Polycystic ovarian syndrome and miscarriage in IVF: systematic revision of the literature and meta-analysis.

PURPOSE: To evaluate the risk of miscarriage in IVF cycles in women with PCOS. METHODS: Systematic review and meta-analysis. Systematic search of MEDLINE, EMBASE and Google Scholar. The language search was restricted to English, Spanish and French, from 2000 to 2019, with crosschecking of references from relevant articles. Inclusion criteria were: (1) IVF cycles (2) a group of patients with PCOS was considered separately, (3) the miscarriage rate was reported, (4) there was a control group, (5) definition of PCOS according the Rotterdam criteria. Exclusion criteria were been excluded from the meta-analysis: (1) publication prior to the year 2000, (2) animal studies, (3) reviews, (4) abstracts or conference papers, (5) letters, (6) case reports, (7) studies comparing different IVF techniques, (8) studies comparing groups with and without metformin or other treatments, (9) studies on induced abortions. Risk of bias was assessed by the Newcastle-Ottawa score (NOS). All the included studies had a low risk of bias (NOS scores ranging 7-8). The review protocol was registered in PROSPERO (CRD42020186713). Seventeen studies were included in the meta-analysis. There was a total of 10,472 pregnancies (2650 in PCOS and 7822 in controls) of which 1885 were miscarriages (682 in PCOS and 1203 in controls). We considered the miscarriage rate (MR), preclinical MR, early MR, and late MR. RESULTS: In IVF pregnancies the risk of miscarriage was significantly increased when considering miscarriages in total (RR = 1.59; CI = 1.45-1.75), preclinical miscarriages (RR = 1.59; CI = 1.35-1.88), and early miscarriages (RR = 1.44; CI = 1.16-1.79). The increased miscarriage rate persisted in Chinese and Western populations when considered separately. The risk of miscarriage was increased in the subgroup of fresh transfers (RR = 1.21; CI = 1.06-1.39) as well as in the subgroup including either fresh or frozen transfers (RR = 1.95; CI = 1.72-2.22). CONCLUSION: PCOS is linked to an increased MR in IVF pregnancies both of miscarriages in total, and to an increase in preclinical and early miscarriages. PROSPERO NUMBER: CRD42020186713.

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium.

STUDY QUESTION: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.

Aspiration of excess follicles before intrauterine insemination in high response cycles.

Purpose: To assess the outcome of excess follicle aspiration before intrauterine insemination (EFABI) in intrauterine insemination (IUI) cycles with 4-6 follicles ≥14 mm. Methods: A retrospective case-control study with 1559 patients undergoing IUI (donor and husband's sperm), of whom 86 underwent EFABI. We studied also an historical series of 2213 patients before EFABI implementation. For 3.5 years, all women undergoing IUI developing 4-6 follicles ≥14 mm were offered EFABI on the day of hCG administration. Pregnancy rates (PRs), multiple PRs, and adverse effects were measured. Results: EFABI was associated with a similar multiple PR (17.8% vs 17.5% in non-EFABI cases), with no triplets in EFABI patients. Live birth rates were significantly higher in EFABI cycles in IUI overall (25.5% vs 15.2%). When considered separately, the performance of EFABI resulted in significantly increased live birth rates in IUI-donor cycles (32.5% vs 18.5%), whereas the differences in IUI-husband cycles (19.5% vs 12.9%) did not reach statistical significance. The PR was 21.2% during the EFABI implementation period and 19.4% in the pre-EFABI period. Conclusions: EFABI in cycles in which 4-6 follicles reach ≥14 mm is a simple option that reduces cycle cancellation rates, results in higher PRs than cycles with 1-3 follicles, and lowers the risk of multiple pregnancy.

Women's vitamin D levels and IVF results: a systematic review of the literature and meta-analysis, considering three categories of vitamin status (replete, insufficient and deficient).

