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Background: Randomized controlled trials have shown that metformin treatment during pregnancy slows down gestational weight gain (GWG) and reduces the risk of preterm birth in women with polycystic ovary syndrome (PCOS), but these trials have not investigated why metformin treatment produces these effects. Studies of metformin's mechanisms of action have mostly been in-vitro studies of cell lines or animal models, or clinical studies of non-pregnant human populations, and it is unknown whether the results of these studies are applicable to human pregnancy. Neonatal outcomes following metformin treatment have been extensively evaluated against insulin treatment for gestational diabetes mellitus (GDM). However, previous assessments have generally combined results from participants treated with metformin alone and results from those who also required supplemental insulin, which makes it difficult to assess effects of metformin per se, and evaluations against diet and lifestyle treatment are lacking.

Aim: The objectives of this thesis were to explore the potential mechanisms by which metformin treatment during pregnancy slows down GWG, affects fetal growth, and reduces the risk of preterm birth in women with PCOS, and to assess the risk of neonatal hypoglycemia following metformin-treated, insulin-treated, and diet-and-lifestyle-treated GDM.

Method: In Studies I-III, we investigated appetite-regulating hormones and immunological factors in serum and placental tissue obtained from women with PCOS treated with either metformin or placebo. In addition, a group of healthy women with normal pregnancies were included as a reference group. In Study IV, we evaluated associations between metformin treatment and neonatal hypoglycemia, and other neonatal outcomes associated with fetal hyperinsulinemia. We used a register-based approach, and a population-based cohort that consisted of more than 16 000 women with GDM, and their singleton offspring. Metformin as a single adjunctive treatment was assessed separately from metformin combined with insulin treatment.

Results: Women with excessive GWG were more leptin resistant throughout pregnancy, and displayed a lower physiological serum allopregnanolone increase in late pregnancy than women who maintained a healthy GWG (Paper I). Metformin treatment improved leptin sensitivity and counteracted excessive GWG in women with PCOS (Paper I). This treatment effect was uncorrelated with placental leptin and leptin-receptor mRNA expression in women with PCOS (Paper II). Placental leptin mRNA expression correlated positively with the birthweight/placental weight ratio in placebo-treated women with PCOS (Paper II). PCOS status was associated with enhanced decidual immune-cell mobilization, particularly greater abundance of CD4⁺ T cells, and with altered placental IL-18 and IL-5 cytokine mRNA expression (Paper III). Metformin treatment altered the immunological landscape at the maternal-fetal interface in women with PCOS. This was shown by greater abundance of decidual CD56⁺ cells, downregulation of placental IL-4 and IL-18 mRNA expression, fewer placental pro-inflammatory intra-class cytokine mRNA correlations, and different cytokine mRNA expression profiles compared with placebo (Paper III). Offspring exposed in utero to only metformin as a pharmacological treatment for GDM appeared to be at similar risk of neonatal hypoglycemia to infants exposed to diet and lifestyle treatment alone, and at lower risk compared to offspring exposed to insulin, regardless of whether the insulin was administered as monotherapy or in combination with metformin (Paper IV).

Conclusions and implications: Metformin treatment effectively reduces the risk of excessive GWG and appears to counteract physiological leptin resistance during pregnancy in women with PCOS. However, a positive correlation between placental leptin mRNA expression and the placental-efficiency measure ‘birthweight/placental weight ratio’, was erased by metformin treatment. The clinical implication of this finding is unclear, and future research should aim for deeper insight into this mechanism for clarification. Metformin treatment induced complex immunomodulatory effects at the maternal-fetal interface in women with PCOS, but further research is required to determine if these findings can explain why metformin reduces the risk of preterm birth in PCOS. The similar risk of neonatal hypoglycemia to diet and lifestyle treatment is reassuring for all metformin-treated women with GDM that achieve glycemic targets without requiring supplemental insulin. In summary, this thesis contributes to increasing knowledge of how metformin treatment during pregnancy affects metabolic adaptations of importance for maternal weight gain and fetal growth in women with PCOS. Further, it provides some insights into how PCOS status and metformin treatment affect the immunological landscape at the maternal fetal interface; expands previous knowledge of how metformin treatment for GDM associates with neonatal hypoglycemia; and demonstrates the importance of differentiating between metformin with and without supplemental insulin when assessing treatment-associated risk of adverse outcomes.

