Embryo quality, ploidy, and transfer outcomes in male versus female blastocysts

Purpose The goal is to determine if variations exist between male and female blastocysts in preimplantation measurements of quality and ploidy and in vitro fertilization elective single-embryo transfer (eSET) outcomes.

A retrospective chart review was conducted from a private fertility center’s database of blastocysts undergoing preimplantation genetic testing for aneuploidy, along with details of eSET from this screened cohort. Main outcomes included preimplantation embryo quality and sex-specific eSET outcomes.

A total of 3708 embryos from 578 women were evaluated, with 45.9% male and 54.1% female. The majority were High grade. No difference existed between embryo sex and overall morphological grade, inner cell mass or trophectoderm grade, or blastocyst transformation day. Female blastocysts had a higher aneuploidy rate than male blastocysts (P < 0.001). Five hundred thirty-nine eSETs from 392 women were evaluated, with High grade embryos more likely to have implantation (P < 0.001), clinical pregnancy (P < 0.001), and ongoing pregnancy (P = 0.018) than Mid or Low grade embryos. Day 5 blastocysts were more likely to have implantation (P = 0.018), clinical pregnancy (P = 0.005), and ongoing pregnancy (P = 0.018) than day 6 blastocysts. Male and female embryos had similar transfer outcomes, although female day 5 blastocysts were more likely to result in clinical pregnancy (P = 0.012), but not ongoing pregnancy, than female day 6 blastocysts. Male eSET outcomes did not differ by blastocyst transformation day.

Male and female embryos have comparable grade and quality; however, female embryos were more likely to be aneuploid. Ongoing pregnancy rates did not differ by embryo sex. Day 5 embryos had more favorable transfer outcomes than day 6 embryos.

Embryo sex, Blastocyst sex, Aneuploidy, Preimplantation genetic testing

Approaches for offspring sex selection in individuals seeking to conceive date back to the 1970s when the Ericsson method of sperm sorting was introduced. By using column filtration to isolate Y-bearing male sperm from X-bearing female sperm, individuals were able to have offspring of their desired sex up to 70% of the time [1, 2]. Preimplantation embryo sex has more reliably been determined since the 1990s, when preimplantation genetic testing became more available in fertility practices, with aneuploidy screening (preimplantation genetic testing for aneuploidy, PGT-A) being the most common indication [3, 4]. Individuals may seek sex selection of their offspring for a variety of reasons, such as personal preference or family balancing [5]. Upwards of 90% of fertility clinics offer preimplantation genetic testing, with nearly a quarter of those clinics offering sex selection services [6]. While embryo sex can be determined as early as day 2 after fertilization, current evidence indicates that clinical pregnancy and live birth rates are highest with transfer of blastocyst stage embryos, rather than at the cleavage stage that typically occurs on day 2 or 3 [7–9]. While PGT-A is not recommended for all patients, recent evidence indicates a possible benefit of use in screening embryos of women over age 35 [10]. Day 3 embryo transfers tend to result in equal distribution of male and female offspring, but transfer of embryos at the blastocyst stage tends to favor male embryos, as they have been shown to mature and reach this developmental stage quicker than female embryos [11–14]. As sex can now be identified during the in vitro developmental process, efforts have been made to characterize the development of male versus female embryos. Specific studies on the rate of blastocyst transformation show a tendency for increased glucose and amino acid usage in female embryos, which may indicate a need to focus energy usage towards inactivation of an X-chromosome, whereas male embryos are able to direct this energy towards mitotic proliferation [15–18]. At the same time, it has been shown that inactivation of the X-chromosome in female embryos is linked to fewer trophectoderm cells, which could influence blastocyst grading systems based on trophectoderm mass [19]. These findings, however, are