Pregnancy outcome in women with APECED (APS-1) – A multicenter study on 43 females with 83 pregnancies

Context Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED or APS-1) has a severe, unpredictable course. Autoimmunity and disease components may affect fertility and predispose to maternal and fetal complications, but pregnancy outcomes remain unknown. Objective To assess fetal and maternal outcomes and course of clinical APECED manifestations during pregnancy in women with APECED. Design and setting A multicenter registry-based study including five national patient cohorts. Patients 321 females with APECED. Main outcome measure Number of pregnancies, miscarriages, and deliveries. Results Forty-three patients had altogether 83 pregnancies at median age of 27 years (range, 17–39). Sixty (72%) pregnancies led to a delivery, including two stillbirths (2.4%) and five (7.1%) preterm livebirths. Miscarriages, induced abortions, and ectopic pregnancies were observed in 14 (17%), eight (10%), and one (1.2%) of pregnancies, respectively. Ovum donation resulted in five (6.0%) pregnancies. High maternal age, premature ovarian insufficiency, primary adrenal insufficiency, or hypoparathyroidism did not associate with miscarriages. Women with livebirth had on average four APECED manifestations (range 0–10); 78% had hypoparathyroidism and 36% had primary adrenal insufficiency. APECED manifestations remained mostly stable during pregnancy, but in one case development of primary adrenal insufficiency led to adrenal crisis and stillbirth. Birth weights were normal in >80% and apart from one neonatal death of a preterm baby, no serious perinatal complications occurred. Conclusions Outcome of pregnancy in women with APECED was generally favorable. However, APECED warrants careful maternal multidisciplinary follow-up from pre-conceptual care until puerperium.

P–687 Impact of medroxyprogesterone acetate (MPA) as pituitary suppression on oocyte quality and clinical outcomes in egg donation recipients

Study question To compare the impact on oocyte quality and reproductive outcomes in patients who received oocytes from donors stimulated with MPA versus GnRH antagonist protocol. Summary answer Compared to GnRH antagonist, MPA does not exert a major effect on oocyte quality and yields similar reproductive outcomes in egg donation recipients. What is known already Conventional ovarian stimulation (OS) protocols have classically used GnRH analogues, both agonists and antagonists, to avoid premature follicular luteinization. The oral administration of MPA or micronized progesterone during the follicular phase of OS has emerged as an attractive alternative to conventional protocols in the prevention of early luteinization. Compared to progesterone, MPA is characterized by a moderate-strong progestanic action, lower androgenic properties and does not interfere with the measurement of endogenous progesterone. In our group, administration of MPA during the follicular phase of OS has been included in the routine clinical practice of our donor program since late 2019. Study design, size, duration Multicentre, retrospective, observational, cohort study carried out in eleven private university-affiliated IVF centers. The present study included a total of 14,282 fresh ovum donation cycles performed from October 2017 to March 2020. Oocyte donors were recruited and stimulated under either MPA (n = 4,665) or GnRHa (n = 9,617) to suppress the pituitary during the follicular phase of OS, and GnRH agonist was administered to trigger final oocyte maturation in all the participants. Participants/materials, setting, methods Recipients were divided according to the protocol used for premature luteinization prevention during the follicular phase of the ovum donation matched-cycle: Group 1, recipients who received oocytes from donors treated with 10 mg/day of MPA (ProgeveraÒ); Group 2, recipients who received oocytes from GnRH antagonist (FyremadelÒ) down-regulated donor cycles. All the procedures were approved by an Institutional Review Board (1910-VLC–091-JG) and complied with Spanish law on assisted reproductive technologies (14/2006). Main results and the role of chance Regarding donoŕs baseline characteristics, age and antral follicle count were significantly different between groups, but not clinical differences. The length of ovarian stimulation was similar in both groups (10.7 days [95% Confidence Interval (CI) 10.5–10–8] vs 10.5 days [95% CI 10.0–11.00]). Despite slightly higher mean total dose of FSH administered in Group 1 compared to Group 2 (1.841 IU [95% CI 1.813–1.868] vs 1.739 IU [95% CI 1.723–1.754]), there were no differences in the total dose of hMG administered between both groups (967 IU [95% CI 901–1.034] vs 971 IU [95% CI 944–998]). With regard to IVF data, both the number of retrieved oocytes (22.9 [95% CI 22.4–23.4] vs 24.1 [95% CI 23.8–24.3]), and mature oocytes (18.7 [95% CI 18.3–19.1] vs 19.3 [95% CI 19.1–19.6]), were slightly lower in Group 1 compared to Group 2, whereas fertilization rate was significantly higher in Group 1 compared to Group 2 (82.1% [95% CI 81.7–82.6] vs 80.8% [95% CI 80.6–81.2]),. Regarding the clinical outcomes, no differences were observed in either implantation rate (58.7% [95% CI 56.7–60.7] vs 59.3% [95% CI 57.3–61.3]) or clinical pregnancy rate (59.5% vs 59.8%, P = 0.04) between both groups. Limitations, reasons for caution As a consequence of being a retrospective study, only association, and not causation, can be inferred from the results. A further limitation is that donors are healthy young women and do not perfectly match other populations, as infertile patients who may be older, low or high responders to OS. Wider implications of the findings: MPA emerges as an effective oral alternative to GnRH analogues for preventing premature luteinizing hormone surges in donors undergoing OS in ovum donation program. Compared with GnRH antagonists, MPA has advantages of being an oral administration route and providing easy access, yielding similar clinical results. Trial registration number 1910-VLC–091-JG

P–030 A sperm chromatin damage >15% negatively impacts on the quality of embryos obtained from ovum donation ICSI cycles of unselected couples

Study question Is embryo quality downgraded in couples with elevated sperm DNA fragmentation (SDF) in the ejaculated semen of male partner using donated eggs? Summary answer The rate of good quality embryos at day 3 and blastocyst-stage is statistically inferior in males with SDF>15% undergoing ICSI cycles with donated oocytes. What is known already The effect of a damaged paternal chromatin will be shown from the 8-cell stage of embryo development, a time which the genome of the embryo is transcriptionally active. Fertilization with a spermatozoon with fragmented DNA may impair the quality of the embryos obtained per cycle, and therefore reduce the chances of pregnancy. The use of donated oocytes is an ideal model to evaluate the real effect of SDF on embryo quality by standardizing the female factor. In addition, we have a large cohort of ovum donation cases in our history, which allows a more proper evaluation of the effect. Study design, size, duration Retrospective multicentric study including the clinical data of 864 couples of ovum donation program who underwent 1903 ICSI cycles between January 2000 and March 2019. The DNA fragmentation of their ejaculated spermatozoa was measured by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Two study groups were created according to the SDF level: ≤15% (low) (n = 1626) or > 15% (high) (n = 277). Participants/materials, setting, methods Embryos were evaluated throughout embryonic development according to classical morphological parameters at day 3 (D3), on cleavage-stage, and at day 5 (D5), on blastocyst-stage (trophectoderm (TE) and inner cell mass (ICM)), following ASEBIR guidelines, categorized from A to D. Embryos scored as A and B were considered to be good quality. The proportion of embryos was calculated according to the total number of correctly fertilized oocytes or zygotes. A p < 0.05 was considered significant. Main results and the role of chance A total of 6130 embryos were evaluated. The SDF average of ≤ 15% group was 5.9% (95%CI 5.7–6.1) and 24.3% (95%CI 23.2–25.3) in the >15% group. The cycle-related characteristics and the seminal parameters were comparable. The proportion of optimal cleavage-stage embryo (number of A+B embryos at D3) per cycle was 21.7% (95%CI 19.0–24.5) (8.1 average cells number, 0.8 embryo fragmentation average, symmetry 1, mononucleated cells) in ≤ 15% SDF group versus 21.1% (95%CI 13.9–28.3) (8.2 cells number average, 1.3 embryo fragmentation average, symmetry 1, mononucleated cells) (p < 0.001). The blastocyst-stage arrival rate (number of embryos at D5) per cycle was higher in the >15% SDF group (p < 0.001), 53.4% (95%CI 48.8–58.1) (TE quality A:20.5%, B:42.5%, C:22.7%, D:14.8%, and the ICM quality A:26.1%, B:52.1%, C:13.2%, D:6.2%) versus 49.9% (95%CI 48.1–51.6) (TE quality A:21.1%, B:42.8%, C:21.85, D:14.1% and ICM A:26.6%, B:55.5%, C:11.1%, D:4.7%) in the low SDF group. The rate of good quality blastocyst (number of quality A+B embryos in D5) per cycle was significantly higher in the couples with low SDF (24.8% (95%CI 23.6–25.9)) than in those with elevated SDF (23.5% (95%CI20.9–26.2)) (p < 0.001). Accordingly, the A+B blastocyst rate divided by the total number of blastocysts was 59.1% (95%CI 56.7–61.4) versus 55.9% (95%CI 49.9–62.0) (p < 0.001), respectively. Limitations, reasons for caution The main limitation is that retrospective design of the study may not eliminate the potential unaccounted-for bias derived from the clinical practice of multiple centers even though both groups were statistically comparable. Also, the assessment of embryo quality is still remaining highly subjective to embryologists. Wider implications of the findings: Although the effect size is small, it may be useful in clinical practice when an ICSI cycle yields no good-quality embryos, as one of the underlying causes of that fact. Knowing the SDF level can be a helpful tool in making subsequent clinical decisions aimed at improving outcomes for couples. Trial registration number Not applicable