I denna avhandling har vi studerat olika metabola och immunologiska aspekter som skulle kunna förklara varför metformin dämpar viktuppgång och förebygger för tidig (prematur) förlossning vid polycystiskt ovarialsyndrom (PCOS), samt hur metforminbehandling vid graviditetsdiabetes påverkar risken för lågt blodsocker hos nyfödda barn.  

PCOS är en hormonell rubbning som förekommer hos 10-13% av kvinnor i barnafödande ålder. PCOS förknippas med glesa eller inga menstruationer, förhöjda nivåer av manliga könshormoner, nedsatt insulinkänslighet, obesitas och en mild kronisk inflammation. Metformin förbättrar känsligheten för insulin och sänker därmed blodsockret och är den vanligaste läkemedelsbehandlingen vid typ 2 diabetes. Eftersom metformin även sänker nivån av manliga könshormoner, dämpar inflammation och motverkar viktuppgång används det ofta av kvinnor med PCOS för att mildra effekterna av tillståndet och för att öka chansen att bli gravid. Vanligtvis avslutas behandlingen när kvinnan blir gravid. Kvinnor med PCOS tenderar att ha en större viktuppgång än gravida utan PCOS, föder oftare för tidigt och drabbas oftare av graviditetsdiabetes och havandeskapsförgiftning. Tidigare forskning har undersökt om metforminbehandling under graviditeten kan förebygga dessa graviditetskomplikationer och har visat att viktuppgången begränsas och att risken för prematur förlossning minskar hos behandlade kvinnor med PCOS. Varför metformin-behandling ger dessa effekter har man däremot inte studerat. Kunskapen om hur fysiologiska förändringar som uppträder vid en graviditet påverkas av metforminbehandling är bristfällig eftersom studier av metformins verkningsmekanismer främst är genomförda i cell- och djurmodeller, och i kliniska studier med icke-gravida deltagare. För att främja näringstillförseln till fostret anpassas metabolismen och för att det genetiskt främmande fostret inte ska stötas bort anpassas immunförsvaret. Det är därför inte självklart att effekter av metformin blir desamma vid behandling i samband med graviditet.

Vi har i denna avhandling jämfört metforminbehandling vid PCOS mot placebo. Vi visar att metformin ökar känsligheten för mättnadshormonet leptin och minskar risken för ohälsosamt stor viktuppgång under graviditeten hos kvinnor med PCOS. Vi fann också att de som gick upp för mycket i vikt var mindre känsliga för leptin redan i början av graviditeten än de som begränsade sin viktuppgång. I motsats till vad vi förväntade oss förefaller däremot inte dessa effekter vara medierade av genuttrycket av leptin i moderkakan. Hos kvinnor med PCOS fann vi tecken på förhöjd immunaktivering i moderkakan i form av en ökad mängd vita blodkroppar i vävnaden, jämfört med moderkaksvävnad från friska gravida, vilket skulle kunna vara en bidragande orsak till den förhöjda risken för graviditetskomplikationer vid PCOS. Metforminbehandling föreföll ge upphov till en komplex påverkan på immunaktiveringen i moderkakan och dämpade både faktorer som bidrar till och motverkar inflammation. För att kunna fastställa om dessa effekter förklarar varför metformin minskar risken för prematurbörd vid PCOS krävs dock ytterligare studier.

