The Copenhagen Analgesic Study

Study Overview
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Brief Summary
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Fundamental aspects of reproductive function are established in fetal life and there is a present increased awareness of the potential effects of fetal exposures on reproductive health of offspring. Experimental studies strongly suggest detrimental effects of prenatal exposure to mild analgesics such as acetaminophen (e.g. paracetamol) and non-steroidal anti-inflammatory drugs, NSAIDs (e.g. ibuprofen and acetylsalicylic acid) on male as well as female gonadal development. Declining fertility has become a growing problem in developing countries, potentially resulting in severe socioeconomic challenges, and fetal exposure of mild analgesics causes part of these alarming observations.This is the first prospective human study designed primarily to assess the effect of fetal exposure of mild analgesics on male and female reproductive function.

Detailed Description
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Fetal gonadal development is essential for adult reproductive health. Experimental studies strongly suggest that maternal use of mild analgesics (e.g. paracetamol and non-steroidal anti-inflammatory drugs (NSAIDs)) during pregnancy affect fetal gonadal development with possible severe reproductive repercussions. In rodents, paracetamol and NSAIDs administered in therapeutic doses in early and mid-pregnancy are endocrine disruptive in the fetus causing reduced prostaglandin synthesis and delayed transition from germ cell mitosis to meiosis resulting in fetal germ cell apoptosis in both female and male gonads. Female offspring were born with reduced ovarian weight and concerning reduction (40-50%) in number of ovarian follicles. Females are born with a defined number of follicles that depletes throughout their reproductive lifespan, inevitably leading to menopause. Establishment of the primordial follicle pool during fetal life is therefore essential for female reproductive health and disruption of this process has important and lasting consequences. Although spermatogenesis is not restricted to fetal life, essential aspects of male gonadal development are tightly regulated in utero and in rodents exposure to mild analgesics causes decreased testosterone production and decreased fertility in male offspring. In adulthood, exposed animals exhibited longer time to conceive and gave birth to fewer pubs per litter compared with controls. Furthermore, studies of rodents suggest that in both males and females, adverse reproductive effects are passed on to the next generation indicating altered genetic programming, i.e. epigenetic changes. Analgesics are sold over the counter and up to 56% of pregnant women use mild analgesics during pregnancy. The bioavailability of acetaminophen is high (app. 90%), and the reactive metabolite passes freely over the placenta to the fetus. Declining fertility has become a growing problem in developing countries, potentially resulting in severe socioeconomic challenges. The anogenital distance (AGD) is defined as the distance from the anus to genital tubercle and is strongly affected by androgens in fetal life resulting in a longer AGD in males than in females. The AGD has shown to be a sensitive marker of androgen exposure in fetal life, and remains the most sensitive parameter when evaluating prenatal exposure to endocrine disruptive environmental agents. Therefore, AGD has been identified as an endpoint in the US Environmental Protection Agency guidelines for reproductive toxicity studies. In humans, use of mild analgesic during the first and second trimester was associated with reduced male AGD, congenital cryptorchidism and hypospadias suggestive of insufficient androgenic action. In male infants born with hypospadias, the reduction in AGD can be seen as early as in the third trimester where fetal AGD is below the fifth percentile compared to normative fetal AGD data. Thus, fetal AGD may assist in early detection of insufficient androgenic action and genital abnormalities. In adult life, consequences can be impaired testosterone production, sub- and infertility as well as testis cancer. Assessment of reproductive function in early life - minipuberty Minipuberty is a term used to describe the transient activation of the hypothalamic-pituitary-gonadal (HPG) axis during infancy in both boys and girls and is a window of opportunity for diagnosis of endocrine disorders as well as future reproductive function. Reproductive hormones exert effects on target tissue resulting in follicle maturation, growth of breast tissue and thickening of uterine endometrium (females) as well as testicular- and penile growth (males). The minipuberty is followed by a quiescent period during mid childhood until pubertal reactivation of the HPG axis at pubertal onset. To date, no prospective human studies have assessed the effect of analgesic exposure on reproductive function. The few retrospective studies that are published are hampered by recall bias and/or lack of thorough reproductive evaluation, and no studies have in detail assessed human female reproductive function after the use of mild analgesics during pregnancy.

Official Title
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The Copenhagen Analgesic Study

Conditions
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Gonad Regulating Hormone Adverse Reaction, Analgesic Adverse Reaction

Intervention / Treatment
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* Other: Observational


Participation Criteria
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Eligibility Criteria
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Inclusion criteria: Infants: Singleton pregnancies Term pregnancy (week 37+0 to 42+0) Parents: Maternal and paternal Caucasian origin Maternal pre-pregnancy BMI between 18 and 35 kg/m2 Exclusion criteria: Infants: • Fetal malformations or chromosomal disorders Parents: Serious maternal illness, including pre-existing maternal diabetes or thyroid gland diseases Gestational diabetes

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Controls<br>Children born from mothers with no consumption of mild analgesics 3 months before or during pregnancy | |
| Exposed<br>Children born from mothers with consumption of mild analgesics 3 months before or during pregnancy | Other: Observational<br>* Maternal consumption of mild analgesics<br>* Other names: Acetyl salicylic acid;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Ovarian volume (female infants) | Ovarian volumen, measured by abdominal ultrasound | 2.5 months old | 
| Ovarian follicle count (female infants) | Ovarian follicle count, measured by abdominal ultrasound | 2.5 months old | 
| Blood sample (female infants) | Serum metabolites Anti Müllarian Hormone (AMH) | 2.5 months old | 
| Testes volumen (male infants) | Testes volumen, measured by ultrasound | 2.5 months old | 
| Blood sample (male infants) | Serum metabolites testosterone, free testosterone. | 2.5 months old | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Length (male and female infants) | Length in cm | 2.5 months old | 
| Weight (male and female infants) | Weight in kilograms | 2.5 months old | 
| Head circumference (male and female infants) | Head circumference measured with a measurement tape, mm. | 2.5 months old | 
| Abdominal circumference (male and female infants) | Abdonimal circumference measured with a measurement tape, mm. | 2.5 months old | 
| Height (fathers) | Height in cm, by stadiometer (Holtain Ltd, Crymych, UK) with a precision of 0.1 cm | Gestational week 12 | 
| Weight (fathers) | Weight in kilograms, by digital scale with a precision of 0.1 kg (SECA delta, model 707) | Gestational week 12 | 
| Biceps skinfold (father) | Skinfold measured above the biceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | Gestational week 12 | 
| Triceps skinfold(father) | Skinfold measured above the triceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | Gestational week 12 | 
| Flank skinfold (father) | Skinfold measured at the flank, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | Gestational week 12 | 
| Scapula skinfold (father) | Skinfold measured below the scapula all on the left side, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | Gestational week 12 | 
| Biceps skinfold (male and female infants) | Skinfold measured above the biceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | 2.5 months old | 
| Triceps skinfold (male and female infants) | Skinfold measured above the triceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | 2.5 months old | 
| Flank skinfold (male and female infants) | Skinfold measured at the flank, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | 2.5 months old | 
| Scapula skinfold (male and female infants) | Skinfold measured below the scapula all on the left side, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK) | 2.5 months old | 
| Asphyxia, adverse events (newborn) | Asphyxia (yes/no) | Retrieved from patient files postpartum within 1 year of study completion | 
| Meconium, adverse events (newborn) | Meconium in amionic fluids (yes/no) | Retrieved from patient files postpartum within 1 year of study completion | 
| Partus mode | Partus mode (vaginal delivery, cesarean section, instrumental delivery) (yes/no) | Retrieved from patient files postpartum within 1 year of study completion | 
| Birth weight (newborn) | Birth weight, grams | Retrieved from patient files postpartum within 1 year of study completion | 
| Birth length (newborn) | Birth length, cm | Retrieved from patient files postpartum within 1 year of study completion | 
| Gestational age (newborn) | Gestational age at birth, weeks and days | Retrieved from patient files postpartum within 1 year of study completion | 
| Drug intake (mother) | Pre- and perinatal drug intake, filled in by mother during the whole prengancy every two weeks online | Retrieved from patient questionnaire postpartum within one year | 
| Pregnancy outcome, preeclampsia (mother) | Preeclampsia (yes/no) | Retrieved from patient files postpartum within one year | 
| Pregnancy outcome, gestational hypertension (mother) | Gestational hypertension (yes/no) | Retrieved from patient files postpartum within one year | 
| Pregnancy outcome, induction of labor (mother) | In duction of labor (yes/no) | Retrieved from patient files postpartum within one year | 
| Medical history and exposure (parents) | General- and reproductive health, the pregnancy, own birth weight, lifestyle, drinking and smoking habits from questionnaire | Retrived from questionnaire within a half year | 
| Pubertal history (parents) | Pubertal history including age at menarche, pubertal timing with regard to peers, age at menopause of the mother of the parents etc. from questionnaire | Retrived from questionnaire within a half year | 
| Blood sample (mother) | Blood samples will be drawn from an antecubital vein and will be measured for steroid hormone metabolites and metabolites of reproductive hormones. Testosterone, androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), Estradiol, Estrone, Progesterone, 17-hydroxyprogesterone: in-house mass-spectrometry; Turboflow (LC-MS/MS). Luteinizing hormone (LH), follicle stimulating hormone (FSH), Sex hormone Binding Globulin (SHBG): Time-resolved immuno- flouroimmunoassay; Delfia, Turko, Finland. Inhibin B: Specific enzyme-linked immunosorbent assay; Beckman Coulter GenII. Anti- Müllerian hormone (AMH): Specific enzyme immuno-metric assay; Immunotech Beckman Coulter. INSL3: Time-resolved immuno- flouroimmunoassay. IGF-I and IGFBP-3 will be analyzed using an immunoassay (iSYS, iDS). | Gestational week 12 and 2.5 months postpartum | 
| Urine sample (mother) | The urine sample will be collected in a cup and analyzed for: Steroid hormone metabolites using in-house mass-spectrometry; Turboflow (LC-MS/MS). Glycoprotein hormones, specifically FSH and LH, using immunoassays. Endocrine disrupting chemicals, specifically phthalates, phenols, perfluorinated compounds and parabens also using in-house mass-spectrometry; Turboflow (LC-MS/MS). | Gestational week 12 and 2.5 months postpartum | 
| Blood sample (father) | Blood samples will be drawn from an antecubital vein and will be measured for steroid hormone metabolites and metabolites of reproductive hormones: Testosterone, androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), Estradiol, Estrone, Progesterone, 17-hydroxyprogesterone: in-house mass-spectrometry; Turboflow (LC-MS/MS). Luteinizing hormone (LH), follicle stimulating hormone (FSH), Sex hormone Binding Globulin (SHBG): Time-resolved immuno- flouroimmunoassay; Delfia, Turko, Finland. Inhibin B: Specific enzyme-linked immunosorbent assay; Beckman Coulter GenII. Anti- Müllerian hormone (AMH): Specific enzyme immuno-metric assay; Immunotech Beckman Coulter. INSL3: Time-resolved immuno- flouroimmunoassay. IGF-I and IGFBP-3 will be analyzed using an immunoassay (iSYS, iDS). | Gestational week 12 | 
| Urine sample (father) | The urine sample will be collected in a cup and analyzed for: Steroid hormone metabolites using in-house mass-spectrometry; Turboflow (LC-MS/MS). Glycoprotein hormones, specifically FSH and LH, using immunoassays. Endocrine disrupting chemicals, specifically phthalates, phenols, perfluorinated compounds and parabens also using in-house mass-spectrometry; Turboflow (LC-MS/MS). | Gestational week 12 | 
| Anogenital distance (AGD) (male and female infants) | Distance from anus to genital tubercle, measured in mm with a ruler | 2.5 months old | 
| Anogenital distance (AGD) (male and female infants) | Distance from anus to genital tubercle, third trimester ultrasound | App. gestational age 30 weeks | 
| Blood sample (female infants) | (estradiol and inhibin B, luteinizing hormone (LH)/follicular stimulating hormone (FSH) ratio) | 2.5 months old | 
| Blood sample (male infants) | Serum metabolites of reproductive hormones (AMH, inhibin B levels, ratios of inhibin B/FSH and LH/FSH) | 2.5 months old | 
| Classification of external genitalia with an external masculinization score (EMS) (male and female infants). EMS provides an objective aggregate score of the extent of masculinization of the external genitalia. | It is an individual score with a maximum of 12 points. The following is assessed: Classification of genital tubercle, measured length with a ruler (mm) >30mm = 3 points, 21-30mm = 2 points, 11-20mm = 1 point,< 10mm = 0 points) Location of gonads, (objectively assessed): labioscrotal = 1,5 points, inguino-scrotal = 1, inguinal = 0,5 points, impalpable = 0 points Site of the urinary meatus (objectively assessed): typical male = 3 points, coronal/glandular = 2,5 points, penile = 2 points, peno-scrotal = 1,5 points, perineal = 0,5 points, typical female = 0 points. Labia/scrotal fusion (objectively assessed): fused = 3 points, posterior fusion = 1,5 points, unfused = 0 points. | 2.5 months old | 
| Pubertal staging (male and female infants) | Pubertal staging using Tanners classification (including testicular size in boys assessed by Prader's orchidometer) | 2.5 months old | 
| Penile measurements (male infants) | Penile measurements with a ruler | 2.5 months old | 
| Epigenetic profiling (male and female infants) | Epigenetic variation of loci regulating hormone signalling | Single determination, 2.5 months old | 
| Urine sample (10 mL) (male and female infants) | Steroid hormone metabolites using in-house mass-spectrometry; Turboflow (LC-MS/MS). Glycoprotein hormones, specifically FSH and LH, using immunoassays. Endocrine disrupting chemicals, specifically phthalates, phenols, perfluorinated compounds and parabens also using in-house mass-spectrometry; Turboflow (LC-MS/MS). | 2.5 months old | 
| Medical report (mother) | Specific medical report on medicine consumption incl. analgesics | Every 2 weeks from enrollment in early pregnancy to birth | 
| Genetic profiling (male and female infants) | Genotyping of different genetic loci (genetic variation of loci regulating) hormone signalling, e.g. FSHB, etc. | Single determination, 2.5 months old | 
| Endometrial thickness (female infants) | Endometrial thickness, measured by abdominal ultrasound | 2.5 months old | 
| Uterine volume (female infants) | Uterine volume, measured by abdominal ultrasound | 2.5 months old |