Abstract
Purpose
To determine if pre-implantation genetic testing (PGT) shifts the sex ratio (SER), the ratio of male to female births in a population normalized to 100 and typically stable at 105, following in vitro fertilization (IVF).
Methods
Data from 2014 to 2016 was requested from the Society for Assisted Reproductive Technologies (SART) database including fresh and frozen transfer cycles. Women with a singleton live birth following a fresh or frozen autologous embryo transfer of a PGT blastocyst, non-PGT blastocyst, or non-PGT cleavage stage embryo were included. The SER between groups was compared using chi-square tests. Modified Poisson regression modeled the relative risk (RR) of having a male compared to a female among PGT blastocyst transfers versus non-PGT cleavage and blastocyst transfers adjusting for age, BMI, smoking status, race, parity, number of oocytes retrieved, and clinic region.
Results
The SER was 110 among PGT blastocyst offspring, 107 among non-PGT blastocyst offspring (p = 0.005), and 99 among non-PGT cleavage offspring (p < 0.001). The risk of having a male infant was 2% higher among PGT blastocyst transfers compared to non-PGT blastocyst transfers (RR 1.02; 95% CI: 1.01, 1.04). The risk was 5% higher among PGT blastocyst transfers compared to non-PGT cleavage transfers (RR 1.05; 95% CI: 1.02, 1.07). The association between PGT and infant gender did not significantly differ by region (p = 0.57) or parity (p = 0.59).
Conclusion
Utilizing PGT shifts the SER in the IVF population from the standard of 105 to 110, increasing the probability of a male offspring.
Keywords: PGT, Sex ratio, In vitro fertilization, Blastocyst
Introduction
The SER is defined as the ratio of male to female births in the population normalized to 100, and it is typically standard at 105 in the USA [1]. Without manipulation, the sex ratio remains relatively constant; fluctuations are minor between 104.6 and 105.9 across a 60-year time period in the USA [1–4]. Notable alterations in the sex ratio occur in times of war, labor migration, and in populations with son preference [1, 3, 5, 6]. Ramifications of a skewed sex ratio include increased heightened masculinity of young adult male populations, increased male to female death rates at older ages, and sustained patriarchy [3, 6].
Approximately 1.7% of all infants born yearly in the USA are conceived using assisted reproductive technology (ART) [7]. This accounts for over one million live births since 1987 [8]. In keeping with the increase in ART in the USA, the utilization of pre-implantation genetic testing (PGT) has increased from 4% of all cycles in 2005 to over 22% in 2015 [9]. While commonly used to assess for aneuploidy and single gene disorders, chromosomal sex is also revealed, enabling couples to select the offspring’s sex when multiple euploid embryos are available for transfer in the USA.
The American Society of Reproductive Medicine (ASRM) Ethics Committee released guidelines surrounding sex disclosure when incidentally revealed after PGT, stating that patients should be asked whether they wish for the embryo sex to be disclosed [10]. In addition, if clinics implement policies disallowing sex as a factor in selecting which embryos to transfer, then patients should be aware of such policies prior to initiating treatment [10, 11], stating it is “unlikely that providing patients with information about embryo sex will have broader social consequences, even in small social subgroups [10].” However, there is currently no evidence to refute or support this claim.
There is evidence that there has been an increase in non-medical sex selection practices among ART clinics [12, 13]. In addition, the process of extended culture itself may alter the male to female offspring ratio; older studies dismiss this theory [14–16], while newer studies support it [17–20]. Given the controversy around non-medical sex selection and the possible impact of extended culture on offspring sex, we sought to determine the extent to which the use of PGT shifts the SER following in vitro fertilization and whether this association differs by clinic region or parity.
Material and methods
Approval was granted by the Duke Medicine Institutional Review Board for this retrospective cohort study. National IVF data from 2014 to 2016 was requested from SART including both fresh and frozen transfer cycles. SART data included whether or not PGT was performed on the transferred embryo. Women who had a singleton live birth following a fresh or frozen autologous embryo transfer of a (1) PGT blastocyst, (2) non-PGT blastocyst, or (3) non-PGT cleavage stage embryo were included. We excluded transfers in which the PGT status or offspring sex at birth was unknown.