To investigate the influence of vitamin D status on in vitro fertilisation (IVF) results, a meta-analysis of 15 cohort studies of 3711 women undergoing IVF was performed. Women were classified into three groups according their vitamin D levels (≥30 ng/mL considered replete/sufficient; 21-29 ng/mL insufficient and <20 ng/mL deficient). Three different meta-analyses were performed: (i) sufficient vs deficient; (ii) sufficient vs 'insufficient + deficient'; (iii) 'sufficient + insufficient' vs deficient. Comparing IVF outcomes in sufficient versus deficient groups (considering autologous and donor oocyte cycles together), we found women with sufficient vitamin D had significantly higher biochemical pregnancy (OR = 1.43 [1.06-1.95]), ongoing pregnancy (OR = 1.29 [1.02-1.64]), and live birth (OR = 1.74 [1.31-2.31]) rates, with a non-significant trend to a higher clinical pregnancy rate (OR = 1.31 [0.94-1.82]), whereas implantation and miscarriage rates were similar. When the meta-analysis was restricted to autologous oocytes, the parameters which had been significant in the joint analysis remained significant, and differences in implantation (OR = 1.64, [1.17-2.29]) and clinical pregnancy (OR = 1.47 [1.2-1.69]) rates became significant. No significant differences were found when considering only cycles with donor oocytes. The sufficient + insufficient vs deficient and sufficient vs 'insufficient + deficient' comparisons identified significant differences in live birth rate. The meta-analysis shows that sufficient vitamin D status is associated with better outcomes in IVF. Nonetheless, there are many demographic, geographic and clinical parameters that may be related to vitamin D status that need to be ascertained before concluding that the better results are due to the higher levels of vitamin D.

Antibacterial and Antifungal Activity of the Human Endometrial Fluid during the Natural Cycle.

Purpose: Some microbiota patterns have been associated with favorable IVF prognosis and others with pathological conditions. The endometrial fluid aspirate (EFA) contains antibacterial proteins that are enriched in implantative IVF cycles, but the antimicrobial effect of EFA has not been addressed. We aimed to evaluate the antimicrobial activity of the human endometrial fluid during the natural cycle. Methods: EFA was obtained through an embryo transfer catheter in 38 women, aged 18-40 years, with regular cycles attending to a fertility clinic. The antimicrobial activity of EFAs was tested against two strains of Staphylococcus aureus; one strain each of Streptococcus agalactiae, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae; and three yeasts (Candida albicans, Candida glabrata, and Candida krusei). Results: All samples exhibited antibacterial activity against S. aureus. In addition, 32.4% of EFAs were active against one of the other microorganisms assayed, 16.2% against two, and 5.4% against four of them. In contrast, none exhibited antibacterial activity against E. coli or K. pneumoniae. The antimicrobial activity differs considerably between EFA samples, and we failed to observe a cycle-related pattern. Conclusions: EFA presented two antimicrobial activity patterns: (a) one common to all the samples, exhibiting activity against S. aureus and lack of activity against E. coli and K. pneumoniae, and (b) an individualized pattern, showing activity against some of the other microorganisms tested. The intensity of antibacterial activity differs between EFA samples. Our data suggest that the uterine microbiota is controlled by means of endometrial fluid components.

Meta-analysis of the embryo freezing transfer interval.

BACKGROUND: The decision of whether frozen embryo transfer (FET) should be performed in the cycle immediately after OPU or at least one cycle later is controversial. FET could improve pregnancy rates in IVF; however, how much time is needed for the endometrium to return to optimal receptivity after ovarian stimulation is not known. METHODS: Electronic search in MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials to identify studies providing data on the influence of the interval between embryo freezing (or OPU) and FET in FET cycles published between January 1, 2007, and February 1, 2020. MAIN FINDINGS: Data analyzed indicated that in the immediate FET cycles, there was a trend to an increased biochemical pregnancy rate (RR = 1.08; CI = 1.00-1.18), whereas the clinical pregnancy rate was somewhat higher, but without reaching statistical significance (RR = 1.07; CI = 0.99-1.15). The live birth rate was similar in the two groups (RR = 1.05; CI = 0.95-1.15), as was the implantation rate (RR = 0.98; CI = 0.83-1.16). Stratifying by embryo stage or FET type (freeze-all or FET after failed fresh transfer) showed no differences. CONCLUSION: Systematically delaying FET does not offer benefits to IVF outcomes. In addition, immediate transfer is associated with a nonsignificant trend to better clinical pregnancy rate and it also avoids the psychological effects of prolonging the stress on prospective parents.

