Open Access

Changes in sex ratio from fertilization to birth in assisted-reproductive-treatment cycles

  • Juan J Tarín1Email author,
  • Miguel A García-Pérez2,
  • Carlos Hermenegildo3 and
  • Antonio Cano4
Reproductive Biology and Endocrinology201412:56

https://doi.org/10.1186/1477-7827-12-56

Received: 7 April 2014

Accepted: 10 June 2014

Published: 23 June 2014

Abstract

Background

In Western gender-neutral countries, the sex ratio at birth is estimated to be approximately 1.06. This ratio is lower than the estimated sex ratio at fertilization which ranges from 1.07 to 1.70 depending on the figures of sex ratio at birth and differential embryo/fetal mortality rates taken into account to perform these estimations. Likewise, little is known about the sex ratio at implantation in natural and assisted-reproduction-treatment (ART) cycles. In this bioessay, we aim to estimate the sex ratio at fertilization and implantation using data from embryos generated by standard in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in preimplantation genetic diagnosis cycles. Thereafter, we compare sex ratios at implantation and birth in cleavage- and blastocyst-stage-transfer cycles to propose molecular mechanisms accounting for differences in post-implantation male and female mortality and thereby variations in sex ratios at birth in ART cycles.

Methods

A literature review based on publications up to December 2013 identified by PubMed database searches.

Results

Sex ratio at both fertilization and implantation is estimated to be between 1.29 and 1.50 in IVF cycles and 1.07 in ICSI cycles. Compared with the estimated sex ratio at implantation, sex ratio at birth is lower in IVF cycles (1.03 after cleavage-stage transfer and 1.25 after blastocyst-stage transfer) but similar and close to unity in ICSI cycles (0.95 after cleavage-stage transfer and 1.04 after blastocyst-stage transfer).

Conclusions

In-vitro-culture-induced precocious X-chromosome inactivation together with ICSI-induced decrease in number of trophectoderm cells in female blastocysts may account for preferential female mortality at early post-implantation stages and thereby variations in sex ratios at birth in ART cycles.

Keywords

Blastocyst-stage transfer Cleavage-stage transfer Preimplantation embryo development Sex ratio X-chromosome inactivation

Background

In Western gender-neutral countries, the sex ratio at birth is estimated to be ≈ 1.06 (for a review, see Hesketh and Xing [1]). This ratio is lower than the estimated sex ratio at fertilization which ranges from 1.07 to 1.70 depending on the figures of sex ratio at birth and differential embryo/fetal mortality rates taken into account to perform these estimations (for a review, see Pergament et al. [2]). Likewise, little is known about the sex ratio at implantation in natural and assisted-reproduction-treatment (ART) cycles. Nonetheless, implantation is a critical process that many embryos do not get through and, therefore, this event should be considered as important as fertilization or birth when analyzing changes in sex ratio through different stages of embryo/fetus development.

Fortunately, data from embryos generated by standard in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in preimplantation genetic diagnosis (PGD) cycles may be used to estimate not only the sex ratio at fertilization (primary sex ratio) in a more accurate way than previous studies (for a review, see Pergament et al. [2]) but also the still-unknown sex ratio at implantation. In this bioessay, we use data from IVF and ICSI embryos analyzed in PGD cycles as a proxy for estimating the sex ratio at both fertilization and implantation. Thereafter, we compare the sex ratios at implantation and birth (secondary sex ratio) in cleavage- and blastocyst-stage-transfer cycles to propose molecular mechanisms accounting for differences in post-implantation male and female mortality and thereby variations in sex ratios at birth in ART cycles.

Methods

A literature review based on publications up to December 2013 identified by PubMed database searches using the following key words: sex ratio, preimplantation genetic diagnosis, cleavage-stage transfer, blastocyst-stage transfer, IVF, ICSI, biochemical pregnancy, fetal mortality, X-chromosome inactivation (XCI). This literature search retrieved a limited number of studies and put in evidence the absence of well-designed controlled randomized trials analyzing the concomitant effect of both insemination technique (IVF versus ICSI) and developmental stage at the time of embryo biopsy/transfer (cleavage versus blastocyst stage) on sex ratio of embryos/newborns. Notably, only one article [3] compiling the chromosomal sex of 117 IVF 4- to 8-cell embryos from PGD cycles was identified in our literature search. This is not surprising because during the early nineties, before the advent of ICSI, PGD technology was in its infancy, and patients and PGD laboratories were limited. For instance, the article by Griffin et al. [3] is a compendium of 27 PGD cycles performed in 4 separated series at the Hammersmith Hospital, London, over a 2-year period in 18 couples at risk of transmitting X-linked recessive disorders. Oocytes and embryos were cultured in Earle’s Balanced Salt Solution (EBSS) supplemented with 10% heat-inactivated maternal serum and biopsied blastomeres analyzed by fluorescent in situ hybridization (FISH). Consequently, estimates of sex ratios at fertilization and implantation based on data shown in Table 1 should be considered as relative values, not as absolute and precise figures. Estimates of sex ratios at birth from Table 2 are based on larger sample sizes and therefore are more robust than estimates of sex ratios at fertilization and implantation in IVF cycles. In any case, comparisons between groups in this bioessay should be performed in a qualitative way, not in a quantitative/statistical mode using meta-analysis or statistical inference methods.
Table 1

Sex ratio (XY/XX) of genetically diagnosed preimplantation embryos according to the method of fertilization applied and embryo developmental stage

Method of fertilization

Four- to 8-cell embryosa

Day-5 blastocysts

References

IVF

1.50b (60/40)

 

[3]

 

Total: 1.50b (60/40)

  

ICSI

0.88 (123/140)

 

[4]

  

0.98 (225/229)

[30]

 

1.18 (741/629)

 

[14]

 

0.86 (96/112)

 

[7]

 

Total: 1.09 (960/881)

Total: 0.98 (225/229)

 

aIn the IVF group, a total of 25 (17 males and 8 females) and 75 (43 males and 32 females) embryos were analyzed at the 4- and 8-cell stage, respectively. In the ICSI group, all the embryos were analyzed at the 8-cell stage.

bSex ratio of 4- to 8-cell embryos would be 1.29 (66/51) if we consider 17 extra embryos that exhibited abnormal number of X and Y signals in the biopsied cell(s).

