P–693 Gonadotropin stimulation reduces the implantation and live birth but not the miscarriage rate – a study based on the comparison of stimulated and unstimulated IVF

Study question Does gonadotropin stimulation in conventional IVF (cIVF) affect the implantation, miscarriage and live birth rates? Summary answer Gonadotropin stimulation negatively affects the implantation and live birth but not the miscarriage rate in IVF treatments. What is known already Literature hypothesizes that embryos derived from unstimulated, natural cycle IVF (NC-IVF) have a higher implantation potential compared to embryos from cIVF. In NC-IVF, recruitment of the leading follicle is based on natural selection. Hormonal stimulation might not only affect the embryo but also endometrial function. It’s possible to compare outcomes of NC-IVF and cIVF if cIVF is performed without embryo selection, in other words, if only those zygotes, which will be transferred 1–2 days later, are left in culture and all other zygotes are cryopreserved. To test this hypothesis, we compared success rates in NC-IVF and in cIVF. Study design, size, duration We performed a cohort study from 2011–2016 including data on IVF cycles with transfer of fresh embryos on day 2–3 at a University based infertility center. Our sample consisted of 640 women with 1482 embryos transferred in 996 cycles. We defined implantation rate as the number of sonograhically detected amniotic sacs per transferred embryos. Data originated from the Swiss ART registry “FIVNAT” and the Bern IVF Cohort and was completed using medical and delivery records. Participants/materials, setting, methods We defined NC-IVF as IVF without stimulation of follicular growth and cIVF as IVF with gonadotropin stimulation ≥75 IE/d and >3 retrieved oocytes. We performed zygote, but not embryo selection and transferred embryos on day 2–3. We calculated implantation and live birth per transferred embryo as binary outcomes using bi- and multivariable multilevel logistic regression models accounting for two clusters; the women and the cycle; and adjusting for maternal and infertility characteristics using STATA. Main results and the role of chance Age of women (p = 0.531), parity (p = 0.194) and type of infertility (primary vs secondary) (p = 0.463) did not differ between women undergoing NC-IVF or cIVF. In NC-IVF, 468 (31.6%) embryos were transferred, 450 as single, 18 as double transfers. In cIVF, 1014 (68.4%) embryos were transferred, 91 as single, 830 as double and 93 as triple transfers. Implantation rate was higher in NC-IVF. In NC-IVF 80 (17.1%) and in cIVF 132 (13.0%) embryos developed into an amniotic sac (OR 1.58; 95% CI 1.01–2.46; p = 0.042). After adjustment for maternal age (p < 0.001), parity (p < 0.001), type of infertility (p = 0.037), duration of subfertility and indication for IVF, aOR for implantation per transferred embryo increased to 1.87 (95% CI 1.21–2.91; p = 0.005). Miscarriage rate was similar. In NC-IVF and cIVF 25% (n = 20; n = 33) miscarried and 75% (n = 60; n = 99) ended in a live birth, respectively (OR 0.91; 95% CI 0.32–2.60; p = 0.855; aOR 1.0; 95% CI 0.42–2.36; p = 1.000). Live birth rate per transferred embryo was increased in NC-IVF; 60 of 468 (12.8%) embryos in NC-IVF compared to 99 of 1041 (9.8%) embryos in cIVF resulted in a live birth (OR 1.51; 95% CI 0.92–2.49; p = 0.106); and became significantly higher after adjustment (aOR 1.85; 95% CI 1.16–2.95; p = 0.010). Limitations, reasons for caution This study analyses observational data from a clinic offering NC-IVF and cIVF treatment as equivalent options. NC-IVF is a model for natural fertility and allows us to study the impact of gonadotropins. However, it is not a randomised study and therefore prone to selection bias. Wider implications of the findings: The study suggests that gonadotropin stimulation might reduce the implantation potential and subsequently live birth rates, by possibly affecting embryo and endometrium quality. Clinicians should consider lower gonadotropin doses for stimulation. Trial registration number Not applicable

P–768 Childhood growth of singletons conceived following in vitro fertilization (IVF) - does gonadotropin-stimulation matter?