P–144 Undisturbed culture in time-lapse systems improves embryo development and quality

Study question Does culture in integrated time-lapse systems (TLS) improve embryo development and blastocyst quality compared to conventional benchtop incubators (CI), within the same IVF laboratory? Summary answer Under similar conditions, culture in TLS resulted in a significant increase in blastocyst rate, top quality blastocyst rate and proportion of biopsied embryos per treatment What is known already Integrated TLS have the potential of delivering a stable and undisturbed environment throughout the whole embryo culture, avoiding taking them out for assessment. However, there is still lack of quality evidence of the performance of these incubators compared to CI at supporting embryo culture until blastocyst stage. Studies abording this issue are still scarce, heterogeneous and have a small sample size. Although some authors have reported an improvement in embryo development and quality using TLS, global results are inconsistent. To our knowledge, the present study evaluates the effect of TLS on embryo quality on the largest sample size yet. Study design, size, duration Unicentric retrospective cohort study including 14248 ICSI treatments from 2016 to October 2020, with both autologous and donated oocytes. We compared blastocyst rate (BR) and proportion of top-quality blastocysts (TQB=Morphology ASEBIR score A) per treatment between those using TLS (N = 7500) and CI (N = 6748), and the proportion of embryos biopsied (EB) in cycles with pre-implantation genetic testing (PGT-A; N = 2642). We performed a sub-analysis in treatments using single-step culture medium (N-TLS=4398, N-CI=1140) in both types of incubators. Participants/materials, setting, methods Embryo cohorts were cultured until blastocyst stage in one of 3 TLS: EmbryoScope, EmbryoScope Plus (Vitrolife,) and Geri (Genea Biomedx), or in a CI (ASTEC). Embryo quality was assessed following ASEBIR morphological criteria. Culture protocols and media changed during the included time period. For that reason, we did a sub-study in the treatments performed since the implementation of Gems® (Genea Biomedx) single-step (SS) culture medium in all incubators. Statistical analysis was done using ANOVA tests. Main results and the role of chance Treatments were differently distributed and heterogeneous in terms of number of oocytes obtained per patient, so we stratified the analysis according to ovum origin and compared mean rates per cycle instead of total number of embryos per group. BR was statistically higher (P < 0,001) in the TLS group, in both autologous (62,98±29,37% vs 59,49±31,09% in CI) and oocyte donation treatments (69,25±22,07% vs 66,27±23,28% in CI). Proportion of TQB was also significantly higher in the TLS in both types of cycles (P < 0,001): 3,60±12,29% in TLS vs 2,27±9,71% in CI in autologous cycles, 8,68±15,31% in TLS vs 7,32±14,02% CI in ovum donation cycles. Results were corroborated in the SS media sub-study (P < 0,05): BR was 63,87±29,23% in TLS vs 57,53±30,61% in CI with autologous oocytes, and 70,76±21,63% in TLS vs 67,39±22,68% in CI with donated oocytes; TQB rates were 3,66±12,06% in TLS vs 2,05±9,26% in CI in autologous treatments and 8,81±15,21% in TLS vs 6,84±12,91% in CI in ovum donation treatments. Regarding PGT-A treatments, we found no significant difference in the biopsy rate in the total comparison, although the rate significantly increased in the TLS group since the implementation of single-step medium (52,36±24,69% in TLS vs 48,63±22,56% in CI; P = 0,007) Limitations, reasons for caution Not only culture conditions varied over time, but also the number of TLS in the laboratory, which increased lately. Hence, even though the most recent treatments included in the all-SS sub-study are more homogeneous in terms of culture conditions, they are unbalanced regarding the distribution among incubators. Wider implications of the findings: Our results demonstrate the superiority of TLS coupled with single-step culture media against traditional embryo culture systems at supporting embryo development. The optimal environment provided by TLS enhances embryo development until blastocyst stage as well as their quality, increasing the cumulative chances of getting a life-birth for each patien. Trial registration number Not applicable

