Initiation of metformin in early pregnancy results in fetal bioaccumulation, growth restriction, and renal dysmorphology in a primate model

Abstract

Background: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, and (most recently) preeclampsia. However, with its expanded use, concerns of unintended harm have been raised.

Objective: This study developed an experimental primate model and applied ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology.

Study design: Within 30 days of confirmed conception (defined as early pregnancy), 13 time-bred (timed-mated breeding) Rhesus dams with pregnancies designated for fetal necropsy were initiated on twice-daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet. Metformin or placebo vehicle control was delivered in various treats while the animals were separated via a slide. A cesarean delivery was performed at gestational day 145, and amniotic fluid and blood were collected, and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry in selected reaction monitoring against standard curves.

Results: Among 13 pregnancies at gestational day 145 with fetal necropsy, 1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 μmol metformin/kg maternal weight or fetal or placental tissue), whereas a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight of 248.32 g [<1%]) and was suspected of having a fetal congenital condition. After excluding these 2 fetal pregnancies from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4: 3 female and 1 male fetuses) or 10 mg/kg metformin (n=7: 5 female and 2 male fetuses) were available for analyses. Among dams initiated on metformin at gestational day 30 (regardless of maternal diet), significant bioaccumulation within the fetal kidney (0.78-6.06 μmol/kg; mean of 2.48 μmol/kg), liver (0.16-0.73 μmol/kg; mean of 0.38 μmol/kg), fetal gut (0.28-1.22 μmol/kg; mean of 0.70 μmol/kg), amniotic fluid (0.43-3.33 μmol/L; mean of 1.88 μmol/L), placenta (0.16-1.00 μmol/kg; mean of 0.50 μmol/kg), fetal serum (0.00-0.66 μmol/L; mean of 0.23 μmol/L), and fetal urine (4.10-174.10 μmol/L; mean of 38.5 μmol/L) was observed, with fetal levels near biomolar equivalent to maternal levels (maternal serum: 0.18-0.86 μmol/L [mean of 0.46 μmol/L]; maternal urine: 42.60-254.00 μmol/L [mean of 149.30 μmol/L]). Western-style diet feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta, or fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (P<.05), collectively driving lower delivery weight (P<.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness. This renal dysmorphology was not accompanied by structural or functional changes indicative of renal insufficiency.

Conclusion: Our study demonstrates fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology after maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

Keywords: fetal bioaccumulation; fetal programming; insulin resistance; metformin use; obesity.

Figure 1.. Metformin does not undergo first pass metabolism and bioaccumulates in maternal-equivalent concentrations in fetal kidney, fetal urine and amniotic fluid as assessed by mass spectrometry.

(A) Renal clearance without first-pass metabolism in a non-pregnant male Rhesus macaque. A standard dose of 10 mg/kg was orally administered to an adult male Rhesus macaques every 9 hours, with urine concentrations at log-fold higher than serum or plasma. (B-E) Maternal metformin bioaccumulates in the fetus. Dams were initiated on 10 mg/kg twice daily metformin or vehicle control by gestational day 30 (G30), and fetuses were delivered via Cesarean at G145. Amongst the n=11 included dams, metformin was not detected with vehicle alone (not shown), and levels did not differ by maternal diet (chow diet vs maternal Western style diet; WSD). *Spearman’s correlation, dashed colored lines for 95% CI, shows significant separation from 1:1 line of identity (dashed). Males are represented as triangles, females as circles. Means −/+ SD are shown. Main effects from ANOVA are shown.

Figure 2.. Initiation of maternal metformin use by G30 and continued duringdruingduring pregnancy twice daily does not result in maternal nor fetal anemia nor hypoglycemia.

Umbilical artery (fetal) blood was collected at G145 (n=2–4/group). Blood gasses, hematocrit, BUN, lactate, and glucose were measured. Maternal glucose concentrations were collected and measured the day prior to Cesarean surgery prior to fasting. Results were analysed by two-way ANOVA. Main effects and measure of significance shown by p value. Males are represented as triangles, females as circles. Means −/+ SD are shown. Chow diet (CD, white bars) or Western style diet (WSD, grey bars)

Figure 3.. Maternal metformin initiation and use during pregnancy reduces fetal visceral and body weight.

Fetuses were delivered via Cesarean at G145 (n=2–4/group) and (A) fetal weight and fetal organ and tissue weights were measured (B-F). Fetal crown-rump length (CRL) (F) and biparietal diameter (BPD) (G) were also directly measured. Results analysed by two-way ANOVA. Main effects and measure of significance shown by p value. Males are represented as triangles, females as circles. Means −/+ SD are shown. Chow diet (CD, white bars) or Western style diet (WSD, grey bars).

Figure 4.. Maternal metformin initiation and use during pregnancy reduces fetal skeletal muscle mass.

Cross-sectional area (CSA) was measured at the muscle midbelly in the fetal medial gastrocnemius at G145 from dams treated with twice daily metformin or a vehicle control and fed either a chow diet (CD, white bars) or Western style diet (WSD, grey bars), n=2–4 per group. (A) Representative images of muscle from each group taken at 20x magnification with scale bar. (B) CSA was calculated for each animal using a minimum of 500 fibers. Results were analyzed by two-way ANOVA with main effects and measure of significance shown by p value. Data are the mean ± SD. Males are represented as triangles, females as circles. (C) Frequency distribution for fiber size was calculated per animal and frequency per bin averaged by group. Data sets were compared to test if data fit same or different best-fit curves, with the p value indicating that best fit curves are different across groups. Graph shows frequency of data at each 100 unit bin and the best fit curve of the data.

Figure 5.. Representative images of fetal renal histology and ultrastructure with maternal metformin initiation and use during pregnancy.

(A, D, G, J) H&E staining of the renal cortex from capsule (outer edge) to the corticomedullary junction. Glomeruli (G) develop from the capsular edge and travel toward the corticomedullary junction during development, following the path indicated in panel A (dashed line) between tubule bundles (T). Animals exposed to metformin exhibited greater disorganization, reduced generations, and greater proliferation of glomeruli near the renal capsule. (B,E,H, K) H&E stains at greater magnification to increase mesangial proliferation within the glomeruli of metformin exposed fetuses (arrows). (C,F,I,L) Electron microscopy ultrastructural images of renal glomeruli (dashed line) including podocytes, with no evidence of ultrastructural disruptions with fetal metformin exposure in utero.

Figure 6.. Quantitative analyses of fetal renal histology.

(A) The number of fetal glomerular generations present varied significantly in association with both maternal metformin use and WSD feeding. (B) Within each generation, there was increased proliferation of glomeruli in association with maternal metformin exposure, but not with maternal WSD. The disordered patterning (C) and generational proliferation (D) (as compared to absence of proliferation) of fetal glomeruli was observed with maternal metformin exposure during pregnancy independent of maternal diet as blindly assessed by a pediatric pathologist. The significance was assessed by two-way ANOVA (A&B) and Fisher’s exact test (C&D).