not universally recognized. Other studies comparing progression of male versus female embryos have indicated that embryo development rate is independent of sex, and live birth rates are unaffected when transfer of morphologically and genetically normal blastocyst stage embryos is carried out [20–22]. Evidence exists that prevalence of euploidy is similar between male (42.2%) and female (38.5%) embryos, though higher embryo grade favors male embryos [23]. It has been seen in some studies that there are improved pregnancy rates and live births with transfer of day 5 blastocysts compared to day 6 blastocysts, but rarely are sex-specific outcomes detailed [24–28]. Findings on embryo transfer outcomes are similarly conflicting with some studies reporting higher male offspring rates with blastocyst-stage transfers [11, 29] and others reporting equal rates of males and females [20, 30]. Assessing features such as TE has also shown varying results with some studies demonstrating a sex difference [19, 23] and others showing no difference [13]. Previous studies are lacking in information on specific preimplantation characteristics as relate to embryo sex and the relationship between known sex of transferred embryos and in vitro fertilization (IVF) elective single-embryo transfer (eSET) outcomes. In all, data that exists regarding the specific relationship between embryo sex, embryo quality, day of blastocyst transformation, and transfer outcomes remains incomplete and inconclusive. Our goal is to determine if differences exist between male and female embryos in preimplantation measures of embryo quality and development and subsequent eSET outcomes. Materials/Methods A university institutional review board (IRB) determined that this study was not a “human subject research” study and did not require IRB approval or oversight. A retrospective chart review was conducted of patients who underwent PGT-A (n = 3708) with elective single-embryo transfer (eSET) of frozen embryos (n = 539) between June 2007 and December 2018. Women with more than one embryo transferred or who used donor eggs were excluded. Deidentified data was collected from the database and electronic medical record of a single private fertility clinic. Ovarian stimulation and trophectoderm biopsies Patients received controlled ovarian stimulation per standard protocols. A human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone agonist (GnRH-A) trigger was given when there were at least two ovarian follicles that measured 18 mm. Intracytoplasmic sperm injection (ICSI) was used for fertilization in the presence of standard culture conditions and media. On culture day 5, 6, or 7, contingent on the rate of embryo development, laser-assisted blastocyst trophectoderm (TE) biopsy took place. Preimplantation genetic testing for aneuploidy Embryonal trophectoderm cells were lysed and DNA extracted. With phi 29 polymerase, a modified whole genome amplification (WGA) protocol was carried out. Between 50 and 100 ng of amplified DNA went through library preparation (Thermo Fisher Scientific, Waltham, MA). DNA fragments of approximately 200 base pairs were formed through enzymatic shearing of pooled DNA samples. These DNA fragments were then purified with an AMPure Bead (Beckman Coulter, Sharon, IL). Next, each end of the purified DNA fragment was ligated with adapters and barcodes to establish stability, with size chosen using an additional AMPure Bead wash. Individual DNA fragments were adhered to an ion sphere particle (ISP) and amplified with PCR emulsion reaction. Dynabeads MyOne Streptavidin CI bead washes (Invitrogen, Carlsbad, CA) were employed to recover template positive ISPs. Sequencing primers and Ion Hi-Q sequencing polymerase were subsequently included with the samples, following recovery. This was then loaded onto a sequencing chip which was analyzed at a 1× depth throughout the full genome by a Personal Genome Machine (PGM) (Thermo Fisher Scientific, Waltham, MA). Preliminary sequencing data was issued by a Torrent Browser Server (Thermo Fisher Scientific, Waltham, MA). This confirmed the established quality assurance measures were fulfilled. Expanded data analysis was provided through the use of