Study question Is there a difference in growth or weight gain of children conceived after IVF with or without gonadotropin-stimulation compared to standard growth references? Summary answer: We observed no difference in growth between children conceived after IVF with or without gonadotropin-stimulation and spontaneously conceived children. What is known already In recent studies, singletons conceived after IVF cycles had lower birth weight than spontaneously conceived singletons. The etiology of the impaired intrauterine growth is unclear, but insufficiency of placental function or possible epigenetic effects is discussed. Data regarding normalization or continuation of reduced birth weight are controversial. The growth of children born after unstimulated natural cycle IVF (NC-IVF) has never been studied. Study design, size, duration Single-center, university based cohort study. 139 singletons born after NC- IVF and children born after conventional gonadotropin stimulated IVF (cIVF) in 2010 –2017 were studied. Stimulation dosage in cIVF was ≥150 IU/d human gonadotropin. Participants/materials, setting, methods We collected weight, length and head circumference at birth and at one, two, four, six, 12, 18 and 24 months. We calculated standard deviation scores based on national growth references. Growth parameters (weight, length and head circumference) were compared between NC-IVF and cIVF singletons (stimulated with ≥150 IU/d human gonadotropin) using Mann-Whitney U tests. Main results and the role of chance In general, growth of children conceived after IVF did not differ from national references. Of the 139 singletons conceived, 98 singletons were conceived after NC-IVF and 41 after cIVF. The parents did not differ in ethnicity, age, BMI or health status between groups, and there was no significant difference in gestational age, pregnancy complications and smoking or breastfeeding habits either. The median birth weight in NC-IVF children was 3.4kg (0.1 standard deviation score, SDS) and in cIVF 3.3kg (–0.3 SDS) (p = 0.53). Median length at birth in NC-IVF was 50cm (–0.5 SDS) and did not differ from cIVF children 50cm (–0.8 SDS) (p = 0.52). At age 12 months, the median weight was 9.3kg (0.0 SDS) for NC-IVF children compared to 9.0kg (–1.7 SDS) for cIVF children (p = 0.44). Median lengths was 75cm (0.1 SDS) in NC-IVF versus 71cm (–1.6 SDS) in cIVF children (p = 0.89). At age 24 months, median weight in NC-IVF children was 12.3 kg (0.3 SDS) versus 10.5 kg (–1.2 SDS) in cIVF (p = 0.72) and median lengths 87.5cm (0.1 SDS) in NC-IVF versus 87.6 cm (0.1 SDS) in cIVF children. These discrete non-significant differences in weight and length gain compared to standardized growth curves and between the two groups are reassuring. Limitations, reasons for caution Willingness to participate is prone to selection bias. Further studies with larger samples are needed to confirm these findings. Wider implications of the findings: This is the first study investigating weight and length gain in children after unstimulated IVF. Growth is an important proxy for the health of children. These reassuring results are of imminent importance for the children born after IVF and their parents. Trial registration number BASEC (ID 2015–00235)

The Effects of Adiponectin on Infertile Women Undergoing IVF/ICSI Treatment and on Human Granulosa Cells

Adiponectin, one of the most abundant adipocyte-secreted protein, has been involved in female reproductive regulation. This study aimed to 1) compare serum adiponectin levels in various phases of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment including Phase I (the beginning of gonadotropin stimulation), Phase II (around 8 days after gonadotropin stimulation), and Phase III (on the day of ovum pick-up) between success and unsuccess subjects; 2) compare follicular fluid (FF) adiponectin levels between success and unsuccess groups; 3) compare serum adiponectin levels among different phases of IVF/ICSI treatment in success and unsuccess groups; 4) compare the levels of adiponectin between serum and FF; and 5) investigate the effects of adiponectin on mRNA expressions of follicle stimulating hormone receptor (FSHR) and CYP19A1 (aromatase) in human granulosa-like tumor cell line (KGN) (n=3). In the human study, recruited participants (n=30) with age of 26-40 years were enrolled between April 2018 - May 2019. Blood samples were collected at Phases I, II, and III while FF samples were collected at Phase III. Adiponectin levels were comparable between success and unsuccess subjects in both serum (all phases) and FF (Phase III). Furthermore, serum adiponectin levels were comparable among Phase I, II, and III in success and unsuccess groups. In Phase III, serum adiponectin showed positive correlations with serum adiponectin in Phase I and II and serum FSH in Phase I. Interestingly, adiponectin levels in FF were significantly lower than serum at Phase III in unsuccessful pregnancies but were comparable in successful pregnancies. Moreover, FF adiponectin had a negative correlation with serum LH at Phase III in success subjects. In the KGN cell study, adiponectin had no effects on FSHR and CYP19A1 (aromatase) mRNA expression compared with control. In conclusion, high adiponectin levels in serum compared to FF might contribute to unsuccessful IVF/ICSI treatment.

