During the past two decades, several evidences have shown the role of fetal sex in pregnancy outcome. In singleton pregnancies a sex-related increased risk of adverse obstetric and neonatal outcome is well established (Al-Qaraghouli & Fang, Reference Al-Qaraghouli and Fang2017). Male fetal sex is an independent risk factor for preterm birth (PTB) (Brettell et al., Reference Brettell, Yeh and Impey2008; Challis et al., Reference Challis, Newnham, Petraglia, Yeganegi and Bocking2013; Ingemarsson, Reference Ingemarsson2003; Zeitlin et al., Reference Zeitlin, Ancel, Larroque and Kaminski2004), gestational diabetes (GDM), and macrosomia and cord complications (cord prolapse, nuchal cord, true umbilical cord knots) (Sheiner et al., Reference Sheiner, Levy, Katz, Hershkovitz, Leron and Mazor2004; Verburg et al., Reference Verburg, Tucker, Scheil, Erwich, Dekker and Roberts2016). Males also have a higher risk of complications during labor and delivery, such as failure to progress during the first and second stages of labor (Sheiner et al., Reference Sheiner, Levy, Katz, Hershkovitz, Leron and Mazor2004), nonreassuring fetal heart patterns (Dawes et al., Reference Dawes, Dawes, Moulden and Redman1999; Porter et al., Reference Porter, Triebwasser, Tuuli, Caughey, Macones and Cahill2016), cesarean section (CS) delivery (Eogan, Reference Eogan2003; Lurie et al., Reference Lurie, Weissler, Baider, Hiaev, Sadan and Glezerman2004), and neonatal morbidity and mortality (Mondal et al., Reference Mondal, Galloway, Bailey and Mathews2014; Stevenson, Reference Stevenson2000).
Available data support the hypothesis that male sex is an offending factor, whereas female sex a protective one; however, the mechanisms underlying these observations remain unclear. Presumably, the hormonal environment the fetus is exposed to during pregnancy is the main conditioning factor. For instance, the observation that female fetal sex would increase the risk of hypertensive disorders during pregnancy (HDP) is supported by the potential role of elevated levels of maternal serum human chorionic gonadotropin in pregnancies carrying females compared to those carrying males (Zheng et al., Reference Zheng, Deng, Zhong and Shi2016) or the elevated levels of angiotensin at 15 weeks only in pregnancies carrying females compared to controls (Sykes et al., Reference Sykes, Pringle, Zhou, Dekker, Roberts and Lumbers2014). Besides, the placental vascular bed seems to be more responsive to magnesium sulphate in preterm female than male pregnancies, so females would have better fetal nutrient delivery and gas exchange than males (Gray et al., Reference Gray, Vickers, Dyson, Reynolds and Berry2015). These observations show how fetal sex represents an interactive factor between the mother, the placenta, and the fetus (Al-Qaraghouli & Fang, Reference Al-Qaraghouli and Fang2017).
With this background, twin pregnancies represent a unique opportunity to explore the effect of fetal sex in pregnancy complications, given the same-sex pairs and opposite-sex pairs may present also different hormonal phenotypes. Some previous studies analyzed the impact of fetal sex on pregnancy and perinatal outcome among twins, but results are heterogeneous and debatable, considering also the effect of chorionicity (Derom et al., Reference Derom, Derom, Loos, Thiery, Vlietinck and Fryns2005; Esposito et al., Reference Esposito, Cantarutti, Mauri, Franchi, Fedele, Corrao, Parazzini and Persico2023; Funaki et al., Reference Funaki, Ogawa, Ozawa, Hosoya, Okamoto, Urayama, Morisaki and Sago2022; Goldman et al., Reference Goldman, Blumrozen and Blickstein2003; Luke et al., Reference Luke, Hediger, Min, Brown, Misiunas, Gonzalez-Quintero, Nugent, Witter, Newman, Hankins, Grainger and Macones2005; Shinwell et al., Reference Shinwell, Reichman, Lerner-Geva, Boyko and Blickstein2007; Tan et al., Reference Tan, Wen, Mark, Fung, Demissie and Rhoads2004).