Characteristics of women and IVF cycles were summarized by PGT, non-PGT cleavage, or non-PGT blastocyst transfers and compared using ANOVA, Kruskal-Wallis, or Chi-square tests as appropriate. The SER for each group was calculated and compared between groups using Chi-square tests. Subsequently, modified Poisson regression was used to model the risk of having a male infant compared to a female infant among PGT embryo transfers versus both non-PGT cleavage and blastocyst transfers while adjusting for age, BMI, smoking status, race, parity (parous versus nulliparous), number of oocytes retrieved, and clinic region. Separate models for group by parity and group by clinic region interactions were fit to assess for effect modification by these two factors. Wald test was used to test the overall interaction effect. Risk ratios (RR) and 95% confidence interval (CI) were presented for these models. Analysis was conducted using R 3.5.3 [21]. P < 0.05 was considered statistically significant for all other tests.
Results
The demographics of our study population are shown in Table 1. A total of 91,805 embryo transfer cycles were analyzed from 2014 to 2016. Among these, 21,426 (23.3%) were PGT cycles, 14,327 (15.6%) were non-PGT cleavage cycles, and 56,052 (61.1%) were non-PGT blastocyst cycles. The average recipient’s age in this study cohort was 34.1 (standard deviation SD = 4.2) with a mean BMI of 25.3 (SD = 5.5). The majority of the cohort was Non-Hispanic white (45.7%), nulliparous (65.7%), and from the northeast (32.3%). Women utilizing PGT were more likely to be older than women undergoing cleavage stage or non-PGT blastocyst stage transfer. PGT use was also disproportionately higher in the West and lowest in the Midwest.
Table 1.
Demographics of PGT and Non-PGT Patients from 2014 to 2016
| Characteristics | PGT blastocyst (n = 21,426) | Non-PGT cleavage (n = 14,327) | Non-PGT blastocyst (n = 56,052) | Total (N = 91,805) | P value |
|---|---|---|---|---|---|
| Age, years | 35.4 (4.1) | 34.6 (4.2) | 33.4 (4) | 34.1 (4.2) | < 0.001 |
| Body mass index (kg/m2) | 24.4 (4.9) | 25.7 (5.8) | 25.6 (5.6) | 25.3 (5.5) | < 0.001 |
| Current smokera | 263 (1.2%) | 232 (1.6%) | 850 (1.5%) | 1345 (1.5%) | 0.003 |
| Race/ethnicity | < 0.001 | ||||
| Non-Hispanic white | 8747 (40.8%) | 6653 (46.4%) | 26,566 (47.4%) | 41,966 (45.7%) | |
| Non-Hispanic black | 463 (2.2%) | 461 (3.2%) | 2305 (4.1%) | 3229 (3.5%) | |
| Hispanic/Latino | 752 (3.5%) | 808 (5.6%) | 2574 (4.6%) | 4134 (4.5%) | |
| Other | 2658 (12.4%) | 1169 (8.2%) | 5124 (9.1%) | 8951 (9.8%) | |
| Unknown | 8806 (41.1%) | 5236 (36.5%) | 19,483 (34.8%) | 33,525 (36.5%) | |
| Nulliparous | 14,041 (65.5%) | 9884 (69.0%) | 36,357 (64.9%) | 60,282 (65.7%) | < 0.001 |
| Number of prior pregnancies | 1 [0, 2] | 1 [0, 2] | 1 [0, 2] | 1 [0, 2] | < 0.001 |
