Does the sex ratio of singleton births after frozen single blastocyst transfer differ in relation to blastocyst development?

Ideally, human-assisted reproductive technology (ART) aims to achieve a single live birth. Following improvements in blastocyst culture systems and freezing technology [1, 2], single blastocyst transfer (SBT) is now considered the most effective means of avoiding multiple pregnancies subsequent to ART [3‐6]. Gradually, SBT has become the preferred transfer strategy throughout the world [7‐10]. However, blastocyst transfer has some potential limitations, including adverse effects such as a male-biased imbalance in the sex ratio and an increased incidence of monozygotic twinning (MZT) [11‐13].

The sex ratio at birth is often calculated as the proportion of males among all live births or the number of male births per 100 female births [14]. Dean et al. reported a significantly higher sex ratio at birth among infants born after SBT (54.1%) than among those born after cleavage-stage transfer cycles (49.9%) [15]. Another study determined that the higher sex ratio was independent of the fertilization method [16]. However, the details of the relationship between the characteristics of blastocysts and the sex ratio at birth remain unclear.

Blastocyst grading prior to embryo transfer usually occurs on day 5 or day 6 of culture. A detailed blastocyst scoring system is required to identify the blastocysts with the highest implantation potential. Static morphology remains the most commonly used embryo selection method worldwide. This system is used to grade blastocysts according to three different variables: blastocoel expansion, inner cell mass and trophectoderm [17]. Several studies have attempted to identify the individual contributions of these parameters to the implantation potential or live birth rate. In particular, trophectoderm morphology may be predictive of pregnancy outcomes related to single blastocyst transfer [18].

Ebner et al. reported that the embryo sex ratio was skewed significantly in relation to morphology. Male blastocysts had a 2.53-fold higher odds of receiving a trophectoderm quality score of A than female blastocysts [19]. All previous related studies were based on morphological parameters and embryo sex. To our knowledge, no previous study has explored the relationship between blastocyst development and the sex ratio of singleton live births after single-blastocyst frozen embryo transfer (FET) cycles. The objective of this study was to investigate this association and fill this gap in the literature.

Our study results indicate that implantation with high-quality blastocysts, rather than poor-quality blastocysts, during single-blastocyst FET cycles is associated with an increased sex ratio among live births. We further determined that only the trophectoderm grade was associated with a significantly higher sex ratio among singleton live births. Similar observations were not observed for the blastocoel expansion and inner cell mass grades. Therefore, our findings demonstrate that a grade B trophectoderm is associated with a higher probability that a male infant will be born from a single-blastocyst FET cycle.

Single blastocyst transfer was designed to avoid the complications associated with multiple pregnancies and is thus becoming the preferred type of ART. The use of extended blastocyst culture conditions may favor the selection of male blastocysts for transfer, as male embryos are thought to exhibit greater preimplantation developmental rates. In this study, the sex ratio among single-blastocyst FET cycles was 55.1%, which was consistent with the ratios reported in previous studies. For example, a large nationwide longitudinal birth cohort study of 103,099 pregnancies in Japan reported a skewed sex ratio in favor of males after blastocyst transfer relative to spontaneous conception (OR: 1.095; 95% CI: 1.001–1.198). However, the sex ratios in the non-ART treatment and cleavage groups were slightly lower than or equivalent to the ratio in the spontaneous conception group [13]. A meta-analysis of 13 studies also suggested that blastocyst transfer was associated with a male-biased sex ratio when compared with cleavage-stage embryo transfer (OR: 0.89, 95% CI: 0.86–0.93) [26]. This observation may be attributable to the more frequent selection of male embryos for transfer, as male embryos develop more rapidly in vitro and thus may appear more viable at the blastocyst stage [27, 28]. We were interested in determining the mechanism underlying the observed phenomenon of sex deviation in offspring after blastocyst transfer. Accordingly, we retrospectively analyzed the potential effects of morphological parameters on the sex ratio after single-blastocyst FET.

Our study is the first to correlate various blastocyst parameter grades with the sex ratio among singleton live births after single-blastocyst FET cycles. We observed a significant increasing trend in the sex ratio in the context of high-quality versus poor-quality blastocyst transfer, suggesting that the transfer of more advanced blastocysts may increase the proportion of male infants. We note that male blastocysts may grow more rapidly and receive better morphological scores than female blastocysts. These discrepancies may at least partially account for the skewed sex ratio at birth. Mohamad et al. reported a greater likelihood of euploidy among blastocysts with good morphology scores and among embryos that progressed more rapidly to the blastocyst stage [29]. Therefore, differences in the observed sex ratios of live births subsequent to single-blastocyst FET cycles may also be attributable to blastocyst selection and transfer strategies.