Predictors of reproductive and non-reproductive outcomes of gonadotropin mediated pubertal induction in male patients with congenital hypogonadotropic hypogonadism (CHH)

Purpose To investigate predictors of testicular response and non-reproductive outcomes (height, body proportions) after gonadotropin-induced puberty in congenital hypogonadotropic hypogonadism (CHH). Design A retrospective analysis of the puberty induction in CHH male patients, undergoing an off-label administration of combined gonadotropin (FSH and hCG). Methods Clinical and hormonal evaluations before and during gonadotropin stimulation in 19 CHH patients genotyped by Targeted Next Generation Sequencing for CHH genes; 16 patients underwent also semen analysis after gonadotropins. Results A lesser increase in testicular volume after 24 months of induction was significantly associated with: (I) cryptorchidism; (II) a positive genetic background; (III) a complete form of CHH. We found no significant correlation with the cumulative dose of hCG administered in 24 months. We found no association with the results of semen analyses, probably due to the low numerosity. Measures of body disproportion (eunuchoid habitus and difference between adult and target height: deltaSDSth), were significantly related to the: (I) age at the beginning of puberty induction; (II) duration of growth during the induction; (III) initial bone age. The duration of growth during induction was associated with previous testosterone priming and to partial forms of CHH. Conclusions This study shows that a strong genetic background and cryptorchidism, as indicators of a complete GnRH deficiency since intrauterine life, are negative predictors of testicular response to gonadotropin stimulation in CHH. Body disproportion is associated with a delay in treatment and duration of growth during the induction, which is apparently inversely related to previous androgenization.

Gonadotropin Stimulation Has Only a Limited Effect on the Concentration of Follicular Fluid Signalling Proteins: An Antibody Array Analysis

Objective. The follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated in vitro fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF. Method. Twenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of P < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids. Results. Array analysis yielded only a small number of differentially expressed signalling markers by unadjusted P values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities. Conclusion. In contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.

Rate of rebound in follicle growth after cessation of ovarian stimulation in initial non‐responders: a prospective cohort study

Previously anecdotally observed rebounds in follicle growth after interruption of exogenous gonadotropins in absolute non-responders were the impetus for here reported study. In a prospective cohort study, we investigated 49 consecutive patients, absolutely unresponsive to maximal exogenous gonadotropin stimulation, for a so-called rebound response to ovarian stimulation. A rebound response was defined as follicle growth following complete withdrawal of exogenous gonadotropin stimulation after complete failure to respond to maximal gonadotropin stimulation over up to 5–7 days. Median age of study patients was 40.5 ± 5.1 years (range 23–52). Women with and without rebound did not differ significantly (40.0 ± 6.0 vs. 41.0 ± 7.0 years, P = 0.41), with 24 (49.0%) recording a rebound and 25 (51.0%) not. Among the former, 21 (87.5%) reached retrieval of 1–3 oocytes and 15 (30.6%) reached embryo transfer. A successful rebound in almost half of prior non-responders was an unsuspected response rate, as was retrieval of 1–3 oocytes in over half of rebounding patients. Attempting rebounds may, thus, represent another incremental step in very poor prognosis patients before giving up on utilization of autologous oocytes. Here presented findings support further investigations into the underlying physiology leading to such an unexpectedly high rebound rate.