| Number of oocytes retrieved, | 16 [11, 23] | 9 [6, 15] | 15 [11, 22] | 15 [10, 21] | < 0.001 |
| Reporting year | < 0.001 | ||||
| 2014 | 3604 (16.8%) | 5836 (40.7%) | 18,458 (32.9%) | 27,898 (30.4%) | |
| 2015 | 7090 (33.1%) | 4499 (31.4%) | 18,741 (33.4%) | 30,330 (33%) | |
| 2016 | 10,732 (50.1%) | 3992 (27.9%) | 18,853 (33.6%) | 33,577 (36.6%) | |
| Clinic Region | < 0.001 | ||||
| Midwest | 2406 (11.2%) | 3282 (22.9%) | 11,279 (20.1%) | 16,967 (18.5%) | |
| Northeast | 6569 (30.7%) | 5474 (38.2%) | 17,568 (31.3%) | 29,611 (32.3%) | |
| South | 5475 (25.6%) | 3035 (21.2%) | 17,576 (31.4%) | 26,086 (28.4%) | |
| West | 6971 (32.5%) | 2478 (17.3%) | 9597 (17.1%) | 19,046 (20.7%) | |
| Puerto Rico | 5 (0%) | 58 (0.4%) | 32 (0.1%) | 95 (0.1%) | |
Data are presented as the mean (standard deviation) or number (%)
ADefined as smoking within the 3 months prior to cycle initiation
Among all PGT embryo transfers, the calculated SER was 110. Among all non-PGT blastocyst transfers the SER was 107 (p = 0.005), and for non-PGT cleavage stage transfers, the SER was 99 (p < 0.001). More specifically, the proportion of males was significantly different between groups (p < 0.001) with the highest following PGT-blastocyst transfer (52.3%) compared to non-PGT blastocyst (51.6%) and cleavage (49.8%) transfer.
After adjusting for age, BMI, smoker, race, parity, number of oocytes retrieved, reporting year, and clinic region, the risk of having a male infant was 2% higher among PGT blastocyst transfers compared to non-PGT blastocyst transfers (RR 1.02; 95% CI: 1.01, 1.04, p = 0.004). The risk was 5% higher among PGT blastocyst transfers compared to non-PGT cleavage transfers (RR 1.05; 95% CI: 1.02, 1.07, p = < 0.001). The increase in risk did not differ by parity of the recipient (p = 0.59, Table 2). Although the risk of a male infant following PGT appeared to be increased in the West compared to other regions, the differences between regions was not statically significant, as the interaction term was not statistically significant (p = 0.57, Table 3).
Table 2.
Association between PGT embryo transfer and male offspring by parity of the recipient
| Male offspring | |||
|---|---|---|---|
| RR (95% CI)a | RR (99% CI)b | ||
| PGT versus non-PGT cleavage |
p value for the interaction term P = 0.59 |
||
| Nulliparous recipient | 1.04 (1.02, 1.07) | 1.04 (1.01, 1.08) | |
| Parous recipient | 1.06 (1.02, 1.10) | 1.06 (1.01, 1.11) | |
| PGT versus non-PGT blastocyst | |||
| Nulliparous recipient | 1.03 (1.01, 1.05) | 1.03 (1.00, 1.05) | |
| Parous recipient | 1.02 (0.99, 1.05) | 1.02 (0.99, 1.06) | |
Model included parity, age, BMI, smoking status, race, number of oocytes retrieved, reporting year, and clinic region as covariates
(a) Post-hoc pairwise comparisons without multiple testing adjustment
(b) Post-hoc pairwise comparisons with Bonferroni adjustment
Table 3.