Table 2

Sex ratio (XY/XX) at birth of singleton deliveries according to the method of fertilization applied and the day of embryo transfer

Method of fertilization

Day of embryo transfer

Sex ratio

Referencesa

IVF

≤ Day 3b

0.98 (1929/1968)

[23]

  

1.08 (2084/1932)

[24]

 

Total: ≤ day 3b

1.03 (4013/3900)

 
 

> Day 3c

1.22 (1030/846)

[23]

  

1.28 (1088/852)

[24]

 

Total: > day 3c

1.25 (2118/1698)

 

ICSI

≤ Day 3b

0.94 (3047/3236)

[23]

  

0.95 (2414/2542)

[24]

 

Total: ≤ day 3b

0.95 (5461/5778)

 
 

> Day 3c

0.98 (1542/1566)

[23]

  

1.10 (1289/1167)

[24]

 

Total: > day 3c

1.04 (2831/2733)

 

aLarge-sample surveys using United States [23] and Australia and New Zealand [24] assisted reproductive databases.

bCleavage-stage transfer.

cBlastocyst-stage transfer.

Fertilization and preimplantation stages

It has been reported that human ejaculated spermatozoa display a normal Y:X ratio that does not differ from the Mendelian ratio [46]. Nevertheless, Table 1 shows that genetically diagnosed 4- to 8-cell IVF embryos exhibit sex ratios between 1.29 and 1.50. These figures contrast with the sex ratio closer to unity displayed by ICSI 8-cell embryos (1.09). Differences in sex ratios between IVF and ICSI embryos may be due to the fact that ICSI bypasses the zona pellucida and thereby any putative role it may have in selecting X- or Y-bearing spermatozoa (see below). Nevertheless, we should note that the sex ratio of cleavage-stage ICSI embryos is biased towards females when performing sperm selection for normal shaped nuclei, especially under high magnification (0.53, 112/210, in selected sperm injection versus 0.86, 96/112, in standard ICSI) [7] or when using the swim-up technique for preparation of spermatozoa from heavy smokers (0.47, 22/47, in heavy smokers; 0.95, 21/22, in slight-to-moderate smokers; and 1.13, 80/71, in non-smokers) [4].

There are several mechanisms that may account for the relatively elevated sex ratio found in IVF 4- to 8-cell embryos: (i) IVF male embryos may have a developmental advantage over female embryos after fertilization; (ii) the sperm preparation technique (either swim-up or three-layer discontinuous Percoll density gradient centrifugation) used in IVF may increase the proportion of Y-bearing spermatozoa; (iii) the molecular composition of the zona pellucida may render oocytes more susceptible to fertilization by Y-bearing spermatozoa; and/or (iv) Y-bearing spermatozoa may have higher fertilization ability.

Previous studies have reported that the sex ratio of preimplantation bovine embryos may be skewed towards males (i.e., preferential loss of female embryos) by manipulating the culture system including addition of glucose [8, 9] and glucosamine [10]. In contrast, in humans the possibility that IVF male embryos have a developmental advantage over female embryos after fertilization is not supported by data on preimplantation embryo development. Firstly, it is known that ≈ 10% of all human IVF (or ICSI) embryos undergo early developmental arrest [11]. This arrest likely occurs to prevent further development of certain chromosomally abnormal embryos and/or embryos that fail to activate embryonic genome around the 4- to 8-cell stage [12]. Of note, this early developmental block does not seem to depend on sex of embryos. Actually, a non-significant sex ratio of 1.05 (86/82) has been evidenced in arrested embryos that do not pass the 8-cell stage after IVF [13]. And secondly, as shown in Table 1, the sex ratio of both ICSI 8-cell embryos (1.09) and day-5 blastocysts (0.98) is close to unity suggesting that further developmental arrest after the 8-cell stage is not sex dependent. Indeed, the developmental potential of ICSI 8-cell embryos towards the early, full or hatched-blastocyst stage on day 5 is similar between male (23.1%, 110/475) and female (21.6%, 88/408) embryos [14]. Consequently, we can assume that the sex ratio at both fertilization and implantation is between 1.29 and 1.50 in IVF cycles (the sex ratio of cleavage-stage embryos) and 1.07, 1185/1110, in ICSI cycles (this estimate results from combining sex ratios of cleavage-stage and blastocyst-stage ICSI embryos; see Table 1). We should note that the estimates of sex ratios at fertilization and implantation in IVF cycles are not robust due to the relative small number of embryos analyzed (n = 117) and the bias that may be introduced by inferring sex ratios at fertilization and implantation from data of cleavage-stage embryos. We should bear in mind the work by Fiala [15] pointing out that the sex ratio of surviving offspring cannot correctly be used to estimate the primary sex ratio because of the potential sex differential of mortality. Unfortunately, obvious ethical reasons prevent assessing directly sex ratios at fertilization and implantation in human beings.