P–287 Uterine adenomyosis does not affect perinatal outcomes in ART treatments

Study question Is adenomyosis associated with worse clinical and perinatal outcomes in ovum donation cycles? Summary answer Adenomyosis was associated with reduced live birth rate per embryo transfer but not with increased risk of miscarriage or worse perinatal outcomes than controls. What is known already The effect of adenomyosis on IVF/ICSI outcomes are controversial as studies addressing this issue are limited in number and heterogeneous. Conclusions withdrawn from previous works differ regarding the prospective or retrospective design of the study. Two different metanalysis conducted showed that adenomyosis reduced implantation and clinical pregnancy rate and increased miscarriage risk. However, current data regarding perinatal outcomes of assisted reproduction techniques cycles in patients diagnosed with uterine adenomyosis is scarce. Study design, size, duration A retrospective cohort study in which 3307 patients undergoing ovum donation cycles were included. Patients who underwent single embryo transfer (SET) between years 2018 and 2019 were included and divided into two groups: adenomyosis (n = 179) and controls (n = 3218). Participants/materials, setting, methods Inclusion criteria consisted of patients in an oocyte donation program who had fresh SET on day 5 blastocyst stage development. Patients diagnosed with miomas and/or severe endometriosis and those who had undergone previous uterine surgical interventions were excluded from the study. Cases consisted of patients with a history of either focal or diffuse adenomyosis diagnosed via transvaginal ultrasonography (TVUS). Main results and the role of chance Clinical pregnancy rate per embryo transfer was 82/179 (45.8%) in those women diagnosed with adenomyosis versus 1869/3218 (59.8%) in control group (OR = 0.57 95% CI. 0.41–0.78, p < 0.001). Miscarriage rate was similar in the two study groups and differences found were not statistically significant, being 15/82 (18.3%) for adenomyosis and 309/1869 (16.5%) for control group. A lower live birth rate per embryo transfer was observed in women diagnosed with adenomyosis versus control, being 68/179 (38%) and 1560/3128 (49.9%) respectively (OR = 0.615 95% CI 0.44–0.85, p = 0.002). There were no statistically significant differences between childbirth delivery methods (vaginal versus caesarean section). Furthermore, means of gestational age at the time of delivery, newborn size and weight and incidences of low birth weight, preterm birth and admission in neonate intensive care unit (NICU) did not differ between the two groups. In addition, IVF and perinatal outcomes were similar in patients with diffuse adenomyosis compared to focal adenomyosis. Limitations, reasons for caution This is an observational study and thus possible confounders cannot be completely excluded. Diagnostic of adenomyosis is complex and, despite imaging via TVUS is both sensitive and specific, different criteria may be combined in order to fully assess the diagnostic. Wider implications of the findings: Published literature has described how adenomyosis negatively impacts clinical outcomes in ART cycles; however, data regarding perinatal results is scarce. This study is of interest as it provides a first insight for clinicians showing that adenomyosis affects clinical but not perinatal outcomes in ovum donation cycle. Trial registration number Not applicable

P–081 Microfluidic sorting does not improve clinical outcomes compared to magnetic activated cell sorting (MACS) in Assisted Reproduction