Thus, the aim of the study is to establish the role of fetal sex pairing on obstetric and perinatal outcome in dichorionic (DC) and monochorionic (MC) twin pregnancies managed in a single referral center over a 10-year period.
Materials and Methods
A retrospective observational study was conducted on a cohort of 1180 twin pregnancies delivered from 2010 to 2020 at Careggi University Hospital, a tertiary hospital in Florence, Italy. The sample population was restricted to women with twin pregnancies who delivered later than 22 weeks. Triplets and higher order multiple pregnancies were excluded. In addition, pregnancies with at least one infant with a birth defect (6.1%) were not included.
Obstetric digital records were used to extrapolate data regarding maternal characteristics, such as maternal age, body mass index (BMI), parity, mode of conception (i.e., spontaneous conceived [SC] or assisted reproductive technologies [ART]], and complications that occurred during pregnancy. Furthermore, all data regarding labor and delivery, including neonatal outcome, were collected. Women with any missing data regarding fetal sex, birth weight, gestational age at birth, or unreliable data on chorionicity were excluded.
The analysis was performed on monochorionic (MC) and dichorionic (DC) twin pregnancies separately. Twin pregnancies were classified into three groups based on fetal sex pairing (female- female, male-male and female-male pairs), compared in terms of maternal characteristics, pregnancy and perinatal outcomes. The binary sex categorization (male or female) was designated at birth, based on the visible external anatomy of the newborn.
The study aimed to evaluate the following obstetric outcomes, defined according the most recent international guidelines: hypertensive disorders of pregnancy (HDP), gestational diabetes (GDM), fetal growth restriction (FGR), obstetric cholestasis, spontaneous preterm birth (sPTB), premature preterm rupture of membranes (pPROM) and intrauterine fetal demise (IUFD). Pregnancy dating and gestational age at delivery were based on last menstrual period in spontaneous conceived twins, confirmed during the first ultrasound examination. ART twins’ dating was based on the egg retrieval in fresh cycle conceived pregnancies or on the age of the frozen embryo at the time of transfer in frozen cycle conceived pregnancies.
Data on labor and delivery outcome included the onset of labor (spontaneous or induced), mode of delivery, gestational age at delivery, and postpartum blood loss. Neonatal data comprised birth weight, Apgar score at 5 and 10 minutes, arterial pH, need for neonatal resuscitation, and neonatal intensive care unit (NICU) admission.
Data analysis was performed by IBM SPSS version 22 (IBM, Armonk, NY, USA) statistical software package. Data distribution was evaluated by the D’Agostino and Pearson omnibus normality test. All outcomes of DC twins were investigated, comparing the female-female, male-male, and female-male groups by using chi-square test and analysis of variance (ANOVA) test, as appropriate. Data from MC twin male pairs versus female pairs were compared by using Fisher’s exact test or chi-square test and t Student test or Mann-Whitney test, as appropriate. P-values of <.05 represent statistical significance.
Results
Maternal characteristics among DC twin pregnancies (n = 837) and MC twin pregnancies (n = 228) are reported in Table 1 and Table 2 respectively. Among DC twins, 27.9% carried female-female twin pairs, 30.3% male-male twin pairs and 41.8% discordant sex pairs. In the MC twin group 49.1% was represented by female-female twin pairs. More than half of pregnant women included among DC twin pregnancies and 35% among MC twin pregnancies were over 35 years and most were nulliparous. Maternal age, pregestational BMI and parity were similar and there was no statistical difference in terms of gestational age at delivery between fetal sex groups among both DC and MC twin pregnancies.
Table 1. Maternal characteristics according to sex pairing among DC twins (N = 837).
Note: CS, cesarean section; ART, assisted reproductive technology. Continuous variables are expressed as mean ± SD; categorical variables are expressed as n (%). p < .05 indicates statistical significance.
Table 2. Maternal characteristics according to sex pairing among MC twins (N = 228)
Note: MC, monochorionic; CS, cesarean section; ART, assisted reproductive technology. Continuous variables are expressed as mean ± SD; categorical variables are expressed as n (%). p < .05 indicates statistical significance.
