Background
In Japan, there has been a steady decrease in the overall neonatal secondary sex ratio (SSR) since 1970 (male/female: 1.070), with a significant decline to 1.056 in 2016 (Ministry of Health, Labour and Welfare Vital Statistics of Japan 2016) [
1]. A number of biological and environmental factors related to the parents have been shown to reduce the SSR. These include older paternal age [
2], factors causing metabolic alterations in women, such as diabetes [
3], stressors (i.e., war, earthquakes and economic distress), toxins (i.e., smoking, pollutants and pesticides), [
4‐
8] and regionality (race) [
9].
Recent advances in sequential culture media have led to the reliable growth of embryos to the blastocyst stage. Blastocyst transfer (BT) is a well-established method employed throughout the world. BT after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has facilitated selection of top-quality embryos which results in high implantation and pregnancy rates compared with early cleavage stage embryo transfer (CT) [
10]. Blastocyst culture allows selection of those embryos that have embryonic gene activation [
11]. Another potential advantage of BT is better embryo-uterine environment for implantation [
12]. Furthermore, since 2008, the single embryo transfer (SET) policy introduced by the Japan Society of Obstetrics and Gynecology has significantly decreased the multiple pregnancy rate [
13]. In addition, BT facilitates preimplantation genetic testing (PGT) [
14]. However, there remain concerns about BT and its adverse effects with regard to higher SSR and increased incidence of monozygotic twinning (MZT). Many studies have suggested that BT is associated with a male-biased SSR imbalance, compared with CT [
15‐
20]. In contrast, other studies have shown no significant differences in the male-to-female ratio after BT [
21,
22]. Some studies have also indicated higher MZT rates after BT [
16,
23‐
25]. MZT carries additional risks, including higher perinatal mortality and morbidity, and increased risks of developmental anomalies, prematurity and discordant growth [
25]. Vitthala et al. reviewed the wide variation between studies on the reported incidence of MZT after BT (1.4–13.2%) [
25]. However, because of their small sample sizes, these studies did not provide sufficient information on various potential confounders such as maternal age, body mass index (BMI), smoking, and basic characteristics. There are still many unknown factors with regard to the SSR imbalance and MZT incidence.
The Japan Environment and Children’s Study (JECS) is a large, nationwide longitudinal birth cohort study directly funded by the Ministry of the Environment of Japan. In this study, we used data from JECS to investigate the association between BT and SSR and MZT, using spontaneously conceived pregnancies, non-assisted reproductive technology (non-ART) treatment (intrauterine insemination and ovulation induction/timed intercourse) and CT for comparison using multivariable logistic regression analysis.
Discussion
With the development of the currently available culture media, blastocyst transfer has increasingly become a common ART treatment. BT allows for the evaluation and selection of high-quality embryos for transfer. In addition, SET helps prevent multi-zygotic multiple pregnancies. However, adverse effects of BT such as an SSR imbalance and an increase in MZT rate have been reported [
15‐
20]. The basic characteristics of ART patients differ from those of the general population. Whether the variations in the SSR and MZT rate are a result of environmental and biological background of ART patients or BT per se is debatable.
In this study, parental basic characteristics, mean BMI and levels of education, both maternal and paternal, were higher in the non-ART treatment, CT, and BT groups compared with the spontaneous conception group. This may be related to the advanced age of patients receiving ART. In addition, the low proportion of parents with a smoking habit, lower calorie intake, lower levels of alcohol consumption, and higher mental component summary of mothers in the CT and BT groups may indicate higher levels of health consciousness of ART patients. As a demographic difference that changes sex ratio, older parental age has been shown to reduce the SSR [
2]. One possible biological explanation for the decrease in sex ratio with parental age is age-related hormonal changes such as an increase in female gonadotrophin and decline in male testosterone concentrations with age [
30]. It is considered that such age-related hormonal changes may affect differential mortality of XX and XY fetuses and skewed ratio of Y- and X chromosome spermatozoa [
31]. Regarding the MZT, the recent review indicated that although some studies showed an increase in the MZT rate in cases of young maternal age, it can be speculated that the true underlying mechanism might be the oocyte age, or even the quality of the oocyte, rather than maternal age itself [
32].
Some reports have suggested that psychological disorders such as depression are associated with the SSR [
33,
34]. Regarding association between SSR and parental stress, it is indicated that the variation in the SSR may result from alteration in the sex-selective preimplantation embryo loss [
35]. As mechanisms affecting this alteration, sperm abnormality, reduced coitus and perturbations in the female reproductive tract caused by stress before preconception are considered [
36,
37]. Similar reasons may affect the incidence of MZT. Multivariate logistic regression analyses indicated that higher K6 and lower physical health condition scores in mothers led to a lower risk of MZT. Miscarriage caused by stress might be related to the results, but it is not known whether MZT and maternal stress are related.
Analyses also showed that maternal ongoing histories of diabetes, hyperthyroidism and hypothyroidism were highest in the CT and BT groups, and hyperlipidemia was higher in the non-ART treatment, CT, and BT groups than in the spontaneous conception group. These differences in basic characteristics are related to the fertility rate.
Multivariate logistic regression analysis of parental characteristics found a significant difference in sex ratio from blastocyst transfer compared with spontaneous conception groups (
P = 0.047). However, based on the CI of 1.001–1.198, the difference barely achieving statistical significance, which is concerning in the setting of multiple comparisons done in the analyses for this study. Meanwhile, we found that BT caused a significant increase in MZT; results that coincided with previous reports [
16,
23‐
25]. However, there was no significant difference in the SSR or MZT rate among the spontaneous conception, non-ART treatment, and CT groups.
There are thought to be several possible reasons for the higher SSR after BT. First is the selection of embryos for transfer based on morphological assessment by the embryologist. More male blastocysts may be selected for transfer because the cleavage of male embryos is faster than that of female embryos up to the blastocyst stage and sequential media may be preferential for male embryos [
16,
38‐
40]. However, some studies have reported different results [
21,
22]. The second issue is the timing of embryo transfer. Extended culture of embryos up to the blastocyst stage may well alter the properties of the cell surface and/or adhesiveness of embryos at implantation [
41]. BT results in enhanced implantation rates, which may cause selection in favor of male embryos [
19]. However, it remains unclear which factor of BT is involved in the risks of SSR.
We concluded that BT significantly increased the MZT rate, consistent with previous reports [
16,
23‐
25]. The causes suggested in previous studies to explain the observed increase in the MZT rate are as follows: 1) extended time in culture, 2) culture medium composition, and 3) embryology laboratory experience [
42‐
45]. The blastocyst undergoing prolonged culture might experience excess environmental stress, weakening cellular adhesion and increasing MZT, but in shorter culture, embryos might be able to tolerate such circumstances [
46]. Recently, it has been reported that the risk of MZT after BT has declined significantly [
46,
47]. The reason for this is unclear, but it may be because of improvements in culture media. Assisted hatching (AH) is often performed in frozen thawed ET. It is reported that AH could lead to hatching defects of the embryo, and through these defects, an inner cell mass may be pinched off and divide, forming MZT [
43,
47,
48]. However, Nakasuji et al. suggested that AH and maternal age did not significantly affect the incidence of MZT [
49]. Further studies are needed to confirm the findings in large series.
A strengths of this study is due to the fact that, using a population-based data set with comparison to a large number of spontaneous pregnancies and adjustment for biological and environmental factors. However, there are some limitations to our study. Firstly, it had no specific information on infertility type, the number of embryos transferred, embryo morphology, fertilization methods, or the history of ART treatments. We confirmed that the subjects had not been pregnant with twins more than twice. However, family history of twin pregnancy was not included in the questionnaire item. Secondly, the analysis of monozygotic twinning being based on very few cases after CT, should be investigated further. Thirdly, we could not differentiate between fresh embryo transfer (ET) and frozen-thawed ET, however, In Japan, frozen-thawed embryos are predominantly used (71.2%) [
50]. Fourthly, and most importantly, methodological limitations must also be considered with regard to the diagnosis of zygosity using early ultrasound data. Our definition of monozygotic is limited. Fingerprinting and DNA polymorphism analyses are necessary to distinguish dichorionic diamniotic monozygotics of the same sex; however, such analyses were not performed in this study.
In conclusion, we examined the slight risks of SSR imbalance and a high incidence of MZT after BT and found little influence of parental biological and environmental factors. At present, the number of infants born as a result of ART, particularly BT, continues to increase in developed countries, including Japan. Although JECS is a large longitudinal cohort study on children’s health and development from early pregnancy up to their thirteenth birthday, we recommended further longitudinal follow-up studies are still needed to shed light on the benefits and risks of BT.
Acknowledgements
The Japan Environment and Children’s Study is funded by the Ministry of the Environment of the Government of Japan. The members of the Japan Environment and Children’s Study (JECS) as of 2016 were as follows; Toshihiro Kawamoto (principal investigator), Hirohisa Saito (National Center for Child Health and Development, Tokyo, Japan), Reiko Kishi (Hokkaido University, Sapporo, Japan), Nobuo Yaegashi (Tohoku University, Sendai, Japan), Koichi Hashimoto (Fukushima Medical University, Fukushima, Japan), Chisato Mori (Chiba University, Chiba, Japan), Fumiki Hirahara (Yokohama City University, Yokohama, Japan), Zentaro Yamagata (University of Yamanashi, Chuo, Japan), Hidekuni Inadera (University of Toyama, Toyama, Japan), Michihiro Kamijima (Nagoya City University, Nagoya, Japan), Ikuo Konishi (Kyoto University, Kyoto, Japan), Hiroyasu Iso (Osaka University, Suita, Japan), Masayuki Shima (Hyogo College of Medicine, Nishinomiya, Japan), Toshihide Ogawa (Tottori University, Yonago, Japan), Narufumi Suganuma (Kochi University, Nankoku, Japan), Koichi Kusuhara (University of Occupational and Environmental Health, Kitakyushu, Japan), Takahiko Katoh (Kumamoto University, Kumamoto, Japan).