Maternal characteristics and pregnancy outcomes of Chinese women with infertility undergoing in vitro fertilization with embryonic cryopreservation: a retrospective cohort study

Background: To examine differences in the maternal characteristics and pregnancy outcomes of Chinese women with various causes of infertility who underwent in vitro fertilization(IVF) with embryonic cryopreservation treatment. Methods: This retrospective cohort study included women with various causes of infertility who used IVF. In order to reduce the research error, we deliberately removed the fresh ET cycles and ICSI cycles at the beginning, so in our data the IVF-ET method was frozen-embryo transfer.[MOU1] [MOU2] Data on demographic characteristics, medical history, laboratory tests, and delivery were reviewed. Logistic regression analysis was performed to calculate odds ratios and 95% condence intervals for pregnancy and perinatal complications and neonatal outcomes. The multivariable model was adjusted for age, gravidity, parity, pre-pregnancy obesity, birth plurality, and history of previous caesarean section.[MOU3] Results: The IVF treatment group was divided into 5 subgroups according to infertility cause as follows: ovulation disorder, tubal disease, male infertility, endometriosis, and mixed infertility. Among singleton pregnancies, we veried that IVF with embryonic cryopreservation pregnancies are related to increased risks of adverse perinatal outcomes. Besides (cid:0) compared with spontaneous pregnancies, IVF pregnancies were associated with signicant increases in the rates of the following: gestational diabetes mellitus (GDM), preeclampsia, preterm preeclampsia, postpartum haemorrhage, intrahepatic cholestasis of pregnancy, preterm premature rupture of membranes, preterm birth, low birthweight, macrosomia, and neonatal intensive care unit (NICU) admission in the ovulation disorder group; GDM, placenta previa, placenta accreta, postpartum haemorrhage, macrosomia and 5-minute Apgar score ≤ 7 in the tubal disease group; placenta previa, small for gestational age, macrosomia and NICU admission in the endometriosis group; placenta previa and placenta accreta in the male infertility

conceived after ART have a higher prevalence of certain birth defects. Assisted hatching and the diagnosis [MOU3] [MOU4] of an ovulation disorder are marginally associated with increased risks for nonchromosomal birth defects [8][9][10]. However, other research has shown that ART is associated with a slightly elevated risk of birth defects and that the risks vary depending on the exposure [11]. Besides, other studies have concluded that the ART procedures associated with IVF are not responsible for adverse perinatal complications [12]. Because subfertile women who conceived without the aid of ART exhibited an increased risk for these adverse outcomes. Several maternal factors associated with infertility may contribute to adverse obstetric and perinatal outcomes. Despite the widespread application of ART, concerns about potential health implications remain, and the results of previous studies are controversial, partly because of their different study designs, ethnic group compositions, ART protocols and techniques used, and maternal biometric characteristics. [MOU5] The reasons for the increase in adverse pregnancy outcomes with ART are unknown. It is di cult to identify whether the adverse outcomes observed with ART are the direct result of [MOU6] [MOU7] the patients' characteristics, including type of subfertility or other factors such as carviovascular maladaptation . One hypothesis is that an infertility-related diagnosis in a woman undergoing ART contributes directly to adverse outcomes, and excess perinatal morbidities have been associated with the infertility-related diagnosis in both ART-treated and non-ART-treated women [13]. However, after adjustments for maternal characteristics, other studies have reported few cases in which underlying infertility directly contributed to adverse outcomes [14].
Another possibility is that adverse outcomes result from the ART procedure itself, including the arti cial induction of ovulation; exposure of oocytes, sperm, and embryos to the environment outside of the body; and freezing and manipulation of oocytes and embryos. In several prior studies, age-matching of patients between the ART and spontaneous conception groups was not performed. Knowledge of ART pregnancy outcomes in China is limited, and few studies have examined the relationship between infertility causes and pregnancy outcomes.
In order to explore the relationships between the infertility reasons for IVF and adverse outcomes, we therefore conducted this retrospective cohort study The comparisons of our study are two main aspects, [MOU8] [MOU9] the rst one is comparing IVF pregnancies with natural conception to assess the risk of adverse outcomes conceived by IVF with embryonic cryopreservation. The second aims to search the risk of adverse outcomes among different infertility reasons. [ [MOU16] and 4) the infertility diagnosis was an ovulation disorder, tubal disease, endometriosis, male infertility, or mixed infertility(means multiple infertility-related diagnosis). [MOU17] [MOU18] The exclusion criteria were as follows: 1) the use donor oocytes/sperm or embryos, to ensure that all embryos transferred were autologous; 2) the use of preimplantation genetic testing (PGT);[MOU19] [MOU20] 3) the existence of chronic pre-pregnancy complications, to ensure that only patients with complications that occurred during pregnancy were studied; or 4) women who smoked or consumed alcohol during pregnancy, to prevent confounding effects on outcomes by these factors. Overall, a total of 8773 deliveries were subjected to this retrospective analysis. Among the women, 21% (1843)  singleton and 98 gemellary pregnancies. All data, including infertility diagnosis, pregnancy, obstetric and neonatal outcomes, were obtained from records of the patients' visits to hospitals. The demographic and selected maternal characteristics, pregnancy and labour complications and neonatal outcomes were compared between the two groups.

