Successful Clinical Pregnancy Research Articles

Multi-omics PGT: re-evaluation of euploid blastocysts for implantation potential based on RNA sequencing.

In addition to chromosomal euploidy, can the transcriptome of blastocysts be used as a novel predictor of embryo implantation potential? This retrospective analysis showed that based on differentially expressed genes (DEGs) between euploid blastocysts which resulted and did not result in a clinical pregnancy, machine learning models could help improve implantation rates by blastocyst optimization. Embryo implantation is a multifaceted process, with implantation loss and pregnancy failure related not only to blastocyst euploidy but also to the intricate dialog between blastocyst and endometrium. Although in vitro studies have revealed the characteristics of trophectoderm (TE) differentiation in implanted blastocysts and the function of TE placentation at the implantation site, the precise molecular mechanisms of embryo implantation and their clinical application remain to be fully elucidated. This study involved 102 patients who underwent 111 cycles for preimplantation genetic testing for aneuploidies (PGT-A) between March 2022 and July 2023. The study included 412 blastocysts biopsied at Day 5 [D5] or Day 6 [D6] for patients who underwent PGT-A. The biopsy lysates were split and subjected to DNA and RNA sequencing (DNA- and RNA-seq). One part was used for PGT-A to detect DNA copy number variations, whereas the other part was assessed simultaneously by RNA-seq to determine the transcriptome characteristics. To validate the reliability and accuracy of RNA-seq obtained from this strategy, we initially analyzed the transcriptome of blastocysts with chromosomal aneuploidies. Subsequently, we compared the transcriptomic features of blastocysts with different rates of formation (D5 vs D6) and investigated the network of interactions between key blastulation genes and the receptive endometrium. Then to evaluate the implantation potential of euploid blastocysts, we identified DEGs between euploid blastocysts that resulted in clinical pregnancy (defined as the presence of a gestational sac detected by ultrasound after 5 weeks) and those that did not. These DEGs were then employed to construct a predictive model for optimizing blastocyst selection. The successful detection rate of PGT-A was remarkably high at 99.8%. The RNA data may infer aneuploidy for both trisomy and monosomy. Between the euploid blastocysts that formed on D5 and D6, 187 DEGs were predominantly involved in cell differentiation for embryonic placenta development, the PPAR signaling pathway, and the Notch signaling pathway. These D5/D6 DEGs also exhibited a functional dialog with the receptive phase endometrium-specific genes through protein-protein interaction networks, indicating that the embryo undergoes further differentiation for post-implantation development. Furthermore, a modeling strategy using 280 DEGs between blastocysts leading to successful clinical pregnancies or failing to produce clinical pregnancies was implemented to refine the euploid embryo optimization, achieving areas under the curves of 0.88, 0.71, and 0.84 for the random forest (RF), support vector machine, and linear discriminant analysis models, respectively. Finally, a retrospective analysis of 83 transferred euploid blastocysts using the RF model identified three types of euploid embryos with a decreasing trend in implantation potential. Notably, the implantation rate of the good group was significantly higher than that of the moderate group (88.6% vs 50.0% P = 0.001) and that of the moderate group was higher than that of the poor group (50.0% vs 20.8%, P = 0.035). The sample size was insufficient; thus, a prospective study is needed to verify the clinical effectiveness of the above model. Because we did not analyze blastocysts that led only to biochemical pregnancies but failed clinical pregnancies separately, our classification system still must be modified to screen these embryos. Transcriptomic analysis of blastocysts offers a novel approach for predicting embryo implantation potential, which can be utilized to optimize clinical embryo selection. The ranking system may be effective in reducing the times and costs involved in achieving a clinical pregnancy. This study was funded by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (No. 2023C03034), the National Natural Science Foundation of China (82101709), and the National Key Research and Development Program for Young Scientists of China (No. 2022YFC2702300). The authors state no competing interests. N/A.

Successful Sperm Retrieval and Clinical Pregnancies Following Micro-TESE and ICSI Treatments in Patients with Nonobstructive Azoospermia Due to Various Etiologies.

(1) Background: Nonobstructive azoospermia (NOA) etiologies affect the sperm retrieval rate (SRR) by microdissection testicular sperm extraction (micro-TESE) and the clinical outcomes following intracytoplasmic sperm injection (ICSI); (2) Methods: We investigated seven NOA etiologies. The SRR and clinical outcomes of 627 patients were analyzed between November 2017 and July 2022 in the Reproductive and Genetic Hospital of China International Trust and Investment Corporation-Xiangya (CITIC-Xiangya); (3) Results: The overall SRR was 39.4% (247/627). