Effect of Endometrial Sodium Glucose Cotransporter SGLT1 on Embryo Survival
Ellen Miller, Kerry Peterson, University of Wisconsin-Stout
Abstract:
Embryo growth while needs the best possible environment, and is necessary to have an ample amount of endometrial
glycogen for this development and growth. The uptake of glucose, that later forms glycogen, requires Na and glucose-
dependent SGLT1 carrier in the endometrial epithelial cells. In this study, SGLT1-deficiency resulted in lack of glucose
transport, reduced litter size, glycogen content, and weight of their offspring in tested mice. Discovery of the SGLT1-
deficiency (SLC5A1) directly correlated with a consistent miscarriage (5).
Basis for Histiotrophic Nutrition
A Histiotroph is what provides nutrition to an embryo and will gather between the maternal and fetal tissues.
This is different from the other form of nutrition (hemotrophic), in which the nutrition is received via the
blood and from the placenta. Although only short term, Histiotrophic nutrition needs the SGLT1 receptor to
pick up ample glucose (2).
It is now also thought that histiotrophic nutrition prevails while hemotrophic nutrition is starting and thus is
more valuable to the fetus. The first 6 weeks especially use histiotrophic nutrition due to high glycogen
content. Even more, the first 9 weeks uses the glycogen stores and anaerobic glycolysis which, if the SGLT1 is
malfunctioning, could result in pregnancy loss (2).
SGLT1 Significance in glucose transport
SGLT1 is located on the intestinal luminal side of the enterocyte near the brush border (shown in the diagram below)
and requires ATP in order to perform. The job of the SGLT1 transport is to simultaneously transport glucose or
galactose molecules as a Na+ molecule is transported. There will not be a binding site for the glucose molecule if there
isn’t already a Na+ molecule attached. Once they are both inside the cell they are released from the transporter and
when there is plenty of glucose inside the cell, glucose will bind to GLUT2 to be pumped out of the cell and into the
blood to the liver. The Na+ that was pumped in with glucose will leave via the Na+/K+-ATPase pump, requiring
Potassium as well. This system, along with other transporter proteins, like GLUT1 & GLUT3, are necessary for the
survival and development of the growing embryo (3).
-Upper left box indicates the SGLT1
transports
-Right box indicates Na+/K+-ATPase
transport
Anaerobic Glycolysis
By definition, Anaerobic glycolysis means that there isn’t enough reducing agents to get pyruvate to create energy
because of a lack of oxygen or a high cellular metabolism. This will result in a conversion of pyruvate to lactate. The
lactate can be taken to the liver, and there it will produce glucose for energy. Although deoxygenated conditions aren’t
recommended, anaerobic glycolysis is used routinely with erythrocytes and the brain as well as in the GI tract. So it is
obvious where anaerobic glycolysis plays a role in development and in conditions where oxygen and thus glucose can
initially be in short supply (3).
Introduction
A total approximation of miscarriages is around 50%, a very common problem in human pregnancy. The luminal
epithelium prepares for the implantation of an embryo by creating a stable environment. Higher glucose concentration
is pertinent for preparation, if not recurrent pregnancy loss (RPL) results, which is categorized by 3 or more consecutive
pregnancy losses.
It’s important that glucose is effectively transferred from the mother to the embryo. This relies on hypoxic glycolysis, or
oxygen tension rises, for its energy and also needs anaerobic glycolysis, and therefore glycogen for energy and growth.
Glycogen is stored in the endometrial tissue. Glycogen synthesis and intracellular glycogen synthase requires uptake of
glucose and therefore needs a high affinity for the Na+-coupled glucose transporter SGLT1 (Histiotrophic nutrition), and
the gradient is driven by Na+/K+ ATPase.
This study looks at the build up of glycogen, the impact on the growth of the embryo and if a deficiency in SGLT1
receptor is an indicator of miscarriages (5).
Hypothesis:
Mobilization of glycogen to glucose from the endometrium may, in part, determine embryo survival post-implantation
and thus the litter size at birth (5).
References
(1) Aşkan, Ö. Ö., Bozaykut, A., Sezer, R. G., Güran, T., & Bereket, A. (2015). Effect of Maternal Factors and Fetomaternal Glucose
Homeostasis on Birth Weight and Postnatal Growth. Journal Of Clinical Research In Pediatric Endocrinology, 7(3), 168-174. doi:10.4274/jcrpe.1914
(2) Burton, G. J., Watson, A. L., Hempstock, J., Skepper, J. N., & Januniaux, E. (2002). Uterine Glands Provide
Histiotrophic Nutrition for the Human Fetus during the First Trimester of Pregnancy . The Journal of Clinical Endocrinology & Metabolism, 86(6). Retrieved from
https://doi.org/10.1210/jcem.87.6.8563.
(3) Gropper, S. A., Smith, J. L., & Carr, T. P. (2018). Advanced nutrition and human metabolism. Boston, MA: Cengage
Learning.
(4) Meng, C., Lianqiang, C., Jun, W., Mei, Y., Guoqi, S., Zhengfeng, F., & ... De, W. (2014). Effects of maternal over- and undernutrition on intestinal
morphology, enzyme activity, and gene expression of nutrient transporters in newborn and weaned pigs. Nutrition, 30(11/12), 1442-1447.
doi:10.1016/j.nut.2014.04.016
(5) Salker, M. S. (2017). Loss of Endometrial Sodium Glucose Cotransporter SGLT1 is Detrimental to Embryo Survival
and Fetal Growth in Pregnancy. Scientific Reports . Retrieved from https://www.nature.com/articles/s41598- 017-11674-3.pdf.
Materials and Methods
Subjects: SGLT1-containing mice or Slc5al +/+ (wild type) & SGLT1-deficient mice or Slc5al -/-
Timeframe: Endometrial Biopsies were taken 6-10 days after the preovulatory surge
Protocol: The animals used (mice) were approved and treated under the German law and approved by the ethics authority. The
biopsies occurred during ovulatory cycles and without the use of hormones for at least 3 months before. The control group were
those with a delay in conception due to factors other than SGLT1 deficiency. The uteri were isolated from both the SGLT1 mice and
Figure 1: SGLT1-deficient mice. The diets varied: SGLT1 mice got standard feed while SGLT1-deficient mice got sugar-free diet with added
vitamins and minerals. One week before the study they were all fed the sugar-free diet. For four weeks the SGLT1 mice got a low
glucose diet and then pup were counts after 8 weeks.
Measurements:
• Human Endometrial Stromal Cells (HESC): isolated, expanded, decidualized and done before the third cell passage. This was
performed to separate the cells and allow a better look at the contents, including if they contained SGLT1 transporters and and if
so, how many.
• Laser Microdissection coupled to RNA-sequencing: extracted RNA and counted transcripts per million and assessed. Performed
in order to extract RNA as samples from the inside layers of endometrial glands and counted transcripts used to make SGLT1
transports.
• End-point RT-PCR: Uteri were removed from mice: two types of samples of RNA; two from the SGLT1 mice and two from the
SGLT1-deficient mice. cDNA was synthesized from the RNA samples stored for later denaturation and then visualization under UV
light. Performed to assess amount of SGLT1 transports and health of RNA.
• Quantitative Real-time (qRT): extract the RNA from the previously mentioned HESC cultures and were reversely transcribed and
analyzed and showed an amplification in transcription levels. Performed to assess likelihood of transcription and replication of
RNA for aid in growth of embryo.
• Western Blotting: Performed to detect SGLT1 protein availability by separating the proteins equally. There was a safeguard,
overnight incubation, to make sure that nonspecific binding sites were blocked. Three or more cell culture variations were used
to detect the proteins and identified by primary antibodies against human SGLT1.
• Microscopy: Performed to inspect the mouse uteri and measures were again taken to inhibit nonspecific binding of antibodies.
Figure 2: • Ussing Chambers: Performed to analyze the uterine segments from the SGLT1-deficient mice and were saturated with high
glucose concentrations to identify a current of glucose and the effectiveness of the SGLT1-deficient mice and glucose intake.
• PAS staining & H&E: Performed to detect polysaccharides, namely glycogen, in the pseudo-pregnant uteri. This happened with
the use of Schiff’s reagent and Hematoxylin solution to properly identify the presence of glycogen and the nuclei.
• Glycogen concentration: Performed to assess utilization of glycogen in the uteri by use of a glycogen assay kit as well as
enzymatic assay.
• Cellular Glucose uptake: Performed to measure uptake of glucose by flow cytometry as well as detecting SGLT1 inhibition on
glucose uptake and effect of Na+ (5).
Results (All results used Microscopy)
• HESC, Laser Microdissection coupled to RNA-sequencing, End-point RT-PCR, & Quantitative Real-time (qRT): SGLT1, encoded
by SLC5A1 transcript, were found to be highly increased in human endometrium specifically in mid-secretory phase of
pregnancy. Figure 1a & b show an increase in the SLC5A1 transcript and thus SGLT1 protein due to endometrial stromal cells
need. Figure 1c shows SLC5A1 transcripts were only found in SGLT1 mice, there was also no reactivity of SGLT1 in the SGLT1-
deficient mice shown in figure 1d. All in all, SGLT1 was only being made and used in the SGLT1 mice with the help of the SLC5A1
transcripts encoding.
•
Ussing Chambers: Figure 2b show elecrogenic glucose transport across the endometrium was evident only in SGLT1 mice and in
Figure 2a glucose uptake was significantly impaired in human endometrial cells when extracellular Na+ was removed or a SGLT1-
transport inhibitor was added (b).
• PAS Staining & H&E, Glycogen concentration, & Cellular Glucose uptake: Glycogen content was found to be 60% lower in SGLT1-
deficient mice compared to SGLT1 mice (Figure 3a), and therefore glycogen deposits (Figure 3b). In addition, the number of
glands in the cross-section of the uterine wall of a SGLT1-deficient mouse were reduced (Figure 3b).
• Western Blotting: It can be noted that the number of implantation sites (for an embryo) was dramatically lower (Figure 4a) in
SGLT1-deficient mice which equaled lower average litter size (Figure 4b) and lower birth weights (Figure 4c) (5).
Discussion
As observed, a lack of SGLT1 transporter, and thus a lack in glycogen stores, is directly related to miscarriage. In order for there
Figure 3: to be a high concentration of SGLT1 there needs to be sufficient amount of SLC5A1 transcripts to encode SGLT1. A high Na+
gradient is necessary for the SGLT1 glucose transporter to function. A lack of glycogen formation may result in the compromised
health of an embryo. SGLT1 activity is catalyzed by a kinase (SGK1). Without SGK1 there is the potential for failed pregnancy due
to the lack of glycogen or energy to the developing embryo (5).
• A study regarding birth weight and maternal glucose levels explains that the maternal HgbA1c, and thus glucose in blood not
taken up by the SGLT1 and likelihood of Diabetes, was directly related to a larger birth weight. These larger gestational age
babies eventually had a decrease in growth by 6 months of age (1).
• In contrast, a study discussing maternal nutrition (in pigs) affecting the baby during and after pregnancy concludes that there
is not a restricted transcription of SGLT1 transporters in undernourished mothers which could lead to potential catch up
growth of the baby post-birth. However, in over nourished mothers, SGLT1 gene expression was up-regulated in order to
compensate for the rapidly growing baby. It was also stated that lower maternal dietary intake decreases placenta blood flow
causing decreased fetal growth (4).
Figure 4: Summary
The lack of the SGLT1 Na+/ glucose coupled co-transporters has shown up more in the endometrial biopsies
that are having issues in pregnancy, miscarriages, or lower birth weight babies. It is necessary for glucose,
and thus glycogen stores, to be abundant so that the growing fetus can thrive since the SGLT1 transporters
are what carries the glucose across the endometrial membrane to the baby. The sufficient glucose
concentrations and glycogen stores are accomplished by anaerobic glycolysis and the sufficient amount of
SLC5A1 transcripts. Without enough SGLT1 receptors, not enough glucose is brought to the baby and there is
not a positive outcome. The mother will be advised to be tested for enough SGLT1 receptors and if there are
not enough, or she has SGLT1 inhibitors, then she is advised to undergo treatment for Diabetes so that
glucose can be effectively transferred to her and the growing baby to reduce further health issues to the
baby. In this case, she will likely be referred to an RD for assistance in diet and glucose intake.