Cervical pregnancy in assisted reproduction: an analysis of risk factors in 91,067 ongoing pregnancies.

RESEARCH QUESTION: What is the frequency of cervical pregnancy in women undergoing assisted reproductive technologies (ART) and what are the risk factors? DESIGN: Case-control study of women undergoing assisted reproductive technology (ART) at 25 private assisted reproduction clinics run by the same group in Spain. Two control groups (tubal ectopic pregnancies and intrauterine pregnancies) were established. The main outcome measure was frequency of cervical pregnancy. Demographic, clinical factors and IVF parameters were assessed for their influence on cervical pregnancy risk. RESULTS: Thirty-two clinical pregnancies were achieved out of 91,067 ongoing pregnancies, yielding a rate of 3.5/10,000. Cervical pregnancies represented 2.02% of all ectopic pregnancies (32/1582). The main risk factors two or more previous pregnancies (OR 2.68; CI 1.18 to 6.07); two or more previous miscarriages (OR 4.21; CI1.7 to 10.43), one or more previous curettages (OR 3.99, CI 1.67 to 9.56), two or more previous curettages (OR 4.71; CI 1.19 to 18.66) and smoking (OR 2.82 CI 1.14 to 6.94). History of caesarean sections and tubal pregnancy was not associated with an elevated cervical pregnancy risk. Infertility conditions and endometrial thickness were similar across the three groups. The proportion of women from whom fewer than 10 oocytes were retrieved was higher in the clinical pregnancy group than in the IUP group. CONCLUSIONS: In ART, the main risk factors for cervical ectopic pregnancy are a history of at least two pregnancies, miscarriages, at least one curettage and smoking. IVF parameters do not seem to influence the development of clinical pregnancies. Cervical pregnancies are less common in ART than previously reported, attributable to improvements in ART; a publication bias in early IVF reports cannot be ruled out.

Oocytes of women who are obese or overweight have lower levels of n-3 polyunsaturated fatty acids compared with oocytes of women with normal weight.

OBJECTIVE: To ascertain whether the oocytes of women who are obese or overweight have a different fatty acid (FA) profile than women with normal weight. DESIGN: Prospective case-control study. SETTING: Two IVF centers. PATIENT(S): A total of 205 women undergoing IVF and intracytoplasmic sperm injection (ICSI) were included in the study, totaling 922 oocytes. INTERVENTION(S): The unfertilized and the immature oocytes from the women who underwent IVF/ICSI were subjected to FA analysis with capillary gas chromatography. Women were classified according their body mass index (BMI) as normal, overweight, or obese. Germinal vesicle oocytes, metaphase I oocytes, and unfertilized metaphase II oocytes were analyzed separately. MAIN OUTCOME MEASURE(S): Fatty acid profile. RESULT(S): A very different oocyte FA pattern was observed for each BMI. Women with normal weight had higher levels of saturated FAs, and lower levels of monosaturated FAs. Women who were obese had lower levels of n-3 polyunsaturated FA, and the lowest n-6:n-3 ratios. Regarding specific FAs, docosahexaenoic acid levels were lower in women with normal weight than in those who are overweight, and in women who are overweight than in those who are obese. The opposite occurred with eicosapentaenoic acid, with the highest levels in women who have normal weight followed by those who are overweight and lower levels in those women who were obese. When FA analysis was restricted to a subset of oocytes, many of these differences persisted. CONCLUSION(S): Our study shows that oocytes from women who are obese or overweight have a different FA composition. This difference in levels could be related to the IVF poor outcome in these women. Therefore, this different composition could suggest that offspring of women who are obese or overweight have an unfavorable milieu even before conception.

