
| Basal estradiol, pg/ml | 36.2 ± 27.1 | 34.4 ± 21.0 | 0.15 | 36.1 ± 23.3 | 37.4 ± 26.2 | 0.45 |
| The dosage of GnRH-agonist down-regulation, mg | 1.0 ± 0.7 | 0.7 ± 0.6 | 0.02 | 1.1 ± 0.8 | 1.1 ± 0.7 | 0.09 |
| Total Gn days, n | 11.4 ± 2.8 | 11.4 ± 1.5 | 0.89 | 11.3 ± 2.3 | 11.3 ± 1.8 | 0.91 |
| Total Gn dosage, iu | 2886.3 ± 828.4 | 3007.2 ± 706.9 | < 0.01 | 2830.9 ± 851.7 | 2931.6 ± 781.5 | 0.08 |
| Oocyte retrieved, n | 15.3 ± 6.7 | 15.7 ± 7.3 | 0.81 | 15.7 ± 7.2 | 16.2 ± 7.9 | 0.52 |
| MII, n | 12.5 ± 5.8 | 12.6 ± 6.4 | 0.66 | 13.0 ± 6.3 | 13.5 ± 7.0 | 0.31 |
| Cleavage-stage embryos for extended culture, n | 9.6 ± 4.6 | 9.6 ± 5.4 | 0.95 | 9.8 ± 5.1 | 9.9 ± 5.7 | 0.82 |
| D5 biopsied blastocysts, n | 1.9 ± 1.5 | 0.9 ± 1.5 | 0.02 | 2.1 ± 2.2 | 1.1 ± 2.1 | 0.03 |
Fig. 1.
Stacked column diagrams of eligible blastocysts in each period of age
Table 4.
Diagnosis results of biopsied blastocysts
| PGT-M + PGT-A | PGT-SR + PGT-A | P | |
|---|---|---|---|
| Diagnosed blastocyst, n | 1943 | 2238 | < 0.01 |
| Normal/balanced blastocyst, n (%) | 1163 (59.9) | 722 (32.3) | |
| Aneuploidy, n (%) | 445 (22.9) | 1181 (52.8) | |
| Mosaicism, n (%) | 329 (16.9) | 324(27.4) | |
| Polyploidy, n (%) | 6 (0.3) | 11(0.5) |
As for the expectation on the maximum number of usable blastocysts obtained from oocyte retrieved per cycle, curve estimation was conducted. As the number of oocytes retrieved increased, the estimated curve in the inverse model was represented with a rapid rise initially but followed by a leveling off as the increase of oocytes retrieved, and finally reached a plateau with a minimum curvature at the abscissa value of 20.4 and 28.3 in PGT-M and PGT-SR respectively. It indicated that once oocytes retrieved per cycle achieved the number of 20.4 and 28.3, the yields of balanced/normal blastocyst for biopsy were maximized (Fig. 2).
Fig. 2.
Estimated curves by the logarithmic and the inverse models in regards to the yields of normal/balanced blastocysts per aspiration as the increase of oocytes retrieved
Discussion
In this study, we demonstrated that balanced carriers had a greater chance of poor blastocyst development and less usable blastocyst for transfer in comparison with non-carriers.
Till now, the extent and timing of selection against the embryos with chromosomal unbalanced genome has still been elusive. Even though several checkpoints exited to control the proper alignment of chromosomes, the birth of children with physiological or mental abnormality attributed to the unbalanced chromosomal complements implied that these inspections were not effective enough and might be delayed till the postnatal stage. Since the application of PGT for aneuploidy, it has been widely accepted that the inchoative stage of selection or self-correction for chromosomally normal or balanced embryos occurred at the transitional stage from morula till blastocyst. However, it has still been contradictory to draw a conclusion whether the presence of selection affect the viability of the blastocyst formation. Some declared that a chromosomally unbalanced genome had no adverse effect on the development of embryos [16, 17]. Based on the evidences that those chaotic embryos, a representation of extreme aberrations in genome, were still able to develop into blastocysts, they believed that it was too early to draw a firm conclusion on the direct correlations among aneuploidy, morphology, and blastocyst development. However, in the present study, a comparatively 12.7% lower proportion of embryos classified as suitable ones for biopsy in balanced carriers was detected in comparison with the non-carriers for PGT-M, as one published study similarly concluded [10]. The designs were modified in our study, which let the evidence being more convincing. On the one hand, the biopsy stage was quite different, as polar body and blastomere biopsy were performed in that study but in ours, biopsy was conducted completely at the blastocyst stage, which left the embryos self-selected spontaneously without any redundant manipulations. This stage of biopsy was verified to be non-invasive to the inner cell mass and be compatible for embryo development to term [18]. On the other hand, the cycles with standard ICSI were set as the control in that published study, which let the impacts from biopsy negligible. Therefore, the findings that less qualified blastocyst for biopsy were obtained by extended culture from cleavage-stage in the present study was inclined to give a supportive view on the profound impacts on the embryonic viability from misalignment of chromosomes in the stage of the cleavage to blastocyst transition.