Association between PGT embryo transfer and male offspring by region of the country
| Male offspring | |||
|---|---|---|---|
| RR (95% CI)a | RR (99.4% CI)b | ||
| PGT versus non-PGT cleavage | p value for the interaction term P = 0.57 | ||
| Midwest | 1.02 (0.97, 1.08) | 1.02 (0.95, 1.10) | |
| Northeast | 1.04 (1.01, 1.08) | 1.04 (0.99, 1.10) | |
| South | 1.03 (0.99, 1.08) | 1.03 (0.97, 1.10) | |
| West | 1.08 (1.03, 1.14) | 1.08 (1.02, 1.16) | |
| PGT versus non-PGT blastocyst | |||
| Midwest | 1.00 (0.96, 1.04) | 1.00 (0.94, 1.06) | |
| Northeast | 1.01 (0.99, 1.04) | 1.01 (0.98, 1.05) | |
| South | 1.03 (1.00, 1.06) | 1.03 (0.99, 1.08) | |
| West | 1.04 (1.01, 1.07) | 1.04 (0.99, 1.08) | |
Model included parity, age, BMI, smoking status, race, number of oocytes retrieved, reporting year, and parity as covariates
(a) Post-hoc pairwise comparisons without multiple testing adjustment
(b) Post-hoc pairwise comparisons with Bonferroni adjustment
We performed a sensitivity analysis to account for those intentionally pursuing PGT for the purposes of sex selection. After excluding transfers for which PGT was performed for sex selection (n = 1508), the increase in risk was slightly smaller in PGT versus non-PGT cleavage transfers (RR 1.04; 95% CI: 1.02, 1.06), but remained the same for PGT versus non-PGT blastocyst (RR 1.02; 95% CI: 1.00, 1.03) transfers.
Discussion
In this analysis of national US data, we found a SER of 110 in PGT blastocyst offspring, a SER of 107 in non-PGT blastocyst stage offspring, and a SER of 99 in non-PGT cleavage stage offspring. Performing PGT was associated with a higher probability of a male offspring, even after adjusting for confounders. Some of this increase in risk appears to be attributable to extended culture to blastocyst and some to the PGT itself. While the increase in risk of a male offspring appeared to be greatest in the West and lowest in the Midwest, differences between regions were not statistically significant. The parity of the recipient also did not influence the risk of PGT resulting in a male infant.
We noted that the SER among couples undergoing PGT (110) is higher than the population SER of 105. We believe this to be a marked change considering the stability of the population SER around 105 since the 1940s in the USA [2]. It is unlikely that IVF with PGT currently significantly changes the population SER, as approximately 1.7% of births in the USA are due to IVF annually [7], and only about 50% of ART cycles included PGT in 2016. However, our study revealed an increase in utilization of PGT from 16.8% in 2014 to 50.1% in 2016. Given the increasing uptake of ART and increased utilization of PGT, we believe these technologies could impact the population SER over time.
We found that the risk of a male offspring was increased with PGT. The increase in risk due to PGT could be due to either increased selection of male embryos for transfer, or lack of implantation or increased loss of female embryos. Genetic factors portending a higher likelihood of male embryo survival include possible advantages encoded on the Y chromosome and imprinting errors on the X chromosomes of the female embryo [22]. Furthermore, it has been reported that female embryos have a higher rate of aneuploidy [23]. For instance, Turner’s syndrome has an incidence of 22.2 out of 100,000, which could further explain a preselection for unaffected male embryos [24].
Some of the increase in risk associated with PGT appears to be due to the extended culture. Extended culture possibly favors male embryo development given that early male embryos have been shown to uptake pyruvate and glucose faster than their female embryo counterparts [25], making the female embryo more susceptible to stressful conditions. Another theory is an inadvertent selection bias toward females at the cleavage stage. A cleavage stage transfer could imply that the cycle is in some way suboptimal given that a transfer may have been performed to avoid the risk of no embryos for blastocyst transfer. Female embryos have been theorized to grow slower than male embryos [25], which could increase the likelihood of transfer at the cleavage stage.
Our study was the first national study to show an association between PGT embryo transfer and male offspring. A recent study including fresh cycles from 2006 to 2014 registered in the Centers for Disease Control National Assisted Reproductive Technology Surveillance System (NASS) did not find a statistically significant increase in live born males following PGT blastocyst transfer cycles compared to non PGT blastocyst transfer cycles (53.5% vs. 50.6%) [17]. Their study was limited to data prior to 2014, when PGT was less commonly performed on blastocyst embryos. The authors also noted that blastocyst transfer was positively associated with male sex when compared with cleavage-stage transfer in all live-born infants (aRR = 1.03; 95% CI, 1.02–1.04), a finding that was mirrored in our study [17].