an Ion Reporter Server, version 5.2 (Thermo Fisher Scientific, Waltham, MA). PGM sequencing was executed to a depth of 0.8× for the full genome with at least 3.5 million reads and a median sequencing fragment length of 186 bp. Blastocyst morphological classification An adaptation of the Gardner blastocyst grading system was used to classify embryos as High (3 to 6 AA and 4 to 6 AB), Mid (any BB, 1 to 3 AB, and 1 to 2 AA), or Low grade (any AC, CA, BC, CB, or CC) based on overall morphological grade (see footnote Table 2) [31, 32]. The inner cell mass (ICM) and trophectoderm (TE) grades were also independently classified as High (A), Mid (B), and Low (C). The primary outcome from the preimplantation parameters was to evaluate embryo quality in relation to sex. Secondary outcomes from preimplantation data evaluated embryo sex in relation to day of blastocyst transformation and ploidy. Analyses were repeated after removing embryos with a sex chromosome aneuploidy (n = 3438 remaining) and again for only euploid embryos (n = 1809 remaining), for certain parameters. Embryos from the screened population that underwent eSET were analyzed for relationships between embryo sex and eSET outcomes. Secondary analysis for this subset looked at eSET outcomes based on day of blastocyst transformation and embryo grade. Pearson’s chi-squared was used for statistical analysis with P < 0.05 considered significant. Table 2. | Male (n, %) | Female (n, %) | P-value | | |---|---|---|---| | All screened embryos (n = 3708) | ||| | Ploidy | ||| | Euploid | 898 (52.8) | 911 (45.4) | < 0.001 | | Aneuploid | 804 (47.2) | 1095 (54.6) | | | Day of blastocyst transformation | ||| | 5 | 1079 (63.4) | 1302 (64.9) | 0.566 | | 6 | 622 (36.6) | 702 (35.0) | | | 7 | 1 (0.1) | 2 (0.1) | | | Grade* | ||| | High | 1067 (62.7) | 1191 (59.4) | 0.057 | | Mid | 329 (19.3) | 396 (19.7) | | | Low | 306 (18.0) | 419 (20.9) | | | Inner cell mass | ||| | A | 979 (58.3) | 1235 (60.9) | 0.257 | | B | 514 (30.6) | 576 (28.4) | | | C | 187 (11.1) | 217 (10.7) | | | Trophectoderm | ||| | A | 722 (43.0)) | 829 (40.9) | 0.079 | | B | 741 (44.1) | 886 (43.7) | | | C | 217 (12.9) | 313 (15.4) | | | Sex chromosome aneuploidy omitted (n = 3438) | ||| | Ploidy | ||| | Euploid | 898 (53.9) | 910 (51.4) | 0.135 | | Aneuploid | 768 (46.0) | 862 (48.7) | | | Day of blastocyst transformation | ||| | 5 | 1059 (63.6) | 1164 (65.7) | 0.363 | | 6 | 606 (36.4) | 606 (34.2) | | | 7 | 1 (0.) | 2 (0.1) | | | Grade | ||| | High | 1047 (62.9) | 1063 (60.0) | 0.094 | | Mid | 323 (19.4) | 344 (19.4) | | | Low | 296 (17.8) | 365 (20.6) | | | Euploid only (n = 1809) | ||| | Day of blastocyst transformation | ||| | 5 | 630 (70.2) | 625 (68.6) | 0.485 | | 6 | 268 (29.8) | 285 (31.3) | | | 7 | 0 (0.0) | 1 (0.1) | | | Grade | ||| | High | 640 (71.3) | 626 (68.7) | | | Mid | 156 (17.34) | 166 (18.2) | | | Low | 102 (11.4) | 119 (13.1) | 0.432 | *The Gardner blastocyst grading system denotes scoring parameters for each blastocyst according to stage of development, inner cell mass, and trophectoderm. Stages of development: (1) blastocoel size less than half the volume of the embryo; (2) blastocoel size equal to or greater than half the volume of the embryo; (3) blastocoel entirely fills embryo; (4) blastocyst expanded larger than embryo with thinning of the zona pellucida; (5) hatching from shell; (6) hatched from shell. Inner cell mass: (A) many tightly packed cells; (B) several loosely packed cells; (C) few cells. Trophectoderm: (A) many cells organized in epithelium; (B) several cells organized as loose epithelium; (C) few cells. Overall Grade (High) 3 to 6 AA and 4 to 6 AB; (Mid) any BB, 1 to 3 AB, 1 to 2 AA; (Low) any AC, CA, BC, CB, or CC Defining transfer outcomes A chemical pregnancy was defined as a positive beta human chorionic gonadotropin (beta-hCG) without a gestational sac seen on ultrasound. A clinical pregnancy was defined by the presence of a gestational sac on ultrasound. Implantation was considered to have occurred if either a chemical or clinical pregnancy was present. Ongoing pregnancy was defined as presence of a live fetus at the time the patient was released for routine obstetrical care at approximately 12 weeks gestational age.

