The global obesity epidemic has led to detrimental consequences on human bodily functions including reproductive health (1), with obesity being recognized as one of the leading causes for the decrease in fertility rate. Infertility affects between 8% and 12% of the reproductive age group population, and in certain regions, such as the Middle East and North Africa, a region that includes Qatar, the infertility rate is estimated to be as high as 30%. During the last decade, Qatar has seen a steady rise in the overweight/obese population and a decline in fertility rates among women (2). Obese women have a higher incidence of menstrual dysfunction, anovulation, pregnancy complications, polycystic ovary syndrome, and fertility issues. Furthermore, the rate of miscarriages increases as BMI increases (3). Excess adipose tissue in obese women secretes elevated inflammatory adipocytokines that alter the hormonal balance and cause reproductive disturbances. Elevated levels of inflammatory cytokines, together with elevated insulin secretion, induce hyperandrogenemia and promote granulosa cell apoptosis, which may affect both ovaries (4) and endometrium (5), causing fertility problems.

Complement proteins are secreted by the immune cells, which contribute to danger sensing by activating complement receptors on their target cells. Such activation participates in the type and magnitude of the immune response together with signaling pathways activated in response to pattern recognition receptors (6).

Adipsin, also known as complement factor D, is a member of the trypsin family of peptidases and is secreted mainly from adipocytes, monocytes, and macrophages (7, 8). It plays a critical role in the development of the C5–C9 membrane attack complex and the production of several signaling molecules, including the anaphylatoxins, C3a and C5a (9–11). It is also involved in the first step of activation of the alternate complement pathway ( Supplementary Figure 1 ), where it produces the C3bBb complex (C3 convertase) by factor B and C3b. C3 convertase is responsible for cleaving C3a from C3 and releasing C3b (12). Adipsin regulates adipose tissue homeostasis and elevates glucose secretion (9). It also regulates the differentiation of adipocytes and promotes the accumulation of lipids, which is hypothesized to be a potential cause for the association of adipsin with metabolic disorders (13). Indeed, elevated adipsin levels have been associated with ischemia perfusion (14) and sepsis (15). Furthermore, increased levels of circulating adipsin were closely associated with polycystic ovary syndrome (16), mild cognitive impairment in type 2 diabetic mellitus patients (18), and coronary artery disease (17), suggesting adipsin as a promising biomarker for the diseases. C5a, on the other hand, is a very potent complement factor that plays a chemoattractant role by inducing the migration of many cells involved in the immune response and wound healing, including neutrophils and macrophages (19). It also links innate and adaptive immunity, extending its role in inflammation (19). Previous studies have suggested a role of C5a in the risk of diabetic kidney disease (20) and cardiovascular disease (21).

The plasma levels of adipsin and C5a were shown to be significantly elevated prior to delivery in pregnant women with preeclampsia (22); however, in healthy pregnant women, plasma adipsin and C5a were increased from the third trimester (23). As noted above, the main function of adipsin is to catalyze the breakdown of complement factor C3; therefore, adipsin may affect the downstream molecules such as C3a and C5a that have been shown to be increased in pregnancy, indicating that the complement system is activated during normal pregnancy (Karina 24).

The complement system exhibits both damaging and protective roles at the placental level. Activation of complement factors at the fetal–maternal interface protects against infectious agents and removes apoptotic and necrotic cells (25). However, various reports have implicated complement activation in the pathogenesis of adverse pregnancy outcomes (26–30). Despite evidence suggesting involvement of complement factors in obstetrics diseases, no study has investigated their concentrations with regard to in vitro fertilization (IVF) outcome. The hypothesis of this study was that complement factors are dysregulated in obese/overweight women who fail to have successful pregnancy following IVF treatment. To address this hypothesis, levels of 14 complement factors were assessed in the maternal serum and follicular fluid (FF) and compared in relation to IVF pregnancy outcome in overweight/obese women and other inflammatory markers.

