Effects of Nitric Oxide Inhibition on Early Pregnancy Loss in Female Patients with Type 2 Diabetes
According to data accumulated by the US Census Bureau and the World Bank, the fertility rate in America has been on a steady decline since 2007 [Fig.1]. Many studies aim to determine the cause of this decline. The simultaneous rise of obesity and diabetes in America has lead researchers to study the link between fertility and diabetes. Type 2 Diabetes Mellitus (T2DM) is defined as decreased insulin sensitivity and production which lead to eventual pancreatic beta cell failure with diagnosed patients meeting the criteria for HbA1c results greater than 6.5% and a fasting plasma glucose 126 mg/dL [11]. An issue that is getting a little less attention would be the link between diabetes and early pregnancy loss (EPL). EPL is defined as fetal death, miscarriage or stillbirth, by or at 24 completed weeks of pregnancy. Although late pregnancy loss (beyond 28 weeks) has been declining since 1990, EPL has not followed the same trend [Fig.2]. It’s important to understand the connection between fertility and EPL. Fertility, the average number of births per woman, can decrease if there is high EPL but it is unlikely that EPL is, if present, the only factor responsible for low fertility rates in a given population. Diabetes has a strong association with vascular diseases due to its detrimental effect on multiple mechanisms including nitric oxide inhibition [3]. This mechanism has been proven vital to the growth and development of a fetus therefore it’s reasonable to hypothesize that EPL arises when those it is tampered with.
Fig.1 U.S. Census Bureau: International Database
Fig.2 CDC/NCHS, National Vital Statistics System
State of the Art : Polycystic ovary syndrome (PCOS) is one of the most common hormonal disorders affecting women of reproductive age today. Those affected have increased androgens levels due to high levels of luteinizing hormone (LH) or insulin which can in turn cause anovulation. A study in 2008 has shown that PCOS is very common in women with T2DM [11]. Women with PCOS have a 20-35% higher risk of early pregnancy loss which is clinically defined as a miscarriage during the first trimester [8]. It has been hypothesized that this is due to elevated LH levels and hyperandrogenism which also occurs in T2DM [8]. Considering that PCOS and T2DM also share many risk factors such as obesity, hypertension, and hyperinsulinemia it is reasonable to assume that T2DM can affect EPL similarly to PCOS. A swedish study was done from 1965-2004 which compared the fertility rates of women with T1DM to their non-diabetic counterparts and results showed a 20% decrease in fertility for those with T1DM but there has been limited recent studies with T2DM and pregnancy wastage.
Hypothesis : The prevalence of early pregnancy loss in female patients is increased in those with type 2 diabetes mellitus and this can be observed in the reduced levels of nitric oxide synthase.
Clinical need : Understanding the effect T2DM has on post-fertilization viability of a zygote can decrease the early pregnancy loss of female patients
Knowledge gap: Although other microvascular and macrovascular complications are getting mediated, it isn’t exactly known how to increase and improve placental vasculature. It is also unclear exactly how low NOs levels must be to affect pathology.
Perspective : To potentially provide insight on treatment for similar microvascular and macrovascular complications of diabetes.
Fig.3 Biotechnology Advances Vol 33 Issue 8
Nitric oxide is an essential signaling molecule that is involved in the function and metabolism of nearly every organ system. Nitric oxide itself is a highly reactive molecule so it isn’t consumed from the environment but rather made through various pathways in the body. Although endogenous nitric oxide is usually synthesized through the enzymatic pathway, a non-enzymatic pathway does exist but typically is only able to synthesize NO under acidic conditions. In respect to vasculature, NO regulates vascular tone, blood flow, and mitochondrial oxygen consumption. Irregularities in vascular NO result in endothelial dysfunction and other angiogenesis-associated disorders. NO has a very short half life in the blood which is why it is typically measured based in the concentration of its metabolites. Asymmetric dimethylarginine (ADMA) and NG-monomethyl- L-arginine ( L-NMMA) are two important nitric oxide synthase inhibitors. High ADMA has been linked to endothelial dysfunction because it inhibits NO. In order to help prevent atherosclerosis, studies were performed using L-arginine which has been found helpful in improving endothelium-mediated vasodilation but its clinical utility in diabetic patients has not been proven. Studies have also shown serum levels of both inhibitors to be elevated in patients with insulin resistance but once again it remains unclear as to whether they are the cause or simply a casualty of desentization.