By showing developmental arrest on specific points during preimplantation growth, the production of unbalanced translocation in gamete cells from translocated carrier was the main factor emphasized so far. Besides, there may be some other chromosomal abnormalities (unrelated to translocated chromosomes) or micro-aberrations, which, together with the translocation in question, can cause retardation or incompatibility with embryo development [19]. Hints from the latest studies might be utilized to give some explanation. Some believed that responsible mechanisms associated with the fate of cleavage-stage embryos in vitro might not be aneuploidy, but rather than the other [20, 21], such as mosaicism [22]. Also, aberrant separation during meiosis (accounting the overwhelming 27%), 2% was attributed to mitotic errors in blastocysts from female at the average age of 34.4 years old underwent PGT-A[23]. While currently, it still was controversial on the inter-chromosomal effect from balanced chromosomes [9, 24], and whether these cells were more inclined to be chromosomal instability, thus resulting in an unevenly distribution of chromosome complements into daughter cells has still to be interrogated. If a higher incidence of mitotic-originated mosaicism in balanced carriers was identified, it would be hypothesized that poor blastocyst information was relevantly involved, since this kind of mistakes during mitosis may predispose to give rise to cell arrest [25]. In addition, the gene dynamics were also elucidated to be decisive in embryo culture from primates [26]. Concerning the relevant extent of attributions to whether an embryo was capable to develop to the blastocyst stage from disrupted rearrangements due to balanced translocations, further investigations were needed. From another point of view, sperm quality might be another attributed factor [27]. Owing to meiotic misalignment or epigenetic effect, male carriers had a higher chance of inferior sperm, which was regarded to be negatively associated with blastocyst formation [28]. In our study, an inferior sperm concentration and motility were detected in the group of PGT-SR but without statistical differences significantly. Minority proportions of male carriers (21.3%) might be a probable explanation. Even though there were no significant differences in the aspect of morphology, a higher percentage of sperms with normal morphology in PGT-SR might increase the chance of injecting sperms with chromosomal abnormalities or genetic mutations into the cytoplasm of an oocyte during ICSI. Whether the PGT-SR of male was a decisive factor in the process of blastocyst formation and even to fetal development to the term still need to be verified.
Not all unbalanced translocations could be eliminated at peri-implantation period since translocation carriers had a higher risk of recurrent miscarriage, indicating some unbalanced embryos survive but initiate death process at the first trimester.
As for karyotype constitution, the percentage of blastocysts diagnosed as balanced/normal embryos, aneuploidy, mosaicism, or polyploidy were 32.3, 52.8, 27.4 and 0.5% in the PGT-SR group, as there presented 59.9, 22.9, 16.9 and 0.3%, respectively in the PGT-M. Our results indicated that balanced chromosome could result in a relatively higher frequency of gametic aneuploidy, which was consisted with previous studies[24], and meanwhile the proportion of aneuploidy reported in the present study were within the range reported [29–31]. When considering the incidence of mosaicism based on NGS analysis, recent prevalence of estimated mosaicism varied among laboratories, ranging from 2 to 30% of blastocysts when taking all chromosomes into account [32, 33]. Since Greco et al. first declaimed [34], the potential for giving birth to healthy offspring. Mosaic embryos as well as the ranking threshold for clinical reference have been investigated in recent years [35]. In our center, the blastocysts with low mosaicism level (< 50% affected) was alternated to transfer under conditions when there was without any remaining balanced or normal embryos available for transfer in premise of couples on fully informed. Our results could supplement the existing data in the literature to counsel new patients in terms of realistic expectations on the proportion of transferrable blastocysts taking into low mosaic ones into consideration of PGT-SR in comparison with PGT-M.
In the present study, another striking observation for balanced/normal embryos was that ‘more was not always better’. Previous studies had investigated the formation of euploidy blastocysts per oocyte [11, 36]. Thus, it was habitually thinking that the more oocytes retrieved, the higher the chances of obtaining embryos eligible for biopsy. However, this traditional opinion had been challenged against and might be left behind according to our analysis. As represented, the number of oocytes associated with balanced/normal blastocysts per aspiration seemed to plateau at 20.4, 28.3 in PGT-M and PGT-SR group, respectively. It indicated the number of balanced/normal blastocysts from resulting oocytes was not associated positively with size of the oocyte yield all through. Some previous studies had shown that larger oocyte yields were associated with more euploid embryo [37, 38]. If indeed a larger oocyte yield was associated with oocytes of considerable development potential and hence more blastocysts eligible for biopsy, that essentially translated to a higher number of opportunities for the patient to have a pregnancy that would result in a delivery. Nevertheless, concerns were announced by some researchers in the aspects of inferior developmental potential and furtherly reduced pregnancy rates attributing to the larger oocyte yields [39]. Most importantly, recent evidence originating from a randomized controlled trial suggested that a smaller oocyte yield was associated with a higher proportion of euploid embryos in cleavage stage [40]. Others failed to confirm such an association [41, 42]. Possible explanations were postulated. As a matter of fact, the interpretation of any rate was highly dependent on the denominator. Calculating a detrimental effect of high oocyte yield on the number of euploidy or aneuploidy might mask the embryos that was suitable for biopsy, which was not validated previously. In addition, based on an acknowledged hypothesis that only the follicle with the most competent oocyte was supposed to be selected during the natural cycle, the larger size in oocytes yields per induced cycle might be possessed with the higher chances of recruitment for inferior oocytes into the growing pool, thus resulting in poor blastocyst formation in turns. This concept was supported by some evidences, as less interference with ovarian physiology might give rise to a higher proportion of developmentally competent oocytes [40, 43]. Nevertheless, further studies would be necessary to confirm these findings.