We did not find that delayed blastulation had a significant effect on the newborn sex ratio, as similar values were observed for the groups that received day 5 and day 6 embryo transfers. Our results are consistent with those of a study published by El-Toukhy et al., which reported similar live birth rates after the transfer of vitrified embryos on day 5 and day 6 [30]. Desai N et al. used a morphokinetics approach combined with preimplantation genetic screening test models to determine that embryos exhibiting delayed blastulation were more likely to exhibit aneuploidy [31]. Blastocyst morphology, which is based on blastocoel expansion, the trophectoderm and the inner cell mass, must also be considered when evaluating the outcomes of FET. Initially, we observed an association between the degree of blastocoel expansion and the sex ratio among singleton live births in a small sample. However, we did not observe any association between these variables. Samer et al. reported that sex-related differences in development are highly significant, such that male embryos were 2.6-times more likely to produce a grade 5 or 6 blastocyst than female embryos [32]. Our study differed from that study because we focused on the sex ratio at birth rather than in the embryos. The functions of blastocoel expansion involve hatching from the zona pellucida, adhesion to and invasion of the endometrium and establishment of maternal communication [32, 33]. In a recent study, Jing Zhao et al. confirmed that the degree of blastocoel expansion was a better predictor than the trophectoderm or inner cell mass grade in terms of the likelihood of live birth after both single-blastocyst fresh transfer and FET cycles [34]. The degree of blastocoel expansion may be an essential factor in a successful pregnancy and should be prioritized when selecting a frozen blastocyst for transfer [35].

Some previous studies have emphasized the importance of the inner cell mass with respect to pregnancy establishment [36, 37]. Irani et al. applied the same embryo-grading criteria used in the analysis to 477 preimplantation-genetic-testing-confirmed single-euploid blastocyst FET cycles. These authors determined that the overall blastocyst quality and inner cell mass grade were the most effective predictors of a successful pregnancy and suggested the use of both parameters to facilitate the selection of high-quality euploid blastocysts for IVF transfer [29]. In contrast, Ahlström et al. emphasized the higher predictive strength of the trophectoderm grade relative to the inner cell mass grade when selecting the optimal blastocyst for both fresh and frozen thawed cycles [38]. In our study, however, we did not find that the inner cell mass quality had a significant effect on the newborn sex ratio after single-blastocyst FET cycles.

In our analysis, the trophectoderm grade was identified as the most important factor affecting the newborn sex ratio after a single-blastocyst FET cycle. A significant association was observed between grade B trophectoderm and sex ratio among infants conceived via the transfer of blastocysts. In other words, the effect of blastocyst morphology on the sex ratio is only obvious when blastocysts with grade B trophectoderm are selected. The importance of the trophectoderm during the critical implantation phase might be ascribed to its role as a promoter of hatching and endometrial invasion [39, 40]. According to Thomas et al., the observed correlations of trophectoderm performance with infant sex and the rates of implantation, pregnancy and pregnancy loss indicate that this measure will eventually be prioritized over the inner cell mass score [19]. Interestingly, we also demonstrated that after controlling for the inner cell mass, trophoblast grade B was positively correlated with the highest sex ratio among singleton births. Disparities in morphological scores between male and female embryos are unlikely to reflect any difference in competence. However, these variations clearly emphasize the tendency of male embryos to reach the final stages of blastocyst development more rapidly than female embryos [31].

This study has several strengths. First, all cycles and cryopreservation procedures were completed at a single institution according to a strict cycle protocol. Embryologists scored several standardized transfer parameters using a consistent system according to the classification devised by Gardner and Schoolcraft [21]. We also included only single-blastocyst FET cycles that resulted in singleton live births to enable us to control the within-transfer characteristics that are known to affect outcomes. Our aim was to isolate morphological blastocyst parameters as the exposure while regulating several other potential confounders to the greatest extent possible.

However, our study also has some limitations. First, this was a retrospective study of a relatively small number of patients treated at a single center. In the future, a study with a large sample size is needed for validation. Second, further analyses of blastocyst grading according to morphological parameters should investigate dynamic parameters by time-lapse microscopy, which allows the assessment of embryo morphodynamic patterns throughout preimplantation development. Third, our strategy required that the two best-quality cleavage embryos be chosen for fresh cycle transfer or cryopreservation on day 3, with surplus embryos being cultured to the blastocyst stage for possible cryopreservation. The result was that the blastocyst used for culture was a suboptimal embryo. Therefore, we had very few good embryos (AA) and a large percentage of fair BB and poor BC embryos, and the strategy required an inner cell mass grade of B or higher for optimal cryosurvival. Therefore, we were only able to evaluate the outcomes associated with an inner cell mass grade of A or B. Fourth, blastocyst transfer is more advantageous than cleavage-stage embryo transfer in terms of successful implantation从 and pregnancy rates. We prefer high-quality blastocyst transfer to obtain a better clinical pregnancy rate. Although we determined that the transfer of high-quality blastocysts may skew the sex ratio in favor of male embryos, it is difficult to change perceptions regarding popular blastocyst transfer strategies.

In summary, we demonstrated that the morphological criteria associated with higher-quality blastocysts result in a higher sex ratio after single-blastocyst FET. However, we found that only the trophectoderm grade was significantly associated with the sex ratio among offspring. Our findings may elucidate the underlying determinants of the skewed sex ratio after blastocyst transfer. Clinicians should be aware of the effects of certain protocols on sex distribution, given recent trends towards the increased use of blastocyst transfer. In the future, we aim to identify additional markers of embryonic implantation potential, with the aim of maintaining the equilibrium of the sex ratio among offspring without affecting the pregnancy rate.