In all, 3708 embryos from 578 different individuals were evaluated. When weighted to account for multiple embryos from the same individual, there was a mean maternal age of 34.3 ± 4.3 years and body mass index (BMI) of 26.9 ± 5.8 kg/m2. Embryos were mostly from white individuals (53.6%) with the most common infertility diagnoses being multifactorial (24.4%) and ovulatory dysfunction (15.8%) (Table 1). Table 1. | n | 3708 | |---|---| | Age (mean, ±SD) | 34.3 ± 4.3 | | BMI (mean, ±SD) | 26.9 ± 5.8 | | Ethnicity (n, %) | | | White | 1987 (53.6) | | Hispanic | 595 (16.1) | | Black | 434 (11.7) | | Asian | 397 (10.7) | | Other | 295 (8.0) | | Infertility diagnosis (n, %) | | | Ovulatory | 587 (15.8) | | Unexplained | 493 (13.3) | | Male | 450 (12.1) | | Diminished ovarian reserve | 424 (11.4) | | Tubal | 301 (8.1) | | Endometriosis | 117 (3.2) | | Uterine | 114 (3.1) | | Multiple | 904 (24.4) | | Other | 318 (8.6) | Male embryos (N = 1702) accounted for 45.9% of those biopsied and female (N = 2006) 54.1%. No relationship was seen between embryo grade and sex. This finding also held true when considering aneuploid embryos without a sex chromosome aneuploidy and euploid embryos only. Overall, the majority of embryos of the entire screened population were considered High grade (N = 2258, 60.9%), while Mid (N = 725) and Low (N = 725) grades each accounted for 19.6% of embryos, respectively. Neither ICM nor TE grades correlated with embryo sex. The majority of embryos reached the blastocyst stage on day 5 (N = 2381, 64.2%), with day 6 (N = 1324) and day 7 (N = 3) accounting for 35.7% and 0.1% of screened embryos, respectively. There was no difference in day of blastocyst transformation based on embryo sex when evaluating all embryos, those without sex chromosome aneuploidy or only euploid embryos. 51.2% (N = 1899) of the screened embryos were aneuploid, and 48.8% (N = 1809) were euploid. Female embryos were found to be aneuploid at a higher rate than male embryos (54.6% vs 47.2%, P < 0.001). When repeating the analysis with exclusion of embryos with an abnormal sex chromosome number, this difference was no longer present with males and females having more similar aneuploid rates (46.0% vs 48.7%, P = 0.135) (Table 2). Separately evaluating blastocysts from women under age 35 (n = 1891) and age 35 and older (n = 1817) demonstrated similar results to that of the cohort as a whole in that females were more likely aneuploid. No differences were seen in either subgroup based on sex in day of blastocyst transformation or overall grade (data not shown). A total of 539 eSETs took place from 392 different individuals, with the transfer of 294 male (54.6%) and 245 female (45.5%) embryos. In 19 instances (3.5%), embryo selection for transfer was based on desired offspring sex (12 male, 7 female). The remaining embryos were selected for transfer based on the best quality available embryo, irrespective of sex. Rates of ongoing pregnancy did not differ based on sex. In addition, implantation, clinical pregnancy, spontaneous abortion, chemical pregnancy, and ectopic pregnancy did not differ between male and female euploid embryos (Table 3). High grade embryos were more likely to result in implantation (71.8% vs 56.8% and 45.0%, P < 0.001), clinical pregnancy (69.4% vs 55.4% and 42.5%, P < 0.001), and ongoing pregnancy (59.1% vs 47.3% and 40.0%, P = 0.018) than Mid or Low grade embryos, respectively (Table 4). Day 5 blastocysts were more likely to result in implantation (70.8% vs 60.3%, P = 0.018), clinical pregnancy (69.2% vs 56.4%, P = 0.005), and ongoing pregnancy (59.3% vs 48.1%, P = 0.018) than day 6 blastocyst embryos, respectively (Table 4). Female embryos with blastocyst transformation on day 5 were more likely to result in a clinical pregnancy (68.8% vs 52.0%, P = 0.012) and trended towards being more likely to result in an ongoing pregnancy (58.2% vs 45.3%, P = 0.062) than those with blastocyst transformation on day 6. No differences in transfer outcomes were seen in male embryos based on day of embryo blastocyst transformation (Table 4). When considering separately embryo transfers in women under age 35 (n = 261) and those 35 and older (n = 278), no difference in transfer outcome was observed based on embryo sex (data not shown). Table 3. | Male (n, %) | Female (n, %) | p-value | | |---|---|---|---| | Implantation | ||| | Yes | 203 (69.1) | 162 (66.1) | | | No | 91 (31.0) | 83 (33.9) | 0.470 | | Spontaneous Abortion | ||| | Yes | 30 (10.2) | 24 (9. 