The second option, i.e., the sperm preparation technique used in IVF may increase the proportion of Y-bearing spermatozoa, can be also rejected. In fact, it has been shown that the swim-up technique does not selectively enrich either X- or Y-bearing spermatozoa [1618]. As mentioned above, only in heavy smoking men swim-up technique may increase the proportion of X-bearing (instead of Y-bearing) spermatozoa resulting in higher incidence of female embryos after ICSI [4]. Moreover, it is known that the three-layer discontinuous Percoll density gradient selects spermatozoa with better motion characteristics, more hyperactivation, and improved longevity compared with direct swim-up [19]. However, studies aimed to ascertain the efficiency of discontinuous Percoll density gradient centrifugation in sperm sorting show either no significant effect on X:Y ratio of spermatozoa or even an enrichment of X-bearing spermatozoa that seems to be insufficient for clinical use in pre-conceptional sex selection (for references, see Lin et al. [20]).

The third and fourth possibilities, i.e., oocytes may be more susceptible to fertilization by Y-bearing spermatozoa and/or Y-bearing spermatozoa may have higher fertilization ability, are more likely to be true. Indeed, recent evidence strongly suggests that oocytes during a critical time in folliculogenesis may change the molecular composition of the zona pellucida, e.g., a subtle variation in a sperm-binding carbohydrate on the zona-pellucida proteins induced by high levels of follicular-fluid testosterone. This molecular change may render oocytes more susceptible to fertilization by Y-bearing spermatozoa (for a review, see Grant and Chamley [21]). In addition, there are convincing data on the presence of distorter genes, expressed and translated after meiosis in round spermatids and spermatozoa, able to skew the sex ratio by affecting spermatid maturation and fertilizing ability of either X- or Y-bearing spermatozoa (for a review, see Ellis et al. [22]). This fact suggests that human spermatids and spermatozoa may “intrinsically” express distorter genes favoring spermatid maturation and fertilizing ability of Y-bearing spermatozoa.

Implantation and early post-implantation stages before pregnancy becomes clinically recognized

Table 2 shows data retrieved from United States [23] and Australia and New Zealand [24] assisted reproductive databases. We selected these studies because they focused their analyses on large samples of ART singleton deliveries [23] or births resulting from single embryo transfers [24]. Noteworthy, Dean et al. [24] included in the calculation and analysis of sex ratio at birth only one baby from each set of multiple births. This strategy eliminated the potential bias that monozygotic twins may introduce in the calculation of sex ratio at birth. These data indicate that extended embryo culture to the blastocyst stage is associated with higher sex ratio at birth compared with shorter embryo culture to the 4- or 8-cell stage (1.25 versus 1.03 in IVF cycles and 1.04 versus 0.95 in ICSI cycles). Moreover, sex ratio at birth is lower in ICSI cycles than in IVF cycles after cleavage- (0.95 versus 1.03) and blastocyst-stage (1.04 versus 1.25) transfer. These results are qualitatively consistent with a previous systematic review and meta-analysis [25] and previous studies [2629] not included in Table 2 because they did not provide the appropriate information and/or did not control for the potential bias associated with monozygotic twining.

The higher sex ratio at birth evidenced after blastocyst-stage transfer is not likely a consequence of embryo grading systems that prioritize male embryos for transfer as suggested by Alfarawati et al. [30]. Indeed, despite an early study [31] reported that male IVF human preimplantation embryos display increased number of cells and metabolic activity than female embryos, strong evidence shows that human preimplantation male embryos do not cleave faster [3234], exhibit better morphology [32] and/or have higher developmental potential [13, 14] than female embryos. This fact suggests that the human endometrium does not select the sex of implanting embryos as previously hypothesized by Krackow [35] and Tarín et al. [36], or evidenced in mouse embryos displaying sex-dimorphic developmental rates [37, 38]. Instead, we propose that the higher secondary sex ratio found after blastocyst-stage transfer may be due to preferential female mortality at early post-implantation stages induced, at least in part, by abnormal inactivation of one of the two X-chromosomes (mechanism of dosage compensation).

XCI in the mouse model

Two recent reviews by Lee and Bartolomei [39] and Lessing et al. [40] show that in the mouse XCI begins during the first meiotic prophase of spermatogenesis. After completion of meiosis, the X-chromosome does not completely reactivate. Indeed, 85% of X-linked genes remain suppressed through spermiogenesis. Thus, the paternal X-chromosome is passed onto the next generation in a partially inactivated state. At the 2-cell stage, transcription of repetitive elements on the paternal X-chromosome is already suppressed, but transcription of X-linked coding genes is active. At the 8-16-cell stage (morula stage), the silencing of paternal coding genes is initiated, and is completed at the blastocyst stage or later. Gene silencing absolutely requires cis accumulation of a long non-coding Xist RNA that coats the X-chromosome and binds Polycomb repressive complex 2 (PRC2), the epigenetic complex responsible for trimethylation of histone H3 on lysine 27 (H3K27me3), a repressive epigenetic mark that leads to further silencing of the paternal X-chromosome. This is not the case for silencing repetitive elements on the paternal X-chromosome. By the 2-cell stage, although Xist RNA is present, repetitive elements are silenced in a Xist independent manner. The maternal X-chromosome is protected from inactivation through expression of Xist’s antisense repressor, Tsix.

As paternal XCI is heritable through mitosis, the paternal X-chromosome remains inactivated in both the trophectoderm and the primitive endoderm (hypoblast). In contrast, in the inner cell mass (ICM), the paternal X-chromosome undergoes reactivation. We should bear in mind that the trophectoderm gives rise to the fetal portion of the placenta; the primitive endoderm originates the parietal endoderm that contributes to the parietal yolk sac, and the visceral endoderm that contributes to the visceral and intraplacental yolk sacs; and the ICM gives rise to the epiblast lineage that develops into the embryo proper and the extra-embryonic mesoderm that forms the allantois and the mesodermal components of the visceral yolk sac, amnion and chorion (for reviews, see Hemberger [41] and Gasperowicz and Natale [42]).

Starting from the period shortly after implantation, X-chromosomes in the epiblast experience random inactivation, i.e., the maternal X-chromosome is inactive in some cells whereas the paternal X-chromosome is inactive in other cells. Paternal X-chromosome reactivation also occurs in primordial germ cells in preparation for equal segregation during meiosis (for reviews, see Lee and Bartolomei [39] and Lessing et al. [40]).

