Granulocyte colony-stimulating factor in assisted reproductive technology treatment does not increase the risk of adverse perinatal outcomes in twin pregnancies

Abstract The aim of the study is to compare the perinatal outcomes of twin pregnancies resulting from assisted reproductive technology (ART) treatment in which granulocyte colony-stimulating factor (G-CSF) was used with those in which it was not. In this retrospective study, the clinical data of 122 dichorionic diamniotic twin pregnancies were reviewed. Pregnancies were divided into two groups, G-CSF-treated and non-G-CSF treated. Maternal age, gestational week at birth, oligohydramnios, gestational hypertension, pre-eclampsia, preterm birth, first-trimester bleeding, gestational diabetes, rupture of membrane, foetal congenital anomalies, admission to the neonatal intensive care unit, birth weight (BW), small for gestational age, BW discordance, Apgar score and placental weight were compared between the groups. IMPACT STATEMENT What is already known on this subject? Granulocyte colony-stimulating factor (G-CSF) administrations increase pregnancy outcomes and do not have a negative effect on perinatal outcomes in singleton pregnancies. What the results of this study add? This study showed that the perinatal outcome of dichorionic diamniotic twin pregnancies conceived after assisted reproductive technology (ART) treatment was similar in the GSF administrated and non-GSF administrated groups. What the implications are of these findings for clinical practice and/or further research? Using G-CSF to increase the success of ART does not seem to have an adverse outcome in the dichorionic diamniotic twin pregnancies.


Introduction
Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has a glycoprotein structure (Hiby et al. 2008).It belongs to the colony-stimulating factor group of proteins, which are described as enhancers of the migration of primary trophoblast cells (Hiby et al. 2008).Several studies have reported that G-CSF plays an important role in endometrial receptivity, trophoblastic development and placental metabolism (Rahmati et al. 2014).G-CSF helps migration and differentiation of the macrophages and facilitates endometrial regeneration by promoting angiogenesis (Wurfel et al. 2010, Tutdibi et al. 2012).
In recent years, several studies have been conducted indicating that G-CSF administrations increase the success of assisted reproductive technology (ART) treatment, due to its endometrial and placental effects (Kalem et al. 2020, Zeyneloglu et al. 2020).It has been described that this administration, which increases pregnancy outcomes, does not have a negative effect on perinatal outcomes in singleton pregnancies (Cruz et al. 2019).Multiple pregnancy rates have increased with the advancing maternal age and the increase in the use of ART (Ananth and Chauhan 2012).Considering that the perinatal outcomes of twin pregnancies are worse compared to singleton pregnancies, we think that the effect of this administration on perinatal outcomes in twin pregnancies should be clarified.
The present study aimed to assess the effect of G-CSF administration on the perinatal outcomes of twin pregnancies conceived from ART.