8) | | | No | 264 (89.8) | 221 (90.2) | 0.875 | | Ectopic Pregnancy | ||| | Yes | 0 (0.0) | 2 (0.8) | | | No | 294 (100.0) | 243 (99.2) | 0.121 | | Chemical Pregnancy | ||| | Yes | 8 (2.7) | 4 (1.6) | | | No | 286 (97.3) | 241 (98.4) | 0.394 | | Clinical Pregnancy | ||| | Yes | 197 (67.0) | 156 (63.7) | | | No | 97 (33.0) | 89 (36.3) | 0.418 | | Ongoing Pregnancy | ||| | Yes | 169 (57.5) | 133 (54.3) | | | No | 125 (42.5) | 112 (45.7) | 0.456 | Table 4. | Implantation (n, %) | Clinical Pregnancy (n, %) | Ongoing Pregnancy (n, %) | |||| |---|---|---|---|---|---|---| | Grade | |||||| | High | 305 (71.8) | P < 0.001 | 295 (69.4) | P < 0.001 | 251(59.1) | P = 0.018 | | Mid | 42 (56.8) | 41 (55.4) | 35 (47.3) | ||| | Low | 18 (45.0) | 17 (42.5) | 16 (40.0) | ||| | Day of Blastocyst Transformation | |||||| | Day 5 | 271 (70.8) | P = 0.018 | 265 (69.2) | P = 0.005 | 227 (59.3) | P = 0.018 | | Day 6 | 94 (60.3) | 88 (56.4) | 75 (48.1) | ||| | Male | |||||| | Day 5 Blastocyst | 152 (71.4) | P = 0.164 | 148 (69.5) | P = 0.143 | 128 (60.1) | P = 0.142 | | Day 6 Blastocyst | 51 (63.0) | 49 (60.5) | 41 (50.6) | ||| | Female | |||||| | Day 5 Blastocyst | 119 (70.0) | P = 0.054 | 117 (68.8) | P = 0.012 | 99 (58.2) | P = 0.062 | | Day 6 Blastocyst | 43 (57.3) | 39 (52.0) | 34 (45.3) |

Efforts are continually being made to improve IVF-eSET outcomes. Prior studies have evaluated various embryo parameters and variables that may impact ET outcomes, but these analyses have not been stratified by embryo sex. This study is unique in analyzing preimplantation embryo characteristics and eSET outcomes in the same cohort of individuals with particular attention paid to how embryo sex correlates with various findings. Sex ratios at birth have historically favored a slightly higher proportion of males than would be predicted based on the frequency of Y-chromosome-bearing spermatozoa [33]. While we did not observe a difference in screened embryo quality or grade or in eSET outcomes based on sex, we did find variations in aneuploidy rates of screened embryos. Overall, day 5 embryo transfers in our study performed better than day 6 embryo transfers, as did those with higher grade. We speculate that embryos taking longer to develop may have other, non-chromosomal, factors involved in blastocyst development and implantation leading to less favorable outcomes not apparent when only considering ploidy status. We did not find a difference in ongoing pregnancy rates after transfer of frozen blastocyst stage embryos when comparing males and females as a whole. Day 5 females, however, were more likely to result in a clinical pregnancy, but not ongoing pregnancy, than day 6 females. Otherwise, we found no difference in transfer outcomes of male or female embryos based on day of blastocyst transformation. This could possibly be due to sex chromosomes being less influential in transfer success of known euploid embryos than factors such as morphology and rate of development. Prior studies are varied in findings related to frequency of offspring sex after eSET. These studies have focused on outcomes as measured by live birth or ongoing pregnancy rates in IVF transfers as a whole but not evaluated as outcomes specific to known sex of the blastocyst transferred. Studies tend to show higher rates of male offspring with blastocyst transfers versus a more equal sex distribution with cleavage stage eSETs. Two separate meta-analyses (Milki et al. and Chang et al.) support this finding of mostly equal sex distribution with cleavage stage embryos, whereas males are more common with blastocyst stage embryo transfers [11, 29]. This is corroborated by our findings in that at the blastocyst stage, a larger proportion of female blastocysts are aneuploid compared to male blastocysts and would be unlikely to have a successful embryo transfer. Shaia et al. found that the use of PGT-A pushed the ratio further towards males in blastocyst transferred IVF patients compared to untested blastocyst or cleavage stage transfers, but the authors did not specify the type or indication for PGT nor did they specify the sex