Variables of interest, and de nition of main outcomes
The selected maternal and pregnancy characteristics and pregnancy outcomes included the following: delivery after at least 28 weeks gestation but no more than 37 weeks gestation)[24], low birthweight (LBW, birthweight <2500 g)[24], macrosomia (birth weight≥4000 g)[25], small for gestational age (SGA, de ned as birth weight below the 10th percentile of a standard optimal reference population for a given gestational age and sex) [26], Apgar score at 1 minute, Apgar score at 5 minutes and neonatal intensive care unit (NICU) admission.

Ethical approval
This study was approved by the local institutional ethics committee , namely, The Beijing Obstetrics and Gynaecology Hospital committee (ethics approval number: 2019-KY-024-01) and is being conducted in accordance with the Declaration of Helsinki. Due to the retrospective study design, consent for participation was not required. Nevertheless, private information was well protected during the study.
Statistical analysis SPSS statistical software (version 20.0) was used for data analysis. We rst compared baseline characteristics between IVF and natural pregnancies. Quantitative data are presented as the mean and SD (mean ± SD). Fisher's exact tests, t tests and Pearson's chi-square tests were performed to evaluate differences in the proportions of categorical variables between two or more groups. Second, we assessed the effect of infertility diagnosis on adverse perinatal and neonatal outcomes by comparing the prevalence of adverse perinatal and neonatal outcomes in different infertility diagnosis subgroups and natural pregnancies. Logistic regression analysis was conducted to calculate approximate relative risks of adverse outcomes and to identify possible predictors of pregnancy complications. The multivariable model was adjusted for maternal age, gravidity, parity, andpre-pregnancy obesity (body mass index≥28 [27], birth plurality, and history of previous caesarean section; the results are reported as adjusted odds ratios (aORs) and 95% con dence intervals (CIs). P values of less than 0.05 were considered statistically signi cant. The methods were carried out in accordance with approved guidelines. Figure 1 shows the ow chart of the participants who were either included in the main analysis or excluded for failing to meet the inclusion criteria. The diagnosis for IVF -treated deliveries included ovulation disorders (N=404), tubal disease (N=803), endometriosis (N=107), male infertility (N=403), and mixed infertility (N=126).

Results
The number of natural pregnancies was 6930. Table 1 summarizes the background characteristics of the women who were included in the main analysis. Women with IVF pregnancies were more likely to have signi cantly higher rates of pre-pregnancy obesity, caesarean section, and multiple pregnancy and a lower rate of previous caesarean delivery than women with spontaneous pregnancies (P<0.001). The spontaneous pregnancy group also had a signi cantly higher number of second gravidity and pregnancies than the IVF group (P<0.001). Table 2 and Table 3 [MOU1] [MOU2] show the pregnancy and perinatal complications due to infertility for singleton pregnancies. Table 4 show the pregnancy and perinatal complications due to infertility for multiple pregnancies. Table 5 and Table 6 show the neonatal outcomes by cause of infertility among singleton pregnancies and multiple pregnancies. The associations between each cause of infertility and maternal/perinatal complication or adverse outcomes were assessed using a logistic regression, with women who conceived spontaneously serving as a reference (Table 7, Table 8, Table 9) . In the same way, the associations between each cause of infertility and neonatal complication or adverse outcomes were assessed using a logistic regression, with women who conceived spontaneously serving as a reference (Table10 and those who conceived spontaneously. However, when gemellary pregnancies were compared with spontaneous pregnancies, only the rates of the following were signi cantly increased: GDM in the ovulation disorder and mixed infertility groups and 1-minute Apgar score ≤7 in the mixed infertility group; the other differences that were signi cantly higher in the singleton pregnancy cohort had narrowed or disappeared in the gemellary pregnancy cohort.