In DC twin pregnancies, those conceived from ART were more likely to have one or both female fetuses (p = .008) and the majority (60.1%) carried opposite-sex pairs. Most were conceived after in vitro fertilization (FIVET; 48.3%), and just a minority after intracytoplasmic sperm injection (ICSI; 7.6%). On the contrary, the mode of conception and the rate of ART-conceived pregnancies among female MC twin pairs were similar to male MC twin pregnancies.
Pregnancy and perinatal outcome of DC and MC twin pregnancies are reported in Table 3 and Table 4 respectively. In the DC pregnancies group, GDM was the most frequent observed complication (19.9%). The incidence of adverse pregnancy outcome such as GDM and cholestasis was similar between the three groups: fetal sex pairing was not independently associated with either of these outcomes.
Table 3. Pregnancy and perinatal outcome according to sex pairing among DC twins
Note: DC, dichorionic; HDP, hypertensive disorders of pregnancy; pPROM, premature preterm rupture of membranes; sPTB, spontaneous preterm birth; CS, cesarean section. Continuous variables are expressed as mean ± SD; categorical variables are expressed as n (%). p < .05 indicates statistical significance.
Table 4. Pregnancy and perinatal outcome according to sex pairing among MC twins
Note: MC, monochorionic; HDP, hypertensive disorders of pregnancy; pPROM, premature preterm rupture of membranes; sPTB, spontaneous preterm birth; CS, cesarean section. Continuous variables are expressed as mean ± SD; categorical variables are expressed as n (%). p < .05 indicates statistical significance.
Hypertensive disorders complicated 7.2% of all DC pregnancies recruited, and the incidence was higher in male-male pairs (11%) than in other groups (p = .01). Particularly gestational hypertension contributes to this result (10.7% of male-male pregnancies), whereas the incidence of preeclampsia was very low (overall 1.1% of all DC pregnancies and 0.4% of male-male pregnancies). Only one case of HELLP syndrome was recorded (in the female-female group). sPTB (before 37 weeks) was observed in 18% of cases and only 4% before 28 weeks; the incidence of pPROM in the study population was 10.5%, with no significant difference in the rate of prematurity between groups. FGR of at least one fetus complicates 15.6% of pregnancies, and the frequency of IUFD was low (0.9%). The incidence of adverse fetal outcomes was similar in the three groups. Half of all cases (50.9%) were delivered by elective cesarean section (CS), with similar percentages for both CS (both elective and emergency) and vaginal birth among the three groups (Table 3).
Among MC twin pregnancies, FGR was the most frequent pregnancy complication (18.8%), and the incidence was similar among female-to-male twin pairs. Only a few cases reported IUFD, but no difference between the two groups was observed, even though a trend was observed in increased rate of FGR, IUFD and sPTB in males than females, and male pairs were more likely to report an Apgar score <7 at 5 minutes. GDM complicated 13.5% of all MC pregnancies, and the comparison between female-female and male-male pairs showed a significantly higher incidence in pregnancies carrying females (p = 0.028). The incidence of other pregnancy complications, such as HDP and obstetric cholestasis, was similar between the two groups. pPROM and sPTB occurred similarly in both female- and male-carrying MC pregnancies. However, a trend was observed in increased rate of FGR, IUFD and sPTB less than 28 and 32 weeks in males than females. Most of the MC twin pregnancies (61.4%) were delivered by elective CS, with no difference between female and male twin pairs. In terms of fetal outcome, sex pairing does not affect the incidence of low arterial pH at birth, whereas it seems to affect the need for neonatal resuscitation. Despite the limited number of cases, both in DC and MC twin pregnancies the first twin of female-female pairs have a worse adaptation to extrauterine life and increased need of neonatal care and resuscitation at birth compared to those from male-male or discordant pairs. Male pairs among MC twin pregnancies were more likely to report an Apgar score <7 at 5 minutes (Table 4).
Discussion
Our study results show a higher incidence of HDP among same-sex DC pairs, while a higher rate of GDM among MC female-female pairs and a worse adaptation to extrauterine life among male-male pairs in MC twins are reported.