Under senare år har det blivit vanligt att metformin används för behandling av graviditetsdiabetes när kostbehandling inte ger tillfredställande blodsockerkontroll. Tidigare forskning som jämfört metformin- och insulinbehandling har inte gjort någon åtskillnad mellan metformin i sig och behandling som kombinerats med insulin. Det saknas även studier där man utvärderat metforminbehandling gentemot enbart kostbehandling. Genom att koppla samman data från olika register och justera för ett antal störfaktorer undersökte vi hur sannolikheten för lågt blodsocker hos det nyfödda barnet påverkas av metforminbehandling. Risken för lågt blodsocker första timmarna efter födelsen var likvärdig när modern behandlats med metformin som tilläggsbehandling eller enbart kostbehandlats. Däremot ökade risken signifikant när modern fått tilläggsbehandling med insulin, enbart eller kombinerat med metforminbehandling. Barn vars mödrar behandlats med insulin vägde mer vid födelsen än de vars mödrar enbart kostbehandlats eller enbart fått metformin. I övrigt fann vi inga säkra samband mellan typ av behandling och andra nyföddhetskomplikationer.

Sammanfattningsvis bidrar denna avhandling till ökad kunskap om hur metforminbehandling under graviditeten påverkar metabola processer som har betydelse för viktuppgång och fostertillväxt hos kvinnor med PCOS, samt ger en inblick i hur både PCOS och metforminbehandling påverkar immunförsvaret i moderkakan. Avhandlingen utvidgar även tidigare kunskap om hur metformin påverkar risken för lågt blodsocker hos nyfödda barn vid graviditetsdiabetes och belyser vikten av att särskilja metformin i monoterapi från kombinationsbehandling med insulin. 

Introduction: Neonatal hypoglycemia is a common complication associated with gestational diabetes and therefore relevant to consider in evaluations of maternal treatment. We aimed to investigate the risk of neonatal hypoglycemia in offspring exposed to metformin treatment alone (MT) or combined with insulin (MIT) in comparison with nutrition therapy alone (NT), and insulin treatment alone (IT). In addition, we investigated MT in comparison with MIT. Secondary outcomes included neonatal anthropometrics, respiratory morbidity, hyperbilirubinemia, 5-min Apgar score, and preterm birth.

Material and methods: This Swedish population-based cohort included 16 181 women diagnosed with gestational diabetes, and their singleton offspring born in 2019–2021. We estimated risk as adjusted odds ratio (aOR) with 95% confidence interval (CI), using individual-level, linkage register-data in multivariable logistic regression models.

Results: In the main analysis, MT was associated with a lower risk of neonatal hypoglycemia versus NT (aOR 0.85, 95% CI: 0.74–0.96), versus MIT (0.74 [0.64–0.87]), and versus IT (0.47 [0.40–0.55]), whereas MIT was associated with a similar risk of neonatal hypoglycemia versus NT (1.14 [0.99–1.30]) and with lower risk versus IT (0.63 [0.53–0.75]). However, supplemental feeding rates were lower for NT versus pharmacological treatments (p < 0.001). In post hoc subgroup analyses including only exclusively breastfed offspring, the risk of neonatal hypoglycemia was modified and similar among MT and NT, and higher in MIT versus NT. Insulin exposure, alone or combined with metformin, was associated with increased risk of being large for gestational age. Compared with NT, exposure to any pharmacological treatment was associated with significantly lower risk of 5-min Apgar score < 4. All other secondary outcomes were comparable among the treatment categories.

Conclusions: The risk of neonatal hypoglycemia appears to be comparable among offspring exposed to single metformin treatment and nutrition therapy alone, and the lower risk that we observed in favor of metformin is probably explained by a difference in supplemental feeding practices rather than metformin per se. By contrast, the lower risk favoring metformin exposure over insulin exposure was not explained by supplemental feeding. However, further investigations are required to determine whether the difference is an effect of metformin per se or mediated by other external factors.

Objective: To explore mechanisms that modulate gestational weight gain (GWG) in women with polycystic ovary syndrome (PCOS) and healthy controls.

Design: Sub-sample of randomised controlled trials (PCOS) combined with a prospective cohort (controls).

Setting: Eleven Norwegian, Swedish, and Icelandic hospitals.

Population: Pregnant women with PCOS treated with metformin (PCOS-M, n = 36) or placebo (PCOS-P, n = 37), and healthy pregnant women (HC, n = 15).

Methods: Serum levels of the appetite regulating hormones leptin, ghrelin, allopregnanolone, and soluble leptin receptor (sOB-R) were determined in the first and third trimesters.

Main Outcome Measures: Excessive GWG (eGWG) relative to body mass index according to Institute of Medicine (IOM) guideline. Serum leptin/sOB-R ratio, or free-leptin-index (FLI), as biomarker of leptin sensitivity. Serum ghrelin and allopregnanolone levels.

Results: The overall prevalence of eGWG was 44% (38/86). Women with eGWG had higher first and third trimester FLI (P < 0.001), and lower third trimester allopregnanolone levels (P = 0.003) versus women with non-eGWG. The prevalence of eGWG was lower in PCOS-M versus PCOS-P (28% versus 62%, odds ratio = 0.4, 95% CI 0.2–0.8, P = 0.005). FLI decreased during pregnancy in PCOS-M (P = 0.01), but remained unaltered in PCOS-P and HC. Ghrelin and allopregnanolone levels were comparable in PCOS-M, PCOS-P and HC throughout pregnancy.

Conclusion: Excessive GWG is associated with enhanced leptin resistance, and attenuated physiological increase in serum allopregnanolone levels during pregnancy. Metformin reduces the risk for eGWG and improves leptin sensitivity in pregnant women with PCOS.

Background: Women with polycystic ovary syndrome (PCOS) have an increased risk of pregnancy complications. Epi-analysis of two previous randomised controlled trials that compared metformin with placebo during pregnancy in women with PCOS showed a significant reduction in late miscarriages and preterm births in the metformin group. The aim of this third randomised trial (PregMet2) was to test the hypothesis that metformin prevents late miscarriage and preterm birth in women with PCOS.

Methods: PregMet2 was a randomised, placebo-controlled, double-blind, multicentre trial done at 14 hospitals in Norway, Sweden, and Iceland. Singleton pregnant women with PCOS aged 18-45 years were eligible for inclusion. After receiving information about the study at their first antenatal visit or from the internet, women signed up individually to participate in the study. Participants were randomly assigned (1: 1) to receive metformin or placebo by computer-generated random numbers. Randomisation was in blocks of ten for each country and centre; the first block had a random size between one and ten to assure masking. Participants were assigned to receive oral metformin 500 mg twice daily or placebo during the first week of treatment, which increased to 1000 mg twice daily or placebo from week 2 until delivery. Placebo tablets and metformin tablets were identical and participants and study personnel were masked to treatment allocation. The primary outcome was the composite incidence of late miscarriage (between week 13 and week 22 and 6 days) and preterm birth (between week 23 and week 36 and 6 days), analysed in the intention-to-treat population. Secondary endpoints included the incidence of gestational diabetes, preeclampsia, pregnancy-induced hypertension, and admission of the neonate to the neonatal intensive care unit. We also did a post-hoc individual participant data analysis of pregnancy outcomes, pooling data from the two previous trials with the present study. The study was registered with ClinicalTrials. gov, number NCT01587378, and EudraCT, number 2011-002203-15.

Findings: The study took place between Oct 19, 2012, and Sept 1, 2017. We randomly assigned 487 women to metformin (n=244) or placebo (n=243). In the intention-to-treat analysis, our composite primary outcome of late miscarriage and preterm birth occurred in 12 (5%) of 238 women in the metformin group and 23 (10%) of 240 women in the placebo group (odds ratio [OR] 0.50, 95% CI 0.22- 1.08; p = 0.08). We found no significant differences for our secondary endpoints, including incidence of gestational diabetes (60 [25%] of 238 women in the metformin group vs 57 [24%] of 240 women in the placebo group; OR 1.09, 95% CI 0.69-1.66; p=0.75). We noted no substantial between-group differences in serious adverse events in either mothers or offspring, and no serious adverse events were considered drug-related by principal investigators. In the post-hoc pooled analysis of individual participant data from the present trial and two previous trials, 18 (5%) of 397 women had late miscarriage or preterm delivery in the metformin group ]compared with 40 (10%) of 399 women in the placebo group (OR 0.43, 95% CI 0.23-0.79; p=0.004).

Interpretation: In pregnant women with PCOS, metformin treatment from the late first trimester until delivery might reduce the risk of late miscarriage and preterm birth, but does not prevent gestational diabetes.