Discussion
As the use of IVF increases and newer technologies continue to push the boundaries of science, it is important to consider the clinical safety of these approaches. Through this retrospective, hospital-based cohort study of pregnant Chinese women, on the one hand, we veri ed that IVFwith embryonic cryopreservation pregnancies are related to increased risks of pregnancy complications, perinatal complications and poor neonatal outcomes. Some possible explanations for these adverse outcomes have been discussed.
[MOU1] First, endometrial preparation and absence of corpus luteum in frozen embryo transfer might predispose to adverse obstetric outcomes such as hypertensive disorders, PE, postpartum haemorrhage, placenta accrete, postterm birth and macrosomia [28,29]. Besides, compared with fresh blastocyst transfer, IVF pregnancies with frozen blastocyst transfer present lower uterine pulsatility index from 7 to 37 weeks and greater fetal growth [30]. On the other hand, diagnostic categories within the IVF population were found to affect maternal and neonatal outcomes among all births. As summarized in Table 7 and Table 10, infertility caused by an ovulation disorder had the worst prognosis. In fact, ovulation disorders were associated with higher risks of preeclampsia (3- Growing evidence demonstrates that PCOS has a negative impact on fertility and pregnancy outcomes, such as GDM, gestational hypertensive disorders, and PB[35]. GDM is evidently related to the delivery of an infant with macrosomia, so the incidence of macrosomia is signi cantly higher for pregnant women with PCOS[34]. In addition, neonates of women with PCOS are at greater risk of neonatal complications, including perinatal mortality, prematurity, SGA, lower birth weight and higher NICU admission [36]. Current evidence also suggests that pre-pregnancy hormonal dysfunction, including hyperandrogenism, progesterone resistance and hyperinsulinism, impairs uterine placentation mechanisms, which may lead to a greater risk of adverse obstetric and neonatal outcomes [36].

[MOU3] [MOU4]
Compared to spontaneous pregnancies, IVF pregnancies in patients who had tubal infertility had an increased risk of GDM (1.5-fold), placenta previa (3-fold), placenta accreta (2-fold), postpartum haemorrhage (2fold), macrosomia (2-fold), and a 5-minute Apgar score≤7 (4-fold). One study reported that infertility, particularly due to an ovulatory disorder or tubal blockage, was associated with an increased GDM risk; speci cally, women with a history of infertility due to tubal blockage had an 83% greater risk[37], consistent with our results. GDM is closely related to the birth of an infant with macrosomia, so the rate of macrosomia in tubal infertility is also signi cantly increased. Tubal-factor infertility is always associated with reproductive in ammation, which may lead to an imbalance in immune-endocrine crosstalk among the endometrium, myometrium and cervix and between the decidua and trophoblasts, predisposing patients to pregnancy complications, such as placenta previa, placenta accreta and postpartum haemorrhage, which could affect neonatal outcomes.
Our data showed that endometriosis was signi cantly associated with placenta previa, SGA, and NICU admission, similar to the ndings of previous studies[38-41]. Endometriosis is a common reason for infertility and may cause chronic in ammation and adhesions in the pelvis of reproductive-aged women.
Moreover, women with endometriosis exhibit defective deep placentation because of defective remodelling of the spiral arteries [42]. These factors may explain why endometriosis is possibly a crucial factor for increased negative outcomes in IVF pregnancy. However, Benaglia L found that women with endometriosis who conceived via IVF do not face an increased risk of preterm birth[43], similar to our nding. In addition, we found that IVF pregnancies in patients with endometriosis had a higher rate of macrosomia (2-fold) than those who conceived naturally. Regrettably, we have not found any literature on the relationship between endometriosis and macrosomia.This controversial result still needs to be further studied by expanding the sample size.
In the male infertility subgroup, the rates of placenta previa and placenta accreta were also increased, but this has not been universally reported. One possible explanation is that the increased risk of placenta previa and placenta accreta is caused by factors related to IVF [44,45]. Indeed, the intrauterine operation and manipulation of embryonic cells in IVF might induce uterine contraction, leading to higher frequencies of implantation in the lower uterine segment, which may increase the risk of placenta previa. The changes to the endometrium wrought by IVF treatment protocols, and the use of hormone therapy to promote embryo implantation, may increase the risk of placenta accreta. In this research, the risk of placenta previa increased in all subgroups except for the ovarian disorder subgroup, which was similar to previous research [44]. Interestingly, there were no signi cant differences in neonatal outcomes between IVF and spontaneous conception in the male infertility subgroup. Vannuccini S found that in uncomplicated term pregnancies following ART, infants born after ART had a similar birthweight, Apgar score and arterial blood pH to those of spontaneously conceived infants [46]. This nding might indicate that the factors associated with infertility are more likely to be associated with adverse neonatal complications rather than the ART procedure itself, which is consistent with a previous study [47]. Overall, the results require further analysis in larger cohorts, adjustments for as many confounders as possible and further preclinical studies.
Our study also showed an increased risk for GDM, placenta previa, chorioamnionitis, PB, and a 1-minute Apgar score≤7 in the mixed infertility subgroup compared with corresponding controls. When there are mixed reasons for parental infertility, pregnancy complications and parental and neonatal outcomes might differ, but perinatal morbidities will always increase. In addition, in gemellary pregnancies, the differences in perinatal and neonatal outcomes between IVF pregnancies and natural pregnancies mostly narrowed or disappeared.
This may indicate that pregnancy outcomes are greatly affected by multiple pregnancies, regardless of whether they are IVF pregnancies or natural pregnancies. This nding may also be the result of a small number of cases.
The major strength of our study is not only the comparison of perinatal and neonatal outcomes of IVF and spontaneous conception but also the assessment of the impact of different infertility diagnosis on pregnancy characteristics and outcomes in China. China has abolished the "one child" policy, and since 2016, it has entered into an era of the two-child policy. As a result, the number of infants is expected to increase greatly, which may promote the demand for IVF [48]. Our ndings have extremely important clinical implications and may provide guidance for couples and obstetricians in determining whether IVF is useful as a rst-line treatment or as a last resort. Moreover, these ndings may help in identifying likely perinatal and neonatal complications and provide information for the underlying pathogenic mechanisms.
There are, however, a few limitations of this study. First, the numbers of stillbirths and neonatal deaths were few; hence, these gures were not included in the main analysis, which may have given rise to the possibility of residual confounding in our results.[MOU5] [MOU6] Therefore, we could not accurately determine the severity of the effects of different infertility diagnosis on neonatal outcomes, nor can we identify the high-risk factors related to the long-term prognosis of the newborn. Another gap in the data that were available was the severity and treatment process of infertility. For example, data on the stage of endometriosis, baseline endocrine level, body mass index, duration of infertility, and ovarian stimulation protocol were incomplete. Although the IVF-ET method in our research was only frozen-embryo transfer, the data includes both the blascocyst transfer and cleavage stage embryos transfer. And each number of blascocyst transfer and cleavage stage embryos transfer respectively was unknown, which would introduce bias in our results.
Besides, since the perinatal period starts at 28 complete weeks in China, the records of infants born before 28 gestational age are relatively incomplete. Therefore only gestational age above 28 weeks are included in this research. As a result, the exclusion of preterm birth between 24 and 28 weeks may lead to a reduction of the prevalence of PTB.
[MOU7] In addition,some information about environmental exposure (educational level, income level) was not included in this study, which may lead to bias. Further studies, particularly systematic reviews of observational studies such as the current study and prospective studies with adjustments for important confounders, will be required to con rm these initial ndings.

Conclusions
Taken together, these ndings indicate that maternal characteristics, in particular type of infertility, appears an additional risk factor for abnormal pregnancy outcomes besides use of IVF techniques. Lower risk is found in male infertility and higher risk for ovulation disorders. Doctors should fully inform patients of possible adverse pregnancy outcomes before they receive IVF. In addition, obstetricians should not only be aware of the increased risk of adverse outcomes with IVF but also pay attention to the speci c complications related to the cause of infertility and provide timely treatment. Further studies, including prospective studies, are needed to con rm the role of the underlying infertility-related diagnosis and severity of infertility in the increase in adverse outcomes with IVF after adjusting for important confounders.

Declarations
Ethics approval and consent to participate All procedures performed in studies involving human participants were approved by the local institutional ethics committee -The Beijing Obstetrics and Gynecology Hospital committee (ethics approval number: 2019-KY-024-01). Due to the retrospective study design, consent for participation was not required. Nevertheless, private information was well protected during the study.

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no con icts of interest.

Funding
This study was not funded.                 Note: *Logistic regression analysis was adjusted for age, gravidity, parity, pre-pregnancy obesity, birth plurality, and history of previous caesarean section. CI=confidence interval; aOR=adjusted odds ratio.
Mixed infertility refers to multiple infertility-related diagnosis; PTD=preterm delivery; SGA=small for gestational age( birthweight below the 10th percentile for gestational age); NICU= neonatal intensive care unit Macrosomia=birth weight≥4000g Bold indicates significant differences; NC=not calculated due to low numbers. Note: *Logistic regression analysis was adjusted for age, gravidity, parity, pre-pregnancy obesity, birth plurality, and history of previous caesarean section. CI=confidence interval; aOR=adjusted odds ratio.
Mixed infertility refers to multiple infertility-related diagnosis; PTD=preterm delivery; SGA=small for gestational age( birthweight below the 10th percentile for gestational age); NICU= neonatal intensive care unit Macrosomia=birth weight≥4000g Bold indicates significant differences; NC=not calculated due to low numbers.