Previous studies found female sex as an independent risk factor for hypertensive disorders in singleton pregnancies, and a higher incidence of HDP in twin pregnancies carrying females than in those carrying males was reported (Funaki et al., Reference Funaki, Ogawa, Ozawa, Hosoya, Okamoto, Urayama, Morisaki and Sago2022; Shiozaki et al., Reference Shiozaki, Matsuda, Satoh and Saito2011). Our results interestingly suggest a more relevant effect of sex pairing rather than sex itself, with a higher rate of HDP, particularly gestational hypertension, in DC same-sex than sex-discordant twins, under the assumption of a worse placentation process for an enhanced hormonal background in same-sex pairs. The high serum androgen levels of women carrying male-male twin pregnancies may have a harmful effect on the vascular endothelium, by activation of inflammatory mechanisms and oxidative stress (Moretti et al., Reference Moretti, Lanzolla, Moretti, Gnessi and Carmina2017). Similarly, in female-female couples, this result could be explained by the elevated levels of maternal serum hCG and angiotensin (Sykes et al., Reference Sykes, Pringle, Zhou, Dekker, Roberts and Lumbers2014; Zheng et al., Reference Zheng, Deng, Zhong and Shi2016). Furthermore, a recent systematic review and meta-analysis demonstrated that twin pregnancies with a female fetus were more likely to be associated with preterm preeclampsia, whereas those carrying a male were more likely to develop term preeclampsia (Broere-Brown et al., Reference Broere-Brown, Adank, Benschop, Tielemans, Muka, Gonçalves, Bramer, Schoufour, Voortman, Steegers, Franco and Schalekamp-Timmermans2020).
In our cohort women carrying female-female MC twin pregnancies showed a significantly higher incidence of GDM compared to those carrying male-male pairs. Probably in female-female pairs, which are known to be more frequent following ART as mode of conception (Scott Sills et al., Reference Scott Sills, Tucker and Palermo2000), the high incidence of dysmetabolic pathologies and insulin resistance, a contributing factor for infertility, may have played a relevant role. Also, a female fetus is thought to be associated with greater maternal insulin resistance during pregnancy (Xiao et al., Reference Xiao, Zhao, Nuyt, Fraser and Luo2014), and higher leptin and C-peptide concentrations were observed in female neonates cord blood (Walsh et al., Reference Walsh, Segurado, Mahony, Foley and McAuliffe2015).
In terms of perinatal outcome, our results show that male pairs from MC twin pregnancies were more likely to report an Apgar score <7 at 5 minutes, confirming previous evidence, where male neonates have a higher rate of respiratory and neurologic morbidity compared to females (Melamed et al., Reference Melamed, Yogev and Glezerman2009). This disadvantage may be partly justified by the androgen conditioning role on fetal lung development (Hanley et al., Reference Hanley, Rassner, Jiang, Vansomphone, Crumrine, Komüves, Elias, Feingold and Williams1996; Tremblay & Provost, Reference Tremblay and Provost2006). As additional proof, a better adaptation and a lower incidence of respiratory distress syndrome, intraventricular hemorrhage (IVH) and convulsions were observed in female neonates from female-female pairs compared with females from unlike-sex twin pregnancies. Thus, the presence of a male rather than a female co-twin would be a risk factor for adverse neonatal outcome (Melamed et al., Reference Melamed, Yogev and Glezerman2009).