Methods
This retrospective study was conducted at Baskent University Hospital, Ankara, Turkey.The study was approved by the Baskent University Institutional Review Board (no.: KA 21/352) in accordance with the Declaration of Helsinki.Twin births between 2017 and 2020 were reviewed.
The chorionicity of the placenta and pregnancy type (ART or spontaneous) was obtained from patients' medical records.In our clinic, the examination protocol includes ultrasonographic studies between 11 and 14 weeks to diagnose multiple pregnancies and chorionicities.Chorionicity was identified based on the presence or absence of the lambda sign at the intertwin membrane.
Patient characteristics such as maternal age, body mass index (BMI), pregestational diabetes mellitus (DM), gestational week at birth, oligohydramnios, gestational hypertension, pre-eclampsia, preterm birth, first trimester bleeding, gestational diabetes and preterm rupture of membrane and neonatal characteristics such as congenital anomalies, admission to neonatal intensive care unit (NICU), birth weight (BW), small for gestational age (SGA), BW discordance, 5th minute Apgar score and placental weight were obtained from electronic and medical records.
Gestational week was determined according to the embryonic age from fertilisation.Preterm birth was defined as deliveries at less than 37 gestational weeks.Gestational hypertension, pre-eclampsia, eclampsia, superimposed preeclampsia and superimposed eclampsia were considered gestational hypertensive disease (GHD).The presence of placenta previa and placenta accreta diagnoses at birth was determined as placentation anomalies.As per the routine of our clinic, twin pregnancy placentas were evaluated histopathologically after birth.Placental weights were obtained from pathology reports.
The commonly used definition of SGA newborn, which is BW less than the 10th percentile for gestational age, was used for either twin.BW discordance was defined as the percentage of discrepancies in BW between the larger and smaller twin and calculated using the following formula: (larger BW -smaller BW)/(larger BW) (Harper et al. 2013).Mean foetal weight was calculated from the following formula: (larger BW þ smaller BW)/2.The cut-off of BW discordance was defined as �20%.
Study population was infertile cases with two or more unsuccessful ART treatments, despite the transfer of good quality embryos.In our centre, we have been using G-CSF for these infertile cases since 2011.All patients for whom G-CSF administration recommended were informed about the offlabel use and possible side effects such as headache, fever, weakness, etc., before they presented their informed consent.According to the policy of our clinic, the intrauterine route of G-CSF was employed on ovulation triggering day; G-CSF (Leucostim, Ankara, Turkey, 30 MIU/1 mL) was provided via an intrauterine insemination catheter after cleaning the cervix (Zeyneloglu et al. 2020).Subcutaneous injections were started on the day of oocyte retrieval and administered for 15 days at 100,000 IU/kg.
Statistical analyses were performed using the SPSS version 22 software package (IBM Corp., Armonk, NY).The variables were investigated using visual (histograms) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk's test) to determine whether or not they are normally distributed.Descriptive analyses were presented using medians for nonnormally distributed and ordinal variables.The Mann-Whitney U-test was used to compare non-normally distributed variables.Chi-square test was used to compare proportions.A p value of <.05 was considered statistically significant.

Results
During the study period, 138 DCDA twin pregnancies conceived after ART were followed up and delivered in our clinic.Dichorionic and diamniotic (DCDA) twin pregnancies conceived after ART (in vitro fertilisation or intracytoplasmic sperm injection) were included in the study.Spontaneous twin pregnancies (n ¼ 4), twin pregnancies with foetal reduction (n ¼ 4) and intrauterine death of the foetus before 24 weeks' gestation (n ¼ 8) were excluded.A total of 122 cases were included in the study.The mean maternal age of the cohort was 31 (range, 24-42) years.Table 1 summarises the demographic and clinical characteristics of the groups.Of the ART pregnancies, 42 (31.8%)received G-CSF (group 1) and 80 (68.2%) did not (group 2).There were no differences between groups in terms of maternal age, gestational week at birth, and mean foetal weight (p ¼ .310,p ¼ .702and p ¼ .866,respectively).When the groups were compared, placental weight was found to be significantly lower in non-G-CSF treated than in G-CSF treated.The comparison of the perinatal outcomes of the groups is presented in Table 2. Placental weight was 914 ± 226 g in non-G-CSF treated and 963 ± 197 g in G-CSF treated (p ¼ .030).There was no significant relationship between the remaining perinatal factors with G-CSF administration.
In G-CSF treated group, one case of multicystic dysplastic kidney and one case of amniotic band syndrome were detected as congenital anomalies.In non-G-CSF treated group, one case of choroid plexus cyst was detected.