ratio of the blastocysts transferred [34]. A series of other individual studies reported similar findings, though with no noted use of aneuploidy screening methods [12, 19, 35–38]. In contrast, Kausche et al. observed no difference in the sex distribution of live births between fresh transfer of blastocyst versus cleavage stage embryos when looking at a sample of 125 patients, and Csokmay et al. considered only transfer of blastocyst stage embryos with normal preimplantation fluorescent in situ hybridization (FISH) testing and found no difference in live birth sex ratios [20, 30]. While variation does exist between our findings and that of Kausche and Csokmay, it should be noted that these studies reported a final endpoint of live birth, whereas we focused on ongoing pregnancy. In terms of day 5 versus day 6 embryos, independent of sex, Barash et al. showed higher ongoing pregnancy rates with day 5 compared to day 6 embryos, though no distinction was made based on sex for these day 5 or 6 embryos [39]. As was noted in prior studies [40, 41], we observed more favorable eSET outcomes for day 5 embryos than with day 6 embryos, with sex being a mostly unrelated factor in our study. We did not observe differences in male and female embryo development with regard to day of blastocyst transformation and overall morphological, ICM, or TE grades. Our findings remained consistent regardless of whether evaluating all embryos, omitting those with an abnormal sex chromosome number, or evaluating only euploid embryos. Eaton et al. similarly concluded that the stage of embryo development was independent of embryo sex on day 5 of culture [22]. Assessment of TE has varied between studies. Tarin et al. suggested that female blastocysts may have poorer transfer outcomes due to having fewer trophectoderm cells. Wang et al. found higher TE grades in male embryos but no other morphologic differences [23]. Our data did not support these TE findings, as both trophectoderm grading and transfer outcomes were similar between males and females [19]. Our findings are also somewhat consistent with Alfarawati et al. who found the highest grade embryos tended to be male and lowest grade female, with no significant difference between sexes on ICM or TE grading [13]. In contrast with our study, their data did suggest male embryos reach the blastocyst stage faster than female embryos. Likewise, Lou et al. found high-quality blastocysts were more likely to be male than poor quality, with higher quality transfers having a higher likelihood of being male than poor-quality embryo transfers [42]. One area where we did find a notable difference was in rates of aneuploidy between male and female embryos as determined on the day of blastocyst transformation. We found female embryos to be significantly more likely to be aneuploid than male embryos. This supports the idea that redirection of cellular resources towards X-chromosome inactivation, or some other to be determined factor, may contribute towards differing aneuploidy rates between male and female blastocysts. In a work by Barash et al., day 5 biopsied embryos had a higher chance of being euploid than did day 6 embryos; however, these results were not stratified by sex [39]. Alfarawati et al. did not demonstrate a relationship between sex and aneuploidy at the blastocyst stage when considering embryos with a normal pair of sex chromosomes [13]. Similarly, Bronet et al. found aneuploidy and sex to be unrelated, after excluding embryos noted to have a “chaotic pattern,” X-chromosome gain, or Y-chromosome gain or loss [14]. Chaotic embryos are those found to have an extreme abnormality of chromosomes such that cells have an apparent random allotment of chromosomes [43]. Similarly, Wang et al. excluded embryos with sex chromosome abnormalities and subsequently found no association between blastocyst stage embryo sex and ploidy [23]. Eaton et al. used a 12 chromosome panel and determined that ploidy was independent of sex when comparing XX and XY embryos. Fragouli et al. found no relationship between aneuploidy rates and blastocyst sex in those with normal sex chromosomes [44]. Omitting sex chromosome aneuploidy from our dataset resulted in similar outcomes of no difference in aneuploidy rates between male and female embryos. This