XCI in humans

Unlike in mice, XIST expression is not imprinted in humans. XIST expression is detected from the 4- to 8-cell stage at the onset of genomic activation [43]. Both ICM and trophectoderm show similar XIST RNA accumulation in their cells. However, XIST upregulation does not result in immediate onset of chromosome-wide XCI even in late (day-7) blastocysts [44]. Recently, Teklenburg et al. [45] using an in-vitro model for human implantation observed that implanting day-8 female embryos had distinct H3K27me3 foci (presumably on the inactive X-chromosome) localized to the trophectoderm lineages and to lesser extend the hypoblast lineages, but not in epiblast cells. These findings indicate that in the majority of the cells of human embryos, silencing of the X-chromosome may occur after the embryo has implanted. This conclusion contradicts data from another study reporting that XIST RNA accumulation is associated with transcriptional silencing of the XIST-coated chromosomal region as early as the morula and the blastocyst stage [43]. Discrepancies between studies may be explained by differences in efficiency of the immunofluorescence/FISH technique in detecting biallelic RNA signals and/or the use of different culture conditions (cited by Okamoto et al. [44]).

Early studies suggested the occurrence of paternal XCI in the fetal side of placentae. These studies analyzed the expression pattern of single X-linked genes. However, other studies using more robust analyses of multiple allele-specific gene expression along the X-chromosome support the notion that XCI in human placentae is random (for a review, see Lee and Bartolomei [39]). Similarly, it is generally accepted that X-chromosomes in the ICM lineage undergo random inactivation (for a review, see Migeon [46]). Notwithstanding, a recent study has shown that the bell-shaped distribution (centered around 50%) of X-inactivation patterns in large populations of normal women fits better a three-allele model of genetically influenced XCI than models of completely random inactivation [47].

We should emphasize that not all the X-linked genes are silenced at X-inactivation. In humans, more than 15% of genes carried on the X-chromosome appear to escape inactivation (for a review, see Brown and Greally [48]). Consequently, differences in gene dosage may explain differences between men and women in developmental programming and disease susceptibility and behavior (for a review, see Aiken and Ozanne [49]). Moreover, although XCI in human epiblast, hypoblast and trophectoderm cells likely occurs during/after implantation (see above), the silencing process may be disrupted during preimplantation stages by any factor that interferes with DNA methylation, histone deacetylation or chromatin modifications. The resulting increased or decreased X-linked gene expression may prevent embryos to either implant or develop normally after implantation (for reviews, see Hemberger [50] and Schulz and Heard [51]). We propose that extended exposure of preimplantation female embryos to suboptimal (non-physiological) culture systems may be “one” of these factors.

Precocious XCI in human embryonic stem cells (hESCs)

It has been reported [52] that the conventional method of hESCs (pluripotent cell types derived from the ICM of human blastocysts) derivation and maintenance under atmospheric O2 conditions (≈20% O2) as well as exposure to other cellular stresses such as harsh freeze-thaw cycles, inhibition of the proteosome, HSP90, gamma-glutamylcysteine synthetase, and treatment with organic peroxide, induces precocious random XCI prior to cellular differentiation. This precocious XCI is associated with either XIST expression in most or all cells, or the absence of XIST expression and failure to reactive XIST expression upon differentiation. This response differs from that found under 5% O2 concentration. In this case, the precocious random XCI in hESCs is prevented, being both X-chromosomes active. Furthermore, hESCs exhibit no XIST expression and retain the ability to activate XIST gene expression upon differentiation.

It is worth mentioning that nowadays in many IVF laboratories gametes and embryos are still exposed to non-physiological culture systems including atmospheric O2 concentrations despite data from a systematic review and meta-analysis [53] suggest that embryo culture to the blastocyst stage under low-oxygen concentration (≈5%) versus high-oxygen atmospheric concentration yields higher live birth rates. Thus, it can be inferred that embryos cultured to the blastocyst stage (embryo transfer on day 5 or 6) under non-physiological environments including atmospheric O2 concentrations are more susceptible to undergo epigenetic changes than embryos cultured for shorter periods of time (embryo transfer on ≤ day 3). Like hESCs, these epigenetic changes may interfere with the normal process of XIST expression and XCI in female embryos. Importantly, in-vitro-produced preimplantation bovine embryos display higher levels of XIST expression than their in-vivo counterparts, suggesting that in-vitro-culture conditions induce premature XCI [54].

We should stress that in the subgroup of hESC lines displaying precocious XCI and XIST expression in most or all cells when exposed to atmospheric O2 conditions [52], XIST expression was unstable and subject to stable epigenetic silencing by DNA methylation. The resulting inhibition of XIST expression reactivated a portion of X-linked alleles on the inactive X-chromosome (12% of X-linked promoter CpG islands became hypomethylated) [55]. Such a reactivation resulted in over-expression of X-linked genes, event that if took place in implanting female blastocysts may produce severe abnormalities in embryonic and extra-embryonic (trophoblast) tissues and early embryonic death (for a review, see Schulz and Heard [51]).