What leads to natural embryo loss:
In order to fully understand how NO might potentially affect early pregnancy loss, natural causes of pregnancy loss must also be examined. Post-conception failure rates in humans are higher than one might expect. Not 45% of all conception fail and out of those that do not, half of the zygotes do not mature into blastocysts, and at least 40% of blastocysts will not implant. Spontaneous abortions (SA), or miscarriages typically occur in one of the 3 following ways; 1. A blighted ovum is defined as a gestational sac but no fetus on ultrasound 2. A missed abortion has a fetal pole or a fetus in the gestational sac but no fetal heart movement on ultrasound 3. A live abortion has “fetal heart movement demonstrated on ultrasound less than a week before the spontaneous abortion”. Most common reasons for SA involve chromosomal error including monosomies, polyploids, and translocations. SA rates also increase when maternal age increases but that is partly because it chances of chromosomal error increase as well. Being a cell signaling molecule that promotes proliferation and survival, it has been reported that NO is critical for the initiation of differentiation. Many of the signalling pathways activated by NO in other cell systems are present in embryonic stem cells.
A critical level of NO is imperative for the establishment of pregnancy. Embryonic implantation is a critical process when pregnancy occurs. Successful nidation of the blastocyst is dependent on synchrony between the embryo development and the endometrial transformation into the tissue that encapsulates the developing embryo, the decidua. Rodent models of NO during nidation have been examined and their findings include increased expression of NO synthase at the implantation site in the first few days of pregnancy. When treated with L-NAME, a synthetic NOS inhibitor, the number of successful implantations decreased significantly, further indicating the importance of NO. Assuming similar parameters in humans, T2DM would essentially mirror the effects L-NAME had on nidation and could be the cause for infertility in this population of women.
The first aim of this study would be to show a reduction of serum nitrite levels in female patients with T2DM during gestation. Nitric oxide synthase (NOs) acts as a vasodilator in uterine arteries and has numerous clinical trials have been done linking low NOs levels to hypertension and preeclampsia [3]. Throughout the gestational period, NOs levels steadily rise until 9-12 weeks postpartum and it has been hypothesized that this NOs is essential in regulating maternal and fetal cardiovascular disturbances [14]. T2DM can cause NOs inhibition which would in turn complicate uterine blood flow. In order to observe this affect firsthand, a cohort study following women seeking to conceive will collect serum samples to analyze the difference in nitrite and nitrate levels, since nitric oxide is spontaneously oxidized to both nitrite and nitrate, in women with or without T2DM throughout their pregnancy. To control for confounding variables, the should have no known previous miscarriages and any previous pregnancies and will have to be documented. Those with reproductive disorders (endometriosis, adenomyosis, polycystic ovary syndrome and uterine fibroids, uterine cancer, cervical cancer) will be labeled high-risk and will not be allowed to participate in the study. This will also serve to explore and determine the normal range of nitrites and nitrates in serum levels to in order to properly attribute a reduction to an effect within fetal pathology.
The second aim of this study would be to attribute a reduction in nitrite levels with impaired UBF and/or PBF which may affect uterine O2 delivery. In order to do so, the UBF (uterine blood flow) of female sheep with clinically low nitrite levels will be measured longitudinally throughout gestation and compared to their unaffected counterparts. Uterine o2 levels will remain at homeostatic levels until UBF falls below 50% if the rate of UBF was optimal before reduction.
Conclusion:
Although it is reported that elevated NO concentrations have been found in pathologic cases in endometriosis and recurrent miscarriages, I believe that, if comprised in a physiological range, increased NO may act at different sites to promote implantation in patients with T2DM. It could contribute to embryonic implantation by increasing: endometrial vascular permeability; prostaglandin mediated decidualization; programmed cell death; extracellular matrix degradation; and uterine relaxation.
Although further larger prospective studies are necessary to comprehend the complex factors that control the reciprocal signalling relationship between maternal and embryonic tissues, it may be concluded that NO is actively produced by the developing embryos and may be deeply involved in embryo nidation.