In a study of 365 women, Zhang et al. found a significantly higher proportion of male neonates following PGT embryo transfer compared to non-PGT embryo transfer (63.0% vs. 45.5%; P = 0.04) in their clinic, located in the western region of the USA [9]. While this was not the primary aim of their study, the sex imbalance was more pronounced in their findings compared to our study. This can be attributed to the smaller sample sizes in their population (n = 177 for IVF with PGT and n = 180 for IVF without PGT), and the location of their clinic in the West. Furthermore, the mean age of patients undergoing IVF with PGT in their study was slightly older at 36.9 (SD = 3.9) years versus our mean age of 35.4 (SD = 4.1) years. The authors found that this imbalance persisted despite removal of PGT for sex selection in their study. Like Zhang et al., we performed a sensitivity analysis to remove those specifically performing PGT for sex selection (n = 1508). The point estimate was reduced (RR 1.04 compared to 1.05) in the PGT versus non-PGT cleavage comparison, but remained the same for the PGT versus non-PGT blastocyst comparison. Only 7% of the PGT cycles were specifically classified as gender determination; thus, this practice is likely under reported.
Utilization of PGT appears to vary by region of the country, and the practice of offering PGT for sex selection has essentially doubled since 2006 [12]. In a telephone survey of 493 ART clinics offering PGT for sex selection, Capelouto et al. found that 72.7% of responding clinics offer sex selection with 81.2% of those performing for solely elective indication. Clinics offering this practice were more likely to be private centers and/or located in the West or Northeast [12]. Our analysis was in keeping with these findings as PGT was more commonly performed in the West. Point estimates suggested that a PGT embryo transfer was more likely to result in a male in this region. Transfer of a tested blastocyst was 4% more likely (RR 1.04; 95% CI: 0.99, 1.08) to result in a male offspring compared to transfer of an untested blastocyst in the West. However, this increase in risk was not statistically different from the increase in risk seen in other regions. Our data includes only clinic region location and does not include whether clinics are private versus non-private, so we cannot compare our findings for this measure.
Our study had several limitations. Given the use of registry data, data was occasionally missing, especially for race. Furthermore, race was not thoroughly categorized, which could underreport its significance. In addition, this analysis did not adjust for ICSI use as we were unable to obtain this data from SART. However, given the widespread use of ICSI in 76.2% of fresh IVF cycles as of 2012 [26], it is clear that most clinics are performing ICSI, especially in conjunction with PGT. We also did not delineate between fresh and frozen transfers when calculating our SER, which could insert potential bias given that frozen transfers have been linked to an increase in female offspring when performed with ICSI [2, 18]. Strengths of our study include that this is the first study to utilize the fresh and frozen transfers in SART to investigate overall alterations in the sex ratio. Because of the information provided to us by SART, we were able to analyze a large sample size of over 90,000 transfers. Another strength is our use of two control groups, non-PGT blastocyst and non-PGT cleavage, which allowed us to better delineate the effect of blastocyst culture from the effect of PGT itself on the SER.
Conclusion
Utilization of PGT shifts the sex ratio from the population standard of 105 to 110, significantly increasing the probability of a male offspring. Utilization of IVF with PGT could potentially alter the sex ratio on a national level, increasing the imbalance of males to females in our society. Further study is needed to determine the extent to which PGT alters the national sex ratio.
Acknowledgments
The authors would like to thank the Society for Assisted Reproductive Technology for providing the data to complete this analysis.
Author contributions
All authors contributed to the study conception and design. Material preparation was performed by all of the authors, and data collection and analysis were performed by Tracy Truong and Carl Pieper. The first draft of the manuscript was written by Kathryn Shaia, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors’ declare that they have no conflict of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Kathryn Shaia, Email: Kathryn.shaia@duke.edu.
Tracy Truong, Email: tracy.truong@duke.edu.
Carl Pieper, Email: carl.pieper@duke.edu.
Anne Steiner, Email: anne.steiner@duke.edu.
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