The placenta is a temporary and sex-specific endocrine organ that regulates maternal–fetal exchanges, with a central role in fetal growth and development. Sex differences in placenta become evident from the beginning of pregnancy. Specifically, a male fetus grows faster than a female fetus, and it invests more in fetal than placental development. Consequently, male placentas are smaller in size and more vulnerable to adverse events during pregnancy than female placentas. In this context, an increasing number of placental noncoding RNAs contributes to fetal development and pregnancy progression, by regulating gene expression in a sexual dimorphic manner.
Pregnancy is a physiological state in which numerous and complex modifications occur in the mother in order to accommodate the growing fetus . According to the theory of the fetal origin of adult diseases (FOAD), hypothesized by David Barker, the intrauterine environment plays a relevant role in fetal growth and development and markedly contributes to the risk of developing diseases later in life . It is well known that fetal growth and development occur in a sexually dimorphic manner . However, growing evidence indicates that fetal sex differently influences the course of pregnancy and associated complications, as well maternal health . Although ethnic differences exist in the maternal response, several data indicate that women carrying a male show an increased risk of gestational diabetes (GDM), placental abruption , and preterm birth , whereas gestational hypertension/preeclampsia (PE) appear to be linked to carrying a female fetus . Genetics, nutrition, and other known and not yet completely known environmental factors contribute to successful pregnancy . Deregulation of the complex interactions between these factors is responsible for the onset of pregnancy-related complications with long-term impact on offspring health until adulthood, as well as on the mother’s health . Despite it being known that the biological differences between sexes become evident early in embryogenesis, the processes involved in these sex differences are not completely known . Knowledge about the mechanisms through which female and male fetuses respond to external and internal stimuli or insults during pregnancy is rather fragmentary . Embryo development requires proliferation and differentiation processes that are tightly regulated in time and duration . Several mechanisms and numerous factors, including an increasing number of noncoding RNAs (ncRNAs), modulate gene transcription at every stage of embryo and fetus development  and pregnancy progression . Non-coding RNAs play a significant role in epigenetic modifications and exhibit dynamic expression patterns in the regulation of biological processes . They are expressed by almost all cells, allowing cell-to-cell interactions, and are detected in body fluids . In recent years, ncRNAs have been studied as promising diagnostic and prognostic factors, as well as being studied as therapeutic targets in tumors  and neurodegenerative and metabolic disorders , thus representing an area of intensive ongoing investigation in the context of human physiology and pathophysiology. The growing evidence of the importance of sexual dimorphism in health and disease imposes a wider knowledge of the biological processes underlying sex differences.
The placenta is a temporary and sex-specific endocrine organ that regulates maternal–fetal exchanges, with a central role in fetal growth and development . Sex differences in placenta become evident from the beginning of pregnancy. Specifically, a male fetus grows faster than a female fetus already in the pre-implantation phase, and it invests more in fetal than placental development . Consequently, male placentas are smaller in size and more vulnerable to adverse events during pregnancy than female placentas . These findings indicate that placental adaption to maternal environmental stimuli can influence fetus programming with long-term effects . Of note, the plasticity of the placenta allows fetal signals to influence in a sex-specific manner maternal health as well . However, the complex interaction between placental function and fetal development is not well known. To this end, in the last few years, several studies have tried to shed light on the mechanisms related to sex-specific fetus adaptation during pregnancy by using omics approaches as well . Metabolomics of mouse placentas showed that sexual dimorphism contributes to a different placental adaptation to nutrition, stress, hormones, and chemicals to ensure normal fetal development . A growing amount of data has shown that placental miRNAs and lncRNAs have a regulatory role in fetal developmental programming  and pregnancy progression , as well as in genomic imprinting and susceptibility to diseases later in life , by influencing many signaling pathways ( Table 1 ).
Female ♀ vs. Male ♂ Fetus
|Overweight and obese pregnant women||miRNAs||♀ (↑) miR-210||♀ (↑) TNFα and NFκB1; mitochondrial dysfunction|||
|Increasing maternal pre-pregnancy BMI||miRNAs||♀ (↓) miR-20a, miR-34a, miR-146a, miR-210, and miR-222||♀♂ Cell proliferation, cell growth and invasion, inflammation, angiogenesis, and oxidative stress|||
|Healthy human pregnancies||miRNAs||♀ (↑) miR-21, miR-34a, miR-146a, miR-210, and miR-222||♀♂ Inflammation, oxidative stress, cell cycle, cellular senescence, and apoptosis
♀ longer placental relative telomere length
|Human low gestational age newborns (<28 weeks gestational age)||miRNAs||♀ (↑) 27 miRNAs, including miR-323b, miR-15a, and miR-223
♂ (↑) 32 miRNAs, including miR-23a, miR-543, miR-495, miR-323a, miR-30a, miR-155, and miR-222
|♀♂ Embryo implantation and development
♀ X chromosome inactivation,
epigenetic control (HDAC1, HDAC8), and cell cycle progression (CDKN1B)
|Human low gestational age newborns (<28 weeks gestational age)||miRNAs||♀ (↑) miR-4747-5p, miR-3674, miR-4252, miR-4461, miR-1285-5p, miR-4421, miR-4728-3p, miR-6081, miR-6890-3p, iR-564, miR-1225-3p, miR-541-3,p miR-3187-5p, miR-1976, miR-548w, miR-5589-5p, miR-889-3p, miR-4734
♂ (↑) miR-4057, (↓) miR-128
|♀♂ Underweight status
♀ No target genes associated with underweight status
♂ regulate 43 genes related to nutrition, growth, and angiogenesis
|Healthy human pregnancies||miRNAs||♀ 76 (↑), and 66 (↓) miRNAs, most significant miR-361-5p
♀ (↑) miR-361-5p, miR-139-5p, miR-4769-3p♀ (↑) miR-520e, miR-518d-3p, miR-331-3p, miR-423-3p, and miR-330-3p
♀ (↑) miR-4287
♀ (↓) miR-1207-5p, miR-4530, and miR-4687-3p
♀ (↓) miR-4299
|♀♂ Metabolic, immunological, apoptotic, and neurogenesis processes underlying sex differences
(↓) CD99 gene
|Healthy pregnancy from women with/without daily supplement with n-3 LCPUFA||miRNAs||♂ (↑) basal miR-99a
♀ (↑) miR-99a following supplementation
|♀♂ Maternal-fetal amino acids homeostasis
♂ ↑ basal mTOR and SLC7A5 mRNA
♀ ↑ mTOR and SLC7A5 mRNA following supplementation
|Healthy pregnancies from major ethnic groups: African Americans, European Americans, South Asians, and East Asians||miRNAs||♂ (↑) 14 miRNAs 371a-5p,372-3p,181b-2-3p, let-7g-3p, 185-3p, 3615, and 3158-3p
♀ (↑) 18 miRNAs
|♂ glutamate receptor signaling and endocrine processes
♀ steroid hormones, estradiol, and glucocorticoid response; differentiation and metabolic processes
↑ Increases; ↓ decreases; ♀ female; ♂ male; BMI, body mass index; CD99, cluster of differentiation 99; CDK16, cyclin-dependent kinase 16; CDKN1B, cyclin-dependent kinase inhibitor 1B; DDX3Y, DEAD-box helicase 3 Y-linked; HDAC, histone deacetylase; KDM6A, lysine demethylase 6A; miRNA, microRNA; mTOR, mammalian target of rapamycin; ncRNA, non-coding RNA; NFκB, nuclear transcription factor kappa B; PCDH11X, protocadherin 11 X-linked; n-3 LCPUFA, n-3 long-chain polyunsaturated fatty acids; SLC7A5, solute carrier family 7 member 5; TNFα, tumor necrosis factor α.
Sex-dependent miRNA and mRNA expression has been observed in placentas from the extremely low gestational age newborn cohort (ELGAN study; < 28 weeks gestational age). Genome-wide RNA-sequencing analysis showed that 59 miRNAs were differentially expressed between sexes. Of these, miR-23a, miR-543, miR-495, miR-323a, miR-30a, miR-155, and miR-222 were upregulated in male, and miR-323b, miR-15a, and miR-223 were increased in female placentas. All of them are differently involved in placental functions. Specifically, miR-23a, miR-495, miR-222, and miR-223 target genes were associated with PE, and miR-323a with ectopic pregnancy. Interactome analysis revealed that the mRNAs upregulated in female placentas included genes involved in epigenetic control (HDAC1, HDAC8) and cell cycle progression (CDKN1B). A large number of these miRNAs are located on the X-chromosome, suggesting for some of them the escape from XCI (X chromosome inactivation). It is worth noting that miR-543, miR-495, miR-323a, and miR-323b are imprinted miRNA genes belonging to the maternally expressed C14CM miRNA cluster involved in prenatal growth and placentation . Recently, the same groups examined sexually dimorphic mRNA–miRNA expression in relation to pre-pregnancy BMI in placentas from ELGAN study subjects. Data showed that 572 mRNAs associated with pre-pregnancy underweight status in male placentas. Almost all of them (96%) were overexpressed and were involved in known regulatory networks related to nutrition, growth, and angiogenesis, including eukaryotic initiation factor 2 (EIF2) signaling, mammalian target of rapamycin (mTOR) signaling, insulin-like growth factor 1 (IGF-1) signaling, and vascular endothelial growth factor (VEGF) signaling. Of note, only 43 of 572 mRNAs were targets of the two miRNA (miR-4057 and miR-128) that were associated with underweight status in male placentas. In female placentas 18 overexpressed miRNA (miR-4747-5p, miR-3674, miR-4252, miR-4461, miR-1285-5p, miR-4421, miR-4728-3p, miR-6081, miR-6890-3p, iR-564, miR-1225-3p, miR-541-3,p miR-3187-5p, miR-1976, miR-548w, miR-5589-5p, miR-889-3p, miR-4734) were associated with underweight status. The differentially expressed genes in male placentas were also expressed in female placentas. However, none of the mRNAs were associated with underweight status, nor were they a target of the 18 overexpressed miRNAs in female placentas. The authors hypothesized that the altered gene expression in male placentas can represent a protection against intrauterine growth restriction. None of the genes were differentially expressed in overweight and obese status. Of note, the authors confirmed the association between miR-210 and overweight status .
Sedlmeier and colleagues revealed a sex-specific response to maternal supplementation with n-3 long-chain polyunsaturated fatty acid (LCPUFA) during pregnancy (INFAT study). They observed that n-3 LCPUFA supplementation had a more evident impact on gene expression in female placentas than in male . The same authors analyzed the effect of n-3 LCPUFA supplementation on placental miRNA expression in a sex-specific manner. They highlighted a differential expression of miR-99a between sexes. MiR-99a can target the expression of mTOR , SLC7A5 , and SLC6A6 genes, encoding the nutrient sensor mTOR and the amino acid transporters LAT1 and TauT, respectively. The authors observed that miR-99a, mTOR , and SLC7A 5 mRNA levels were higher in male placentas compared with female ones, and n-3 LCPUFA supplementation stimulated elevated levels of miR-99a, mTOR, and SLC7A5 mRNA only in female placentas. Amino acids levels were analyzed in maternal plasma (at 15 and 32 weeks of gestation), placenta, and cord plasma. Increased tryptophan (Trp) levels were found in maternal plasma (32 weeks), with significantly higher levels in mothers with female offspring, whereas lower levels of taurine (Tau) were detected in cord plasma of male offspring after supplementation. Significant associations were found for Trp and Tau and for miR-99a mTOR, SLC7A5 mRNA mTOR, and miRNA-99a with offspring body composition. No amino acids changes were detected in placenta after supplementation. The authors concluded that miRNA-99a could be a novel regulator of a complex network in placenta and in maternal and fetal compartments, regulating amino acids homeostasis and offspring’s body composition .
Sex-specific ncRNAs expression in placentas.
Embryo and fetus growth and development are tightly regulated processes involving embryonic genome and transcriptional sexual dimorphism as well as epigenetic and maternal factors that control the sequence of stage-specific changes leading to a fully developed organism . In this complex context, an increasing number of ncRNAs appear to regulate gene transcription at every stage of embryo and fetus development . Several studies have shown the importance of miRNAs and piRNAs in human reproduction . Specifically, they are involved in the regulation of pre- and post-fertilization stages, such as sex-specification, commitment, and maintenance of mammalian gonads; embryonic implantation and early development; silencing of X-linked gene expression; and maternal-embryo cross talk . The increasing knowledge about stage-specific ncRNA profiles during fetal development is expected to take a step forward in improving assisted reproductive technology (ART) programs as well . Various studies have shown that a differential expression of certain miRNAs was associated with implantation failure . Moreover, an aberrant expression of miRNAs has been observed in reproductive cells/tissues in the context of infertility in humans, both in females  and males , indicating a fundamental role of these ncRNAs in the de-regulation of the reproductive system and early embryogenesis ( Table 2 ).
Female ♀ vs. Male ♂ Fetus
|Cryopreserved human embryos (day 5 of development)||miRNAs||♀ (↑) miR-182, miR-206, miR-500, miR-601, miR-604, miR-875-5p
♂ (↑) miR-140-5p, miR-149, miR-151-5p, miR-26b, miR-31, miR-362-3p, miR-512-3p, miR-512-5p, miR-518d-5p, miR-518e, miR-525-3p, miR-886-3p, miR-886-5p, miR-92a
|♀♂ Embryo development, cell cycle, and apoptosis|||
|Mouse embryonic stem cells||miRNAs||♂ (↑) miR-302a, miR-302b, miR-302c, miR-302d, at differentiation day 5
♂ (↓) miR-302, at differentiation day 10
|♀♂ Chromatin remodeling;
proliferation, differentiation, and cell fate determination
|Human feto-placental endothelial cells from healthy pregnancies||miRNAs||♀ (↑) miR-23a-3p, miR-222-5p, miR-181a-3p, miR-151a-3p, miR-4286||♀♂ Endothelial barrier function: adherent junction, ECM receptor interaction, and focal adhesion;|||
|♂ (↑) miR-29b-3p, miR-15b-5p, miR-431-5p, miR345-5p||♂ cells have increased barrier resistance compared with female cells|
↑ Increases; ↓ decreases; ♀ female; ♂ male; ECM, extracellular matrix; miRNA, microRNA; ncRNA, non-coding RNA.
Indeed, different miRNA profiles seem to reflect embryonic ploidy status (i.e., euploid vs. aneuploid), as well as sexual dimorphism, in human blastocysts ( Table 2 ). It has been shown that miR-140-5p, miR-149, miR-151-5p, miR-26b, miR-31, miR-362-3p, miR-512-3p, miR-512-5p, miR-518d-5p, miR-518e, miR-525-3p, miR-886-3p, miR-886-5p, miR-92a, and RNU48 were highly expressed in cryopreserved human male embryos (day 5 of development) from in vitro fertilization (IVF), and miR-182, miR-206, miR-500, miR-601, miR-604, and miR-875-5p were highly expressed in the female ones. Genes targeted by these miRNAs belong to several pathways that are essential for embryo development, cell cycle, and apoptosis, indicating the importance of ncRNAs in these processes and their possible role as diagnostic biomarkers of infertility and prognostic tools of embryo development .
In another complex study, sex-specificity was identified in miRNA expression in mouse embryonic stem cells (ES) at different day (D) of differentiation (i.e., D0, D2, D5). Specifically, the authors highlighted that the miR-302 genomic cluster (i.e., miR-302a, miR-302b, miR-302c, miR-302d) was more expressed in male ES at D5, compared with the female. The authors identified as putative miRNA targets the chromatin remodeling and the E2F-dependent transcription repressors Ari4a and Arid4b, involved in diverse cellular processes including proliferation, differentiation, and cell fate determination. Decreased miR-302 level at D10 in male ES suggested a specific role of this miRNA during a narrow window of male development . By using next-generation miRNA sequencing, sex-dependent miRNA expression patterns were identified in feto-placental endothelial cells (fpEC) obtained from healthy pregnancies. Nine miRNAs were differentially expressed in male and female fpEC; specifically, miR-29b-3p, miR-15b-5p, miR-431-5p, miR345-5p, were overexpressed in male, and miR-23a-3p, miR-222-5p, miR-181a-3p, miR-151a-3p, miR-4286 were overexpressed in female. The analysis of the potential function of these miRNAs indicated their involvement in endothelial barrier function regulation. Specifically, they modulated adherent junctions, extracellular matrix (ECM) receptor interactions, and focal adhesion, eventually suggesting that male cells have increased barrier resistance compared with female ones. The authors hypothesized that the distinct features of endothelial cells already in utero can have a role in sexual dimorphism of endothelial dysfunction and cardiovascular disease in male versus female . However, the advance in high-throughput sequencing technology, together with sexually dimorphic gene expression analysis, represent valuable tools for explaining the basis of sex differences in fetal programming and development and in the susceptibility to diseases.