Data supporting and refuting the hypothesis of occurrence of precocious XCI in human female embryos

The hypothesis of occurrence of precocious XCI in female embryos exposed for extended periods of time to non-physiological culture systems is questioned by (i) the absence of significant differences in miscarriage percentage per couple after cleavage- (8.0%, 86/1069) and blastocyst-stage (9.2%, 97/1058) transfer; and (ii) the higher live-birth percentage per couple after blastocyst-stage transfer (38.9%, 292/751, versus 31.2%, 237/759, after cleavage-stage transfer) (for a systematic review and meta-analysis, see Glujovsky et al. [56]). As a matter of fact, we should expect higher miscarriage percentages and lower live-birth percentages after blastocyst-stage transfer if a given percentage of female embryos undergoes precocious XCI. However, it is generally thought that extended culture selects those embryos that have proven ability to survive and develop to an advanced stage in vitro [although a wide range of blastulation rates has been reported (from 28% to 97%), on average only 46.8% of embryos reach the blastocyst stage (for a systematic review and meta-analysis, see Glujovsky et al. [56])]. This fact together with the presence of an uterine environment that likely is more synchronized compared with cleavage-stage transfers ([57]; for a review, see Bourgain and Devroey [58]) may contribute to the similar miscarriage rates and higher live-birth percentages reported after blastocyst-stage transfer compared with cleavage-stage transfer.

In addition, the incidence of female losses (presumably caused by precocious XCI) is likely higher at early stages of pregnancy before women are aware that they are pregnant than after pregnancy has been clinically recognized (note that early pregnancy losses are not taken into account when analyzing miscarriage percentages). In this context, we should mention that blastocyst-stage transfer is associated with higher percentage of biochemical pregnancy losses per embryo transfer (14.1%, 108/767) [59] than cleavage-stage transfer (8.2%, 154/1888) [60].

Late post-implantation stages after pregnancy becomes clinically recognized

Shortly after pregnancy becomes clinically recognized, females keep displaying a developmental disadvantage compared with males. This disadvantage subsequently vanishes as gestational age increases. In particular, by combining the data reported by Eiben et al. [61] and Yusuf and Naeem [62], sex ratios of chromosomally normal abortions increase from 0.46, 67/147, at 5–9 weeks of pregnancy to 0.79, 137/173, at 10–13 weeks and 1.02, 269/263, at ≥ 13 weeks. A concomitant increase in natural selection against males with gestational age is also evidenced in chorionic villus sampling and amniocentesis material from control pregnant women. In these ongoing pregnancies, sex ratios significantly decrease from 1.28, 791/618, at < 16 weeks of pregnancy to 1.06, 25433/23994, at ≥ 16 weeks [63]. We should bear in mind that human males and females develop at different rates in uterus (and postnatally until the postpubertal stage). Thus, male fetuses have a greater effective exposure to a given insult than female fetuses that undergo fewer cell cycles during the same period of exposure (for a review, see Aiken and Ozanne [49]).

Birth

Table 2 shows that, compared with the estimated sex ratio at implantation (1.29 to 1.50 in IVF cycles and 1.07 in ICSI cycles), the sex ratio at birth is lower in IVF cycles (1.03 and 1.25 after cleavage- and blastocyst-stage transfer, respectively) but similar and closer to unity in ICSI cycles (0.95 and 1.04 after cleavage- and blastocyst-stage transfer, respectively). Note that we should expect lower sex ratios at birth than at implantation if male mortality during pregnancy surpasses female losses. On the contrary, we should expect sex ratios at birth similar to or even higher than sex ratios at implantation if female mortality is comparable or exceeds male mortality.

We should stress that sex ratios at birth are closer to sex ratios at implantation after blastocyst-stage-transfer than after cleavage-stage-transfer. This fact is in consonance with the hypothesis of occurrence of precocious XCI in female embryos cultured in vitro to the blastocyst stage. Likewise, sex ratios at birth are nearer to sex ratios at implantation in ICSI than in IVF cycles. In this context, we should mention the study by Dumoulin et al. [64] reporting decreased number of trophectoderm cells in ICSI female blastocysts compared with ICSI male blastocysts (this effect was not observed in IVF blastocysts). As the trophectoderm lineage gives rise to the fetal portion of the placenta, ICSI female blastocysts may exhibit higher incidence of abnormal trophoblast function and decreased potential for implantation and further development compared with ICSI male blastocysts.

Concluding remarks

Data from genetically diagnosed preimplantation embryos suggest that the sex ratio at both fertilization and implantation is between 1.29 and 1.50 in IVF cycles and 1.07 in ICSI cycles. Embryo exposure to culture media for extended periods of time to the blastocyst stage under non-physiological conditions (e.g., under atmospheric O2 conditions) may induce precocious XCI in female embryos. Such a precocious XCI together with ICSI-induced decrease in number of trophectoderm cells in female blastocysts may account for preferential female mortality at early post-implantation stages and thereby variations in sex ratios at birth in ART cycles. In particular, in IVF cycles the early developmental disadvantage of females would be surpassed by the higher mortality rates of males later in pregnancy resulting in lower sex ratios at birth than at implantation. In contrast, in ICSI cycles early female mortality would be comparable to later male mortality affording similar sex ratios at birth and implantation. Blastocyst transfer in both IVF and ICSI cycles would be associated with higher post-implantation female mortality than cleavage-stage transfer. Consequently, sex ratios at birth would be closer to sex ratios at implantation after blastocyst transfer than after cleavage-stage transfer.

The hypothesis of precocious XCI may be extended to natural cycles to explain, at least in part, some biases of sex ratio at birth observed in human populations/families (for reviews, see James [65, 66]). In particular, disturbances of XCI may be induced by biological (e.g., gametes from reproductive-old women/men and pre- or post-ovulation/ejaculation aged gametes) or environmental (e.g., maternal exposure to nutritional deficits/excesses, physical/psychological/social stresses, medications, social drugs, radiations, environmental pollutants and chemotherapy agents) factors. Certainly, this is a research area that needs further attention.

Abbreviations

5mC: 

Fifth carbon of the cytosine base

ART: 

Assisted reproduction treatment

EBSS: 

Earle’s balanced salt solution

FISH: 

Fluorescent in situ hybridization

H3K27me3: 

Histone H3 on lysine 27

hESCs: 

Human embryonic stem cells

ICSI: 

Intracytoplasmic sperm injection

IVF: 

In-vitro fertilization

PRC2: 

Polycomb repressive complex 2

XCI: 

X-chromosome inactivation.

Declarations

Authors’ Affiliations

(1)
Department of Functional Biology and Physical Anthropology, Faculty of Biological Sciences, University of Valencia
(2)
Department of Genetics, Faculty of Biological Sciences, University of Valencia, Burjassot,and Research Unit-INCLIVA, Hospital Clínico de Valencia
(3)
Department of Physiology, Faculty of Medicine, University of Valencia, and Research Unit-INCLIVA, Hospital Clínico de Valencia
(4)
Department of Pediatrics, Obstetrics and Gynecology, Faculty of Medicine, University of Valencia, and Service of Obstetrics and Gynecology, University Hospital Dr. Peset

References

  1. Hesketh T, Xing ZW: Abnormal sex ratios in human populations: causes and consequences. Proc Natl Acad Sci U S A. 2006, 103: 13271-13275.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Pergament E, Toydemir PB, Fiddler M: Sex ratio: a biological perspective of ‘Sex and the City’. Reprod Biomed Online. 2002, 5: 43-46.View ArticlePubMedGoogle Scholar
  3. Griffin DK, Handyside AH, Harper JC, Wilton LJ, Atkinson G, Soussis I, Wells D, Kontogianni E, Tarín J, Geber S, Ao A, Winston RML, Delhanty JDA: Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization. J Assist Reprod Genet. 1994, 11: 132-143.View ArticlePubMedGoogle Scholar
  4. Viloria T, Rubio MC, Rodrigo L, Calderon G, Mercader A, Mateu E, Meseguer M, Remohi J, Pellicer A: Smoking habits of parents and male: female ratio in spermatozoa and preimplantation embryos. Hum Reprod. 2005, 20: 2517-2522.View ArticlePubMedGoogle Scholar
  5. Bowman M, De Boer K, Cullinan R, Catt J, Jansen R: Do alterations in the sex ratio occur at fertilization? a case report using fluorescent in situ hybridization. J Assist Reprod Genet. 1998, 15: 320-322.PubMed CentralView ArticlePubMedGoogle Scholar
  6. Graffelman J, Fugger EF, Keyvanfar K, Schulman JD: Human live birth and sperm-sex ratios compared. Hum Reprod. 1999, 14: 2917-2920.View ArticlePubMedGoogle Scholar
  7. Setti AS, Figueira RC, Braga DP, Iaconelli A, Borges E: Gender incidence of intracytoplasmic morphologically selected sperm injection-derived embryos: a prospective randomized study. Reprod Biomed Online. 2012, 24: 420-423.View ArticlePubMedGoogle Scholar
  8. Gutiérrez-Adán A, Granados J, Pintado B, De La Fuente J: Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro. Reprod Fertil Dev. 2001, 13: 361-365.View ArticlePubMedGoogle Scholar
  9. Kimura K, Spate LD, Green MP, Roberts RM: Effects of D-glucose concentration, D-fructose, and inhibitors of enzymes of the pentose phosphate pathway on the development and sex ratio of bovine blastocysts. Mol Reprod Dev. 2005, 72: 201-207.View ArticlePubMedGoogle Scholar
  10. Kimura K, Iwata H, Thompson JG: The effect of glucosamine concentration on the development and sex ratio of bovine embryos. Anim Reprod Sci. 2008, 103: 228-238.View ArticlePubMedGoogle Scholar
  11. Betts DH, Madan P: Permanent embryo arrest: molecular and cellular concepts. Mol Hum Reprod. 2008, 14: 445-453.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Braude P, Bolton V, Moore S: Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature. 1988, 332: 459-461.View ArticlePubMedGoogle Scholar
  13. Munné S, Tang YX, Weier HU, Stein J, Finkelstein M, Grifo J, Cohen J: Sex distribution in arrested precompacted human embryos. Zygote. 1993, 1: 155-162.View ArticlePubMedGoogle Scholar
  14. Ben-Yosef D, Amit A, Malcov M, Frumkin T, Ben-Yehudah A, Eldar I, Mey-Raz N, Azem F, Altarescu G, Renbaum P, Beeri R, Varshaver I, Eldar-Geva T, Epsztejn-Litman S, Levy-Lahad E, Eiges R: Female sex bias in human embryonic stem cell lines. Stem Cells Dev. 2012, 21: 363-372.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Fiala KL: On estimating the primary sex ratio from incomplete data. Am Nat. 1980, 115: 442-444.View ArticleGoogle Scholar
  16. Han TL, Flaherty SP, Ford JH, Matthews CD: Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim-up. Fertil Steril. 1993, 60: 1046-1051.PubMedGoogle Scholar
  17. De Jonge CJ, Flaherty SP, Barnes AM, Swann NJ, Matthews CD: Failure of multitube sperm swim-up for sex preselection. Fertil Steril. 1997, 67: 1109-1114.View ArticlePubMedGoogle Scholar
  18. Yan J, Feng HL, Chen ZJ, Hu J, Gao X, Qin Y: Influence of swim-up time on the ratio of X- and Y-bearing spermatozoa. Eur J Obstet Gynecol Reprod Biol. 2006, 129: 150-154.View ArticlePubMedGoogle Scholar
  19. Moohan JM, Lindsay KS: Spermatozoa selected by a discontinuous percoll density gradient exhibit better motion characteristics, more hyperactivation, and longer survival than direct swim-up. Fertil Steril. 1995, 64: 160-165.PubMedGoogle Scholar
  20. Lin SP, Lee RK, Tsai YJ, Hwu YM, Lin MH: Separating X-bearing human spermatozoa through a discontinuous percoll density gradient proved to be inefficient by double-label fluorescent in situ hybridization. J Assist Reprod Genet. 1998, 15: 565-569.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Grant VJ, Chamley LW: Can mammalian mothers influence the sex of their offspring peri-conceptually?. Reproduction. 2010, 140: 425-433.View ArticlePubMedGoogle Scholar
  22. Ellis PJ, Yu Y, Zhang S: Transcriptional dynamics of the sex chromosomes and the search for offspring sex-specific antigens in sperm. Reproduction. 2011, 142: 609-619.View ArticlePubMedGoogle Scholar
  23. Luke B, Brown MB, Grainger DA, Baker VL, Ginsburg E, Stern JE, Society for Assisted Reproductive Technology Writing Group: The sex ratio of singleton offspring in assisted-conception pregnancies. Fertil Steril. 2009, 92: 1579-1585.View ArticlePubMedGoogle Scholar
  24. Dean JH, Chapman MG, Sullivan EA: The effect on human sex ratio at birth by assisted reproductive technology (ART) procedures–an assessment of babies born following single embryo transfers, Australia and New Zealand, 2002–2006. BJOG. 2010, 117: 1628-1634.View ArticlePubMedGoogle Scholar
  25. Chang HJ, Lee JR, Jee BC, Suh CS, Kim SH: Impact of blastocyst transfer on offspring sex ratio and the monozygotic twinning rate: a systematic review and meta-analysis. Fertil Steril. 2009, 91: 2381-2390.View ArticlePubMedGoogle Scholar
  26. Ericson A, Källén B: Congenital malformations in infants born after IVF: a population-based study. Hum Reprod. 2001, 16: 504-509.View ArticlePubMedGoogle Scholar
  27. Bonduelle M, Liebaers I, Deketelaere V, Derde MP, Camus M, Devroey P, Van Steirteghem A: Neonatal data on a cohort of 2889 infants born after ICSI (1991–1999) and of 2995 infants born after IVF (1983–1999). Hum Reprod. 2002, 17: 671-694.View ArticlePubMedGoogle Scholar
  28. Fedder J, Loft A, Parner ET, Rasmussen S, Pinborg A: Neonatal outcome and congenital malformations in children born after ICSI with testicular or epididymal sperm: a controlled national cohort study. Hum Reprod. 2013, 28: 230-240.View ArticlePubMedGoogle Scholar
  29. Maalouf WE, Mincheva MN, Campbell BK, Hardy IC: Effects of assisted reproductive technologies on human sex ratio at birth. Fertil Steril. 2014, 101: 1321-1325.View ArticlePubMedGoogle Scholar
  30. Alfarawati S, Fragouli E, Colls P, Stevens J, Gutiérrez-Mateo C, Schoolcraft WB, Katz-Jaffe MG, Wells D: The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil Steril. 2011, 95: 520-524.View ArticlePubMedGoogle Scholar
  31. Ray PF, Conaghan J, Winston RM, Handyside AH: Increased number of cells and metabolic activity in male human preimplantation embryos following in vitro fertilization. J Reprod Fertil. 1995, 104: 165-171.View ArticlePubMedGoogle Scholar
  32. Richter KS, Anderson M, Osborn BH: Selection for faster development does not bias sex ratios resulting from blastocyst embryo transfer. Reprod Biomed Online. 2006, 12: 460-465.View ArticlePubMedGoogle Scholar
  33. Csokmay JM, Hill MJ, Cioppettini FV, Miller KA, Scott RT, Frattarelli JL: Live birth sex ratios are not influenced by blastocyst-stage embryo transfer. Fertil Steril. 2009, 92: 913-917.View ArticlePubMedGoogle Scholar
  34. Weston G, Osianlis T, Catt J, Vollenhoven B: Blastocyst transfer does not cause a sex-ratio imbalance. Fertil Steril. 2009, 92: 1302-1305.View ArticlePubMedGoogle Scholar
  35. Krackow S: The developmental asynchrony hypothesis for sex ratio manipulation. J Theor Biol. 1995, 176: 273-280.View ArticlePubMedGoogle Scholar
  36. Tarín JJ, Bernabeu R, Baviera A, Bonada M, Cano A: Sex selection may be inadvertently performed in in-vitro fertilization-embryo transfer programmes. Hum Reprod. 1995, 10: 2992-2998.PubMedGoogle Scholar
  37. Krackow S, Burgoyne PS: Timing of mating, developmental asynchrony and the sex ratio in mice. Physiol Behav. 1998, 63: 81-84.View ArticleGoogle Scholar
  38. Jiménez A, Fernández R, Madrid-Bury N, Moreira PN, Borque C, Pintado B, Gutiérrez-Adán A: Experimental demonstration that pre- and post-conceptional mechanisms influence sex ratio in mouse embryos. Mol Reprod Dev. 2003, 66: 162-165.View ArticlePubMedGoogle Scholar
  39. Lee JT, Bartolomei MS: X-inactivation, imprinting, and long noncoding RNAs in health and disease. Cell. 2013, 152: 1308-1323.View ArticlePubMedGoogle Scholar
  40. Lessing D, Lee JT: X chromosome inactivation and epigenetic responses to cellular reprogramming. Annu Rev Genomics Hum Genet. 2013, 14: 85-110.View ArticlePubMedGoogle Scholar
  41. Hemberger M: Epigenetic landscape required for placental development. Cell Mol Life Sci. 2007, 64: 2422-2436.View ArticlePubMedGoogle Scholar
  42. Gasperowicz M, Natale DR: Establishing three blastocyst lineages–then what?. Biol Reprod. 2011, 84: 621-630.View ArticlePubMedGoogle Scholar
  43. van den Berg IM, Laven JS, Stevens M, Jonkers I, Galjaard RJ, Gribnau J, van Doorninck JH: X chromosome inactivation is initiated in human preimplantation embryos. Am J Hum Genet. 2009, 84: 771-779.PubMed CentralView ArticlePubMedGoogle Scholar