It is well known that PTB is a common complication of twin pregnancies, either after spontaneous labor or iatrogenic delivery. In singletons, male sex seems to be a risk factor for PTB (Astolfi & Zonta, Reference Astolfi and Zonta1999; Challis et al., Reference Challis, Newnham, Petraglia, Yeganegi and Bocking2013; Vatten & Skjaerven, Reference Vatten and Skjaerven2004; Zeitlin et al., Reference Zeitlin, Ancel, Larroque and Kaminski2004), particularly sPTB (Verburg et al., Reference Verburg, Tucker, Scheil, Erwich, Dekker and Roberts2016), highlighting a possible association between spontaneous preterm labor and androgen levels (Cathey et al., Reference Cathey, Watkins, Rosario, Vega, Mukherjee, O’Neill, Loch-Caruso, Alshawabkeh, Cordero and Meeker2021; Makieva et al., Reference Makieva, Saunders and Norman2014). However, in the current study we found no significant difference in the incidence of late, very and extremely sPTB in terms of sex-pairing groups, in both DC and MC twins. The available literature suggests that sharing the womb with a male co-twin involves a higher risk of PTB (Derom et al., Reference Derom, Derom, Loos, Thiery, Vlietinck and Fryns2005; Glinianaia, Reference Glinianaia1998; Loos et al., Reference Loos, Derom, Eeckels, Derom and Vlietinck2001; Tan et al., Reference Tan, Wen, Mark, Fung, Demissie and Rhoads2004), and this risk is greater in male-male pairs than in male-female pairs (Funaki et al., Reference Funaki, Ogawa, Ozawa, Hosoya, Okamoto, Urayama, Morisaki and Sago2022; Melamed et al., Reference Melamed, Yogev and Glezerman2009). Nevertheless, in most cases, previous studies present heterogeneous data without differentiating between spontaneous and iatrogenic PTB, whereas in our cohort we accurately identified only cases where delivery occurred after labor started spontaneously.
Another adverse outcome linked to fetal male sex is stillbirth; in singleton pregnancies carrying male fetuses the risk of stillbirth is up to 10% higher than female ones (Wong et al., Reference Wong, Schreiber, Crawford and Kumar2023). In our population study, even though fetal sex pairing does not significantly affect the risk of IUDF, a higher trend may be pointed out among male pairs, confirming the few data currently available on twins (Funaki et al., Reference Funaki, Ogawa, Ozawa, Hosoya, Okamoto, Urayama, Morisaki and Sago2022).
Despite the trend of IUFD among male pairs, no significant difference in fetal growth abnormalities was observed in our cohort based on fetal sex pairing, which seems not to affect the incidence of FGR, confirming some recent evidences (Funaki et al., Reference Funaki, Ogawa, Ozawa, Hosoya, Okamoto, Urayama, Morisaki and Sago2022). Previous data observed that male neonates from male-male pairs have a lower mean birth weight and a higher rate of FGR compared with those from male-female pairs (Melamed et al., Reference Melamed, Yogev and Glezerman2009), but data are still conflicting, given also the mutual hormonal influence of unlike-sex pairs (De Zegher et al., Reference De Zegher, Francois, Boehmer, Saggese, Müller, Hiort, Sultan, Clayton, Brauner, Cacciari, Ibáñez, Van Vliet, Tiulpakov, Saka, Ritzén and Sippell1998; Glinianaia, Reference Glinianaia1998; James, Reference James2002).
A major strength of this study is the inclusion of a large number of twin pregnancies with strict inclusion criteria and reliable information regarding maternal and obstetric outcome. Besides, data were collected from a single referral center, which enabled a consistent evaluation of outcomes, given the uniformity in clinical practice. Among DC twins, the analysis comparing the three fetal sex-pairing groups ensures an accurate evaluation of the mutual influence among unlike-sex pairs of twins. Nevertheless, some limitations need to be acknowledged, as, for instance, the analysis of data from 10 years of observation entails different clinical approaches and management across time. For instance, ultrasound criteria for FGR identification and diagnostic criteria for HDP or GDM changed across this time spam.
In conclusion, fetal sex seems to have a mild effect in twin than in singleton pregnancies, suggesting a more complex set of factors contributing to pregnancy outcome in multiple pregnancies. However, we observed a higher incidence of HDP among same-sex DC pairs, and a higher rate of GDM among MC female-female pairs and a worse adaptation to extrauterine life among male-male pairs in MC twins. More research is still needed to understand the pathophysiological mechanisms underlying the different incidence of pregnancy complications according to fetal sex and sex pairing among twins.
Data availability statement
The anonymized datasets generated during and/or analyzed during the current study can be provided on reasonable request.
Financial support
None.
Competing interests
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. The research protocol involved only existing records, based on information routinely collected and stored in a de-identified dataset and hence was considered exempt from ethical review board approval.
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