Based on their pregnancy outcome, patients were dichotomized into successful (n = 14) and unsuccessful (n = 26) pregnancies. As shown in Table 1 , women who had successful pregnancies were slightly younger (average 4.5 years, p = 0.01) with higher ALT (p = 0.01) and AST (p = 0.004), but lower LH (p = 0.03) and lesser infertility duration (p = 0.04).

It is critical to identify novel biomarkers that may identify those women who will have a favorable outcome and those who will not from those who are undergoing assisted reproductive technology; this may guide the therapeutic strategy. Whereas serum may offer a general indication of the woman’s health, the FF during IVF treatment reflects the intimate environment of the developing cumulus/oocyte complex (34). Pregnancy activates the complement system by triggering the innate immune response, leading to elevation in the levels of C3a, C4a, and C5a. This elevation compensates for the inhibition of adaptive immunity during the normal pregnancy (24) and plays a critical role in pregnancy outcome (35). However, reports have indicated that circulating levels of C3a and C5a pose increased risk of preeclampsia (36). The interface between maternal and fetal tissues is enriched with complement inhibitors to protect the placenta from consequences of complement activation and adverse pregnancy outcomes (37). Complement components are also present in the mucosal secretions of the fallopian tubes, cervix, and uterus (38), suggesting that pregnancy failure could potentially result from excessive complement activation in the pre-implantation stage (35). In this study, we have assessed the levels of complement factors in the pre-IVF serum, serum at the time of oocyte retrieval, and FF samples obtained from women undergoing IVF cycles and compared their levels in relation to IVF outcome (i.e., successful or unsuccessful pregnancy and number of top-quality embryos). Out of 14 assessed complement factors, only adipsin and C5a were significantly associated with IVF outcome. Higher levels of pre-IVF serum and FF adipsin and lower levels of serum C5a were identified in successful pregnancies. In order to understand the potential mechanisms underlying these associations, levels of various cytokines, adipokines, and myokines were determined in the pre-IVF serum and FF samples from the same women and compared in relation to IVF outcome. Serum adipsin levels were positively correlated with circulating levels of vitamin D, glucagon, leptin, resistin, and visfatin, but negatively correlated with total protein. Higher numbers of top-quality embryos were associated with increased levels of C3, properdin, C1q, factors H and B, C4, and adipsin, but with reduced C2 and C5a levels. However, serum taken at the time of FF removal showed no correlation of the complement proteins or with any other parameters, suggesting that the intervention of the exogenous gonadotrophin therapy and the hormonal changes that resulted in oocyte stimulation have modified the complement system abrogating the predictive indices seen in the pre-IVF sera for these patients. The effect of exogenous gonadotrophin therapy on the complement system during oocyte stimulation is not known and needs further specific investigation.

The higher levels of adipsin in the pre-IVF sera and FFs taken from women with successful pregnancies suggest a positive role of adipsin in the pregnancy outcome. The higher serum levels of adipsin prior to implantation could reflect a protective phenotype since serum adipsin levels are negatively associated with insulin resistance, especially in overweight and obese subjects (39). Studies have indicated that adipsin improves the maintenance of β-cell function as adipsin knockout mice exhibit glucose intolerance due to insulinopenia while replenishment of adipsin boosts their insulin secretion (9). Indeed, studies have shown that insulin resistance lowers implantation rate in the in vitro maturation/IVF embryo transfer cycle (40). Therefore, lower adipsin in failed cycles could reflect insulin resistance-mediated lowering of the maturation/IVF embryo transfer cycle. Our data showed that serum adipsin levels were positively correlated with serum vitamin D, glucagon, and various adipokines such as leptin, resistin, and visfatin. However, other studies suggested a negative role for adipsin in pregnancy being associated with metabolic changes during pregnancy (41, 42), including pathogenesis of preeclampsia due to its role in activation of factor B with a direct association with the development of preeclampsia (43, 44).

Several studies have indicated the important physiological role of adipocytokines in metabolism (45). Leptin was suggested to be involved in the control of reproductive functions by acting both directly on the ovaries and indirectly on the central nervous system (46), although other reports have suggested no correlation with pregnancy outcome (47). Reports have also shown that resistin does not correlate with IVF outcome (48). Visfatin, on the other hand, was found to restore ovarian aging and fertility in aging mice (49). Furthermore, previous studies have shown that women with elevated vitamin D had more successful IVF cycles (50). Inflammation is another critical factor for embryo implantation during pregnancy. A proinflammatory environment is essential during embryo implantation (51), followed by suppression of inflammation for the rest of the pregnancy until onset of labor. However, if chronic or acute inflammation persists for a longer duration within the uterine cavity, this may increase the chance of spontaneous abortion or preterm labor. Obese women are likely to have a low-grade inflammatory state throughout pregnancy, which may compromise embryo implantation. This may explain why the success rate of IVF-assisted pregnancy is lower in obese women.

Our data showing higher levels of C5a in the sera taken from women with unsuccessful pregnancies suggest a negative role for C5a in the pregnancy outcome. Our data therefore are in agreement with previous studies implicating elevated C5a levels in trophoblast dysfunction, impaired placental angiogenesis, and adverse pregnancy outcomes (36, 52, 53). Higher levels of C5a were also associated with preterm birth by increasing contraction frequencies (35), cervical remodeling, and fetal brain injury (54–56). Therefore, higher maternal C5a serum levels could be an indicator of failed IVF cycles, as suggested by our data.

Our data also indicate that higher numbers of top-quality embryos are associated with increased levels of C3, properdin, C1q, factors H and B, C4, and adipsin, but with reduced C2 and C5a levels. Levels of specific substances in FF were previously shown to be associated with fertilization outcome and early post-fertilization development, including elevated levels of LH, growth hormone (GH), prolactin, 17β-estradiol (E2), and insulin-like growth factor (IGF)-I and lower IL-1 in women with successful pregnancies. Furthermore, LH and GH levels were higher in follicles, resulting in top-quality embryos with the best morphology and fastest cleavage rate (57). The immunomodulatory role of locally produced complement factors, including C3, properdin, C1q, C4, adipsin, and factors H and B, in immunological tolerance and cellular survival was previously established (58–60). The positive correlation between top-quality embryos and these complement factors could reflect a pro-survival environment for developing oocytes that resulted in better-quality embryos. Future studies are warranted to investigate the functional relevance of these associations in larger independent cohorts.

A strength of this study was that our cohort does not include known causes of infertility, but further studies on specific causes of infertility are needed. The fact that the study utilized pre-IVF and matched serum at the time of oocyte retrieval when the FF samples were taken is also a major strength. Limitations include the fact that our complement cascade panel only had 14 complement-related protein markers and so other crucial proteins involved in the activation of complement cascades were not measured, including protein D hydrolysis and activation mechanisms associated with the MASP proteases. Our pilot study was undertaken in a small cohort of women, and larger studies are needed in the future to confirm and extend our findings. In addition, all the women were Qatari and, therefore, the results may not be generalizable to other ethnic groups.

This study reports, for the first time, higher adipsin and lower C5a levels in the pre-IVF serum in successful IVF-assisted pregnancies and showed positive correlations between complement factors and embryo quality, with a potential utilization as predictive biomarkers of successful pregnancies in obese women.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The study was approved by the Institutional Review Boards of the Hamad Medical Corporation (15101/15) and Weill Cornell Medical College in Qatar (15-00016) research Ethics Committee, and all study participants signed an informed consent form prior to participation. The patients/participants provided their written informed consent to participate in this study.

MR designed the study and performed the measurements. ID and NR analyzed the data and performed statistical analysis and prepared the tables. YD, LA, TA, MS, MB, EE, HB, and SA supervised clinical studies, recruited the patients, and collected demographics. AB prepared pathway analysis for the manuscript. SA, MR, AA-S, and ME supervised the study and interpreted data. ME wrote the manuscript. All authors contributed to the article and approved the submitted version.

Authors MR, TA, MS, MA, HB, and AA-S were employed by Hamad Medical Corporation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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