Influence of Caesarean section-pregnancy interval on uterine rupture risk and IVF pregnancy rates: systematic review and mathematical modelling.

RESEARCH QUESTION: What is the influence of the Caesarean section-pregnancy interval (CSPI) on the risk of uterine rupture, and what are the repercussions on IVF pregnancy rates of prolonging it? STUDY DESIGN: Systematic searches were performed using PubMed MEDLINE to identify studies published up until July 2017 for articles with the following keywords: 'interdelivery interval' and 'uterine rupture'; 'interpregnancy interval' and 'uterine rupture'; 'interpregnancy interval' and 'cesarean section'; and 'uterine rupture' and 'cesarean section'. The search identified 1609 articles, of which six were included (involving 56,419 women). Four reported significantly higher uterine rupture rates in cases of a short CSPI. RESULTS: From the analysis, the uterine rupture rate can be modelled by a formula corresponding to a hyperbolic curve. There is no clear cut-off in uterine rupture in relation to CSPI. The curve showed a sharp decrease in uterine rupture until the 10th month of CSPI (uterine rupture rate 0.7%), then a moderate and steady decrease until the 40th month (uterine rupture rate 0.4%) and afterwards a very mild decrease. From the data it is possible to calculate, according to the age of the woman, the expected reduction in IVF rates and uterine rupture as CSPI increases. CONCLUSION: The risk of uterine rupture in relation to CSPI can be represented by means of a hyperbolic curve. After a 10-month CSPI, the expected uterine rupture rate is close to 0.7%. The impact of prolonging or reducing this interval on IVF pregnancy rates can be easily obtained from the table included in the article. This should be helpful in the decision-making process for both patients and physicians.

Anti-Candidaalbicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans / Los anticuerpos dirigidos contra los tubos germinales de Candida albicans reducen el crecimiento in vitro y la formación de biopelículas de C. albicans

Background: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. Aims: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. Methods: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. Results: CAGTA ≥ 50 μg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥ 80 μg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90 min. Conclusions: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections

Antecedentes: La infección invasora por Candida albicans está asociada a altas tasas de morbimortalidad, en parte debido al retraso en la instauración de una terapia antifúngica adecuada, dificultada a su vez por la falta de un diagnóstico precoz. Objetivos: Evaluar la actividad antifúngica de los anticuerpos contra tubos germinales de C. albicans (CAGTA) obtenidos a partir de un modelo animal de candidemia en conejo. Métodos. El efecto de los CAGTA se evaluó mediante los ensayos colorimétricos XTT y cristal violeta, así como mediante el recuento de unidades formadoras de colonias, tanto en células planctónicas de C. albicans como en distintos estadios de formación y maduración de biopelículas. La viabilidad y la morfología de las células tratadas con CAGTA se determinó mediante microscopía óptica, de fluorescencia o electrónica (SEM). Resultados: Concentraciones de CAGTA ≥ 50 μg/ml generaban una fuerte inhibición del crecimiento de C. albicans, y su actividad se mostró fungicida. Los CAGTA producían alteraciones en la superficie de los tubos germinales desarrollados tanto a partir de células en suspensión como de células en biopelículas. Además, concentraciones de CAGTA ≥ 80 μg/ml redujeron la biomasa de biopelículas de Candida, y este efecto se desencadenaba en los primeros 90min de su formación. Conclusiones: Este es el primer estudio que demuestra la capacidad de los CAGTA para reducir el crecimiento de C. albicans y su actividad metabólica, así como para alterar la formación de biopelículas in vitro. Los antígenos reconocidos por los CAGTA podrían servir de base para el desarrollo de protocolos de inmunización protectores frente a infecciones por Candida

Anti-Candidaalbicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans.

BACKGROUND: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. AIMS: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. METHODS: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. RESULTS: CAGTA ≥50µg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80µg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90min. CONCLUSIONS: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections.