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.
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.
Study Type
OBSERVATIONAL
Enrollment
685
Maternal consumption of mild analgesics
Department of Growth and Reproduction, Rigshospitalet
Copenhagen, Denmark
Department of Obstetrics and Section of fetal medicine, Rigshospitalet
Copenhagen, Denmark
Ovarian volume (female infants)
Ovarian volumen, measured by abdominal ultrasound
Time frame: 2.5 months old
Ovarian follicle count (female infants)
Ovarian follicle count, measured by abdominal ultrasound
Time frame: 2.5 months old
Blood sample (female infants)
Serum metabolites Anti Müllarian Hormone (AMH)
Time frame: 2.5 months old
Testes volumen (male infants)
Testes volumen, measured by ultrasound
Time frame: 2.5 months old
Blood sample (male infants)
Serum metabolites testosterone, free testosterone.
Time frame: 2.5 months old
Length (male and female infants)
Length in cm
Time frame: 2.5 months old
Weight (male and female infants)
Weight in kilograms
Time frame: 2.5 months old
Head circumference (male and female infants)
Head circumference measured with a measurement tape, mm.
Time frame: 2.5 months old
Abdominal circumference (male and female infants)
Abdonimal circumference measured with a measurement tape, mm.
Time frame: 2.5 months old
Height (fathers)
Height in cm, by stadiometer (Holtain Ltd, Crymych, UK) with a precision of 0.1 cm
Time frame: Gestational week 12
Weight (fathers)
Weight in kilograms, by digital scale with a precision of 0.1 kg (SECA delta, model 707)
Time frame: Gestational week 12
Biceps skinfold (father)
Skinfold measured above the biceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK)
Time frame: Gestational week 12
Triceps skinfold(father)
Skinfold measured above the triceps, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK)
Time frame: Gestational week 12
Flank skinfold (father)
Skinfold measured at the flank, measured in mm (Harpenden skinfold Caliper, British Indicators Ltd, London, UK)
Time frame: 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)
Time frame: 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)
Time frame: 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)
Time frame: 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)
Time frame: 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)
Time frame: 2.5 months old
Asphyxia, adverse events (newborn)
Asphyxia (yes/no)
Time frame: Retrieved from patient files postpartum within 1 year of study completion
Meconium, adverse events (newborn)
Meconium in amionic fluids (yes/no)
Time frame: Retrieved from patient files postpartum within 1 year of study completion
Partus mode
Partus mode (vaginal delivery, cesarean section, instrumental delivery) (yes/no)
Time frame: Retrieved from patient files postpartum within 1 year of study completion
Birth weight (newborn)
Birth weight, grams
Time frame: Retrieved from patient files postpartum within 1 year of study completion
Birth length (newborn)
Birth length, cm
Time frame: Retrieved from patient files postpartum within 1 year of study completion
Gestational age (newborn)
Gestational age at birth, weeks and days
Time frame: 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
Time frame: Retrieved from patient questionnaire postpartum within one year
Pregnancy outcome, preeclampsia (mother)
Preeclampsia (yes/no)
Time frame: Retrieved from patient files postpartum within one year
Pregnancy outcome, gestational hypertension (mother)
Gestational hypertension (yes/no)
Time frame: Retrieved from patient files postpartum within one year
Pregnancy outcome, induction of labor (mother)
In duction of labor (yes/no)
Time frame: 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
Time frame: 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
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: Gestational week 12
Anogenital distance (AGD) (male and female infants)
Distance from anus to genital tubercle, measured in mm with a ruler
Time frame: 2.5 months old
Anogenital distance (AGD) (male and female infants)
Distance from anus to genital tubercle, third trimester ultrasound
Time frame: App. gestational age 30 weeks
Blood sample (female infants)
(estradiol and inhibin B, luteinizing hormone (LH)/follicular stimulating hormone (FSH) ratio)
Time frame: 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)
Time frame: 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.
Time frame: 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)
Time frame: 2.5 months old
Penile measurements (male infants)
Penile measurements with a ruler
Time frame: 2.5 months old
Epigenetic profiling (male and female infants)
Epigenetic variation of loci regulating hormone signalling
Time frame: 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).
Time frame: 2.5 months old
Medical report (mother)
Specific medical report on medicine consumption incl. analgesics
Time frame: 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.
Time frame: Single determination, 2.5 months old
Endometrial thickness (female infants)
Endometrial thickness, measured by abdominal ultrasound
Time frame: 2.5 months old
Uterine volume (female infants)
Uterine volume, measured by abdominal ultrasound
Time frame: 2.5 months old
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