  44. Okamoto I, Patrat C, Thépot D, Peynot N, Fauque P, Daniel N, Diabangouaya P, Wolf JP, Renard JP, Duranthon V, Heard E: Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature. 2011, 472: 370-374.View ArticlePubMedGoogle Scholar
  45. Teklenburg G, Weimar CH, Fauser BC, Macklon N, Geijsen N, Heijnen CJ, de Sousa Lopes SM C, Kuijk EW: Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation. PLoS One. 2012, 7: e32701-PubMed CentralView ArticlePubMedGoogle Scholar
  46. Migeon BR: Why females are mosaics, X-chromosome inactivation, and sex differences in disease. Gend Med. 2007, 4: 97-105.View ArticlePubMedGoogle Scholar
  47. Renault NK, Pritchett SM, Howell RE, Greer WL, Sapienza C, Orstavik KH, Hamilton DC: Human X-chromosome inactivation pattern distributions fit a model of genetically influenced choice better than models of completely random choice. Eur J Hum Genet. 2013, 21: 1396-1402.PubMed CentralView ArticlePubMedGoogle Scholar
  48. Brown CJ, Greally JM: A stain upon the silence: genes escaping X inactivation. Trends Genet. 2003, 19: 432-438.View ArticlePubMedGoogle Scholar
  49. Aiken CE, Ozanne SE: Sex differences in developmental programming models. Reproduction. 2013, 145: R1-13.View ArticlePubMedGoogle Scholar
  50. Hemberger M: The role of the X chromosome in mammalian extra embryonic development. Cytogenet Genome Res. 2002, 99: 210-217. ç45View ArticlePubMedGoogle Scholar
  51. Schulz EG, Heard E: Role and control of X chromosome dosage in mammalian development. Curr Opin Genet Dev. 2013, 23: 109-115.View ArticlePubMedGoogle Scholar
  52. Lengner CJ, Gimelbrant AA, Erwin JA, Cheng AW, Guenther MG, Welstead GG, Alagappan R, Frampton GM, Xu P, Muffat J, Santagata S, Powers D, Barrett CB, Young RA, Lee JT, Jaenisch R, Mitalipova M: Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell. 2010, 141: 872-883. ç48View ArticlePubMedGoogle Scholar
  53. Bontekoe S, Mantikou E, van Wely M, Seshadri S, Repping S, Mastenbroek S: Low oxygen concentrations for embryo culture in assisted reproductive technologies. Cochrane Database Syst Rev. 2012, 7: CD008950Google Scholar
  54. Oliveira CS, Saraiva NZ, Cruz MH, Mazeti B, Oliveira LZ, Lopes FL, Garcia JM: HDAC inhibition decreases XIST expression on female IVP bovine blastocysts. Reproduction. 2013, 145: 9-17.View ArticlePubMedGoogle Scholar
  55. Shen Y, Matsuno Y, Fouse SD, Rao N, Root S, Xu R, Pellegrini M, Riggs AD, Fan G: X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. Proc Natl Acad Sci U S A. 2008, 105: 4709-4714.PubMed CentralView ArticlePubMedGoogle Scholar
  56. Glujovsky D, Blake D, Farquhar C, Bardach A: Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev. 2012, 7: CD002118Google Scholar
  57. van der Gaast MH, Classen-Linke I, Krusche CA, Beier-Hellwig K, Fauser BC, Beier HM, Macklon NS: Impact of ovarian stimulation on mid-luteal endometrial tissue and secretion markers of receptivity. Reprod Biomed Online. 2008, 17: 553-563.View ArticlePubMedGoogle Scholar
  58. Bourgain C, Devroey P: The endometrium in stimulated cycles for IVF. Hum Reprod Update. 2003, 9: 515-522.View ArticlePubMedGoogle Scholar
  59. Shapiro BS, Daneshmand ST, Restrepo H, Garner FC: Serum HCG measured in the peri-implantation period predicts IVF cycle outcomes. Reprod Biomed Online. 2012, 25: 248-253.View ArticlePubMedGoogle Scholar
  60. Poikkeus P, Hiilesmaa V, Tiitinen A: Serum HCG 12 days after embryo transfer in predicting pregnancy outcome. Hum Reprod. 2002, 17: 1901-1905.View ArticlePubMedGoogle Scholar
  61. Eiben B, Bartels I, Bähr-Porsch S, Borgmann S, Gatz G, Gellert G, Goebel R, Hammans W, Hentemann M, Osmers R, Rauskolb R, Hansmannt I: Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am J Hum Genet. 1990, 47: 656-663.PubMed CentralPubMedGoogle Scholar
  62. Yusuf RZ, Naeem R: Cytogenetic abnormalities in products of conception: a relationship revisited. Am J Reprod Immunol. 2004, 52: 88-96.View ArticlePubMedGoogle Scholar
  63. Huether CA, Martin RL, Stoppelman SM, D’Souza S, Bishop JK, Torfs CP, Lorey F, May KM, Hanna JS, Baird PA, Kelly JC: Sex ratios in fetuses and liveborn infants with autosomal aneuploidy. Am J Med Genet. 1996, 63: 492-500.View ArticlePubMedGoogle Scholar
  64. Dumoulin JC, Derhaag JG, Bras M, Van Montfoort AP, Kester AD, Evers JL, Geraedts JP, Coonen E: Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF. Hum Reprod. 2005, 20: 484-491.View ArticlePubMedGoogle Scholar
  65. James WH: The human sex ratio part 1: a review of the literature. Hum Biol. 1987, 59: 721-752.PubMedGoogle Scholar
  66. James WH: The variation of the probability of a son within and across couples. Hum Reprod. 2000, 15: 1184-1188.View ArticlePubMedGoogle Scholar

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