There were several limitations. First, this was a retrospective cohort study in a single center, hence the presence of bias could not be excluded. For this reason, the present analysis was grouped and adjusted for the most important patient and treatment characteristics to identify the independent association of the number of oocytes retrieved with the rate of usable blastocyst. Also, quality-controlled laboratory conditions in a constant standard, the uniformed manipulation from the embryologists as well as the same grade on scoring, ranking, and selecting embryos, could reduce confounding factors to a large extent. Secondly, the cohort of control included in this study had an indication for PGT-M, which might not be completely representative of directly reflect the magnitude or direction of the association in the general population. Besides, the effect of parental origin of translocation were not analyzed with respect to the proportion of euploid or normal blastocysts. It was reported that a significantly higher proportion of gametes was derived from adjacent-1 segregation in males than in females [44–46]. However, equal parental contribution to monosomies as well as negligible associations with balanced/normal outcomes of PGT-SR according to the other published studies [1, 47, 48], while the incidence of adjacent‐1 segregation was significantly increased in female carriers aged more than 40 years old. In practice, more attentions were deserted to be paid [1]. The last but not the least, the estimated curve of maximum-output to the generation of eligible blastocysts were modeled depending on the variations of oocytes picked up, the number of which was not in a normal distribution as majority of cycles included were induced to obtain with an optimal number of oocytes ranged from 8 to 15. Also, it had to be declared that any changes of the standards on the embryonic manipulation or grading might affect the outcomes as well. Thus, it was not representable enough for the law of inheritance and was applied as a criterion.
Conclusions
In conclusion, the findings of this study demonstrated that carriers with structural rearrangements was inclined to obtain poorer blastocyst development and significantly fewer usable blastocysts available for transferring in comparison with non-carriers for PGT-M without a history of recurrent miscarriage in their first stimulation cycle. Besides, it was also suggested in the present study that maximizing the oocyte yield might not be beneficial in terms of obtaining more balanced/normal blastocysts.
Acknowledgements
We thank the patients involved in our study. We also gratefully acknowledge all the staff in the department of assisted reproduction in Sun Yat-sen University First Affiliated Hospital for their support and cooperation.
Author contributions
Yizi Wang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Yizi Wang, Yuanlin Ma, Yanling Tan contributed equally to this study. Concept and design: Yizi Wang, Yuanlin Ma, Yanling Tan Acquisition, analysis, or interpretation of data: Yizi Wang, Yanling Tan, Jing Wang, Jiafu Pan, Junli Song, Yali Wang, Yanwen Xu Drafting of the manuscript: Yizi Wang, Yuanlin Ma, Yanling Tan Critical revision of the manuscript for important intellectual content: Yanwen Xu Obtained funding: Yanwen Xu, Yizi Wang Administrative, technical, or material support: Yizi Wang, Yanwen Xu Supervision: Yanwen Xu, Yizi Wang.
Funding
This study was supported by grants from National Key Research and Development Program of China (No. 2018YFC1003102), Key Clinical Technique of Guangzhou (No. 202201011548), Guangdong Province, China (No. 2023P-ZD19), Key-Area Research and Development Program of Guangdong Province (No. 2023B1111020006), Beijing Health Promotion Association Project, Natural Science Found of Guangdong China (No. 2019A1515010991), and Medical Scientific Research Foundation of Guangdong Province, China (A2025203).
Data availability
Data will be made available on request.
Declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Ethical approval
Institutional review board approval was obtained for this study by the institutional review board of the First Affiliated Hospital of Sun Yet-Sen University [Application ID: (2018)029; Date of Approval: January 2018]. This research was conducted in accordance with the Helsinki Declaration. Informed consent was waived due to the retrospective analysis of anonymized data.
Consent to participate
Informed consent was waived due to the retrospective analysis of anonymized data.
Consent to publish
Not applicable.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yizi Wang, Yuanlin Ma, and Yanling Tan have contributed equally to this study.
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Data Availability Statement
Data will be made available on request.