Study question Does microfluidic sorting improve clinical outcomes over magnetic activated cell sorting (MACS) in ovum donation cycles? Summary answer Performing microfluidic sorting does not seem to improve clinical outcomes compared to MACS in ovum donation cycles. What is known already Novel sperm selection techniques, such as magnetic activated cell sorting (MACS), have been described as useful procedures to increase reproductive outcomes when male factor is present. Because of centrifugation steps associated to swim-up or density-gradient can induce sperm DNA fragmentation, microfluidic sperm sorters are being used to isolate motile human spermatozoa based on fluid dynamics and avoiding sample manipulation. This new technology has been shown to reduce the level of sperm DNA damage, especially double strand breaks, but an improvement of clinical outcome by using this technique remain unclear. Study design, size, duration Prospective and observational study to evaluate the efficacy of a sperm sorting technique based on microfluidic technology versus a technique based on the removal of apoptotic spermatozoa by MACS. The study was performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. All men attending for an ovum donation cycle during the aforementioned study period were included. The exclusion criteria were sperm concentration <5 mill/mL and <15% of progressive motile spermatozoa. Participants/materials, setting, methods Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. Subsequently, each of the processed sperm samples was used to microinject half of the oocytes obtained during oocyte retrieval. In all case, a single-embryo transfer was performed. Variables were expressed as mean values and standard deviations. Statistical analysis was performed by ANOVA and Chi-squared where applicable; significance established under 0.05. Main results and the role of chance We included 48 couples in the study; of these, n = 31 transferred an embryo derived from a MACS processed sperm sample, while n = 17 received an embryo from the microfluidic one. Groups were homogeneous in terms of number of transferred embryos and frozen embryos, usable blastocyst rate and fertilization rate; results were as follows for MACS and microfluidic processing respectively: number of transferred embryos (1.0±0.0 vs. 1.0±0.0, p = 0.978); number of frozen embryos (1.6±0.5 vs. 1.4±0.7 p = 0.168); usable blastocyst rate (40.7% vs. 43.1%, p = 0.384); and fertilization rate (80.4% vs. 75.3%, p = 0.075). However, according clinical outcomes, we observed significant differences in implantation rate (74.2% vs. 58.8%, p < 0.001) and in clinical pregnancy rate (74.2% vs. 58.8%, p < 0.001); finally, the miscarriage rate was similar between the two groups of study (6.4% vs. 5.8%, p = 0.876). Limitations, reasons for caution This study has not considered the indication in male factor couples due to a high degree of double-strand DNA fragmentation. Therefore, more specific studies are required to determine in which patients, microfluidics sorter selection would significantly improve clinical outcomes. Wider implications of the findings: In an unselected population, magnetic activated cell sorting significantly improves clinical outcomes compared to a microfluidic technique, so this latter method should not be recommended without a male factor indication associated with sperm DNA damage. The proposed microfluidic technology does not seem to offer a flow-free approach to select spermatozoa. Trial registration number NCT04061486

O-126 Endometrial microbiota composition is associated with reproductive outcome in infertile patients

Study question Is there an association between the composition of the endometrial microbiota and the reproductive outcomes in infertile patients undergoing in vitro fertilization (IVF)? Summary answer The composition of the endometrial microbiota (EM) prior to embryo transfer is associated with the different reproductive outcomes: live birth, no pregnancy or clinical miscarriage. What is known already The investigation of bacterial communities in the female reproductive tract using molecular methods has revealed the existence of a continuum microbiota that extends from the vagina to the upper genital tract. Previous evidence suggests the existence of an association between the vaginal and endometrial microbiome composition with reproductive and obstetrical outcomes. Specifically, the presence of specific pathogens together with low abundance of Lactobacilli has been associated with poor IVF outcomes. Study design, size, duration Multicentre prospective observational clinical study analysing the EM of infertile patients undergoing IVF (with maternal age ≤40) or ovum donation (≤50 years). A total of 452 infertile patients undergoing IVF/ovum donation were assessed for eligibility in 13 reproductive clinics in Europe, America, and Asia. The duration of the study was 30 months and the recruitment period extended between August 2017 and February 2019 (ct.gov 03330444). Participants/materials, setting, methods Endometrial fluid and endometrial biopsy were collected during a hormonal replacement therapy cycle after 5 days of progesterone (P) administration prior to a frozen embryo transfer cycle. Endometrial microbiota (EM) composition was analyzed using 16S rRNA gene sequencing using compositional data to transform scale-invariant values in both sample types. The EM in fluid and biopsy was associated with live birth, biochemical pregnancy, clinical miscarriage, or no pregnancy. Main results and the role of chance Of the 452 patients assessed, 44 did not meet the selection criteria and were excluded for the study and 66 patients were lost to follow-up. Of the 342 remaining patients, 198 (57.9%) became pregnant [141 (41.2%) had a live birth, 27 (7.9%) had a biochemical pregnancy, 2 (0.6%) had an ectopic pregnancy, and 28 (8.2%) a clinical miscarriage], while 144 (42.1%) did not become pregnant. The baseline characteristics, clinical and embryological variables were homogeneous and no bias toward the clinical outcome categories was observed. Our association study showed that the composition of the EM was associated with the reproductive outcome in both endometrial fluid and biopsy. A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was significantly associated with unsuccessful outcomes, especially no pregnancy and clinical miscarriage. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. The EM in endometrial fluid did not fully reflect that in endometrial biopsy, although their association with clinical outcome was consistent. Limitations, reasons for caution The main limitation was the small number of biochemical pregnancy and clinical miscarriage analysed. During transcervical collection of endometrial samples caution was taken to avoid contamination with the cervix although cervical contamination cannot be fully discarded. Wider implications of the findings Our data indicate that EM dysbiosis is associated with poor clinical outcome in ART. Thus, the EM composition before embryo transfer could be a useful biomarker to consider offering an opportunity to further improve diagnosis and treatment strategies. Trial registration number Clinical trials.gov 03330444