supports the notion of an influence of X-chromosome inactivation in XX embryos on aneuploidy rates. To our knowledge, this is the first study to evaluate sex-specific transfer outcomes in a cohort of embryos having undergone PGT-A. While our data did not show a difference in ongoing pregnancy rates as pertains to transfer of a euploid male or euploid female embryo, it is not in disagreement with other studies that do show a difference in sex distribution for ongoing pregnancies or live births [11, 29, 33, 34]. Our data demonstrate a higher aneuploid rate in female blastocysts. Thus, it is plausible that transfer of a given unscreened blastocyst with unknown ploidy status could have a higher likelihood of transfer of a normal male than a normal female. Transfer of an aneuploid female embryo would likely not result in ongoing pregnancy and thus shift the sex outcomes in favor of more viable males. Following findings of prior studies [15–19], it could be postulated that this higher female aneuploidy rate is related to the process of X-chromosome inactivation and its required energy expenditure. The results from this study can be useful when counseling patients undergoing eSET with sex-selective embryos in that day of blastocyst transformation is a better predictor of ongoing pregnancy rates than is embryo sex. Preference should be given to transfer of euploid day 5 embryos over euploid day 6 embryos, regardless of sex, unless sex selection is desired. In addition, it should be noted that for 98 (17%) of the individuals in the screened population, all screened embryos were aneuploid, with a median of 3.5 [range 1–20] aneuploid embryos per individual of this subpopulation. The median age for this population was 38 [range 25–47]. Women with only aneuploid embryos should not undergo embryo transfer and instead be counseled regarding whether or not to attempt another retrieval with PGT-A. Our study is possibly limited by its observational and retrospective nature, with data derived from a single center. Further limitations may also exist given different physician techniques. An additional bias exists in patient selection for PGT-A, as not all patients underwent this testing, and the method of embryo selection for eSET (e.g., specific sex or overall morphology). Patients may elect for PGT-A for reasons such as prior failed transfers, desire for specific offspring sex, or concern for aneuploidy associated with maternal age. We acknowledge that these results are from a select group of patients who elected for PGT-A for specific reasons. We recognize that sex selection of embryos may not be permissible in all areas where IVF is carried out. The vast majority of embryos in our study were transferred based on embryo quality, rather than desire for specific offspring sex. Therefore, we feel our eSET population does fairly represent plausible conditions across a range of practice locations. Finally, there does always exist the possibility of mosaicism of embryos with reported normal PGT-A [45], and patients should be made aware that concern does exist regarding the safety of PGT-A to the embryo [46].

While we did not observe a difference between male and female embryos in grade or quality, there was a significantly higher rate of aneuploidy in females than in males. This information helps to explain the higher rates of male offspring seen in IVF pregnancies and can be used to help counsel patients who may be undergoing eSET without having undergone PGT-A. Further studies are warranted to describe on a cellular and molecular level the specific mechanisms of this aneuploidy difference at the blastocyst stage. Author contribution All authors contributed to the study concept and design. CPM, SEF, JPR, and JRC collected the data. Data was analyzed by CPM, AFS, and JRC. The first draft of the manuscript was written by CPM, and all authors contributed to and approved the final version. Data availability Data will be made available upon request to the corresponding author. Code availability N/A Declarations Ethics approval This study was deemed exempt by the Institutional Review Board of the University of Texas Medical Branch Galveston. Conflict of interest The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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