Discussion
In this study, we found that the perinatal outcomes of twin pregnancies conceived by ART treatment with GSF administration were similar to those that were conceived without GSF administration.To the best of our knowledge, this study is the first publication to evaluate the effect of G-CSF administration on perinatal outcomes in twin pregnancies.We found that the placental weight was significantly higher in the G-CSF treated group than in the control group, although there was no significant difference in terms of perinatal outcomes including, oligohydramnios, GHD, preterm birth, firsttrimester bleeding, gestational diabetes, rupture of membrane, congenital anomalies, admission to the NICU, BW, SGA, BW discordance and Apgar score between the groups.
In the literature, there are a few studies on perinatal outcomes of G-CSF use in ART cycles (Scarpellini and Sbracia 2009, Santjohanser et al. 2013, Cruz et al. 2019).In a randomised controlled trial conducted in singletons, pregnancy complications in 35 patients with recurrent miscarriage treated with G-CSF were compared with a placebo group comprising 33 patients (Scarpellini and Sbracia 2009).It was shown that the groups were similar in terms of gestational week of neonatal weight and there were no pregnancy complications such as pre-eclampsia, pre-term birth, gestational diabetes, pregnancy hypertension, bleeding and thrombosis in the G-CSF group.No congenital malformations were observed in the placebo group, but one case of mild hypertension in pregnancy was detected.In a retrospective cohort study in singleton pregnancies, G-CSF was used for recurrent miscarriages and none of the newborns had any congenital malformations (Santjohanser et al. 2013)  pregnancies using G-CSF for KIR-HLA-C mismatch and recurrent miscarriage (Cruz et al. 2019).As a result, they showed that perinatal outcomes were similar to the control group.In the control group, 2.1% of newborns had some congenital anomalies, but none of the newborns in the G-CSF group had any anomalies.
It is known that the risk of mortality and congenital anomaly in DCDA twin pregnancies is higher than in singleton pregnancies (Rodis et al. 1990, Meyers et al. 1997, Rydhstroem and Heraib 2001).Additionally, ART pregnancies have been associated with an increased risk of placentarelated perinatal outcomes (Qin et al. 2016).In the present study, the incidence of congenital anomalies was similar to that reported in the literature for singleton pregnancies.Additionally, perinatal outcomes and the congenital anomaly incidence were similar in the G-CSF group compared with the control group.Accordingly, we speculate that using G-CSF in the implantation period and early pregnancy seems to be safe in terms of perinatal outcomes and congenital malformations of twin pregnancies.
Low placental weight was associated with poor perinatal outcomes in several studies (Eskild et al. 2009, Shehata et al. 2011, Haavaldsen et al. 2012).Placental weight may be an indicator of placental function, and small placentas are more likely to be dysfunctional (Salafia et al. 2006).In the infertile population, abnormal trophoblast invasion, vascularisation and oxidative stress are thought to be responsible for the presence of abnormal placentation and dysfunctional placenta (Sundheimer and Pisarska 2017).It is unclear whether these effects originate from infertility or result from the in vitro fertilisation procedure (Helmerhorst et al. 2004).Increased placental weight has been reported in ART pregnancies (Daniel et al. 1999, Haavaldsen et al. 2012).However, to our knowledge, there is no publication evaluating placental weight in twin pregnancies after ART treatment.In our study, the mean placental weight was significantly higher in the G-CSF-treated group than in the non-treated group.High placental weight would be expected to be associated with decreased pregnancy complications.Interestingly, although the difference between the groups in terms of placental weight was statistically significant, both groups were similar in terms of mean foetal weight and placenta-related perinatal complication rates.We may speculate that the difference was due to G-CSF-administration, or it may be due to infertilityrelated factors.
The study has some limitations including its retrospective design and the relatively small number of cases.The reason for the relatively small number of cases is that G-CSF is not administered to every patient in IVF/ICSI cycles, and our study group consisted only of dichorionic diamniotic twin patients.It should be noted that the retrospective design of the study may have caused selection bias.However, welldesigned, large-scale, randomised controlled trials are necessary to improve our understanding of the effect of using G-CSF in the implantation period and early pregnancy on perinatal outcomes and congenital malformations in twin pregnancies.
In conclusion, using G-CSF in patients with recurrent implantation failure or recurrent miscarriage for increasing the success of IVF/ICSI does not seem to have an adverse outcome in the dichorionic diamniotic twin pregnancies.

Table 1 .
Demographic and clinical characteristics of the groups.
. In 2019, Cruz et al. analysed perinatal outcomes including congenital malformations, preterm birth and newborn weight of singleton

Table 2 .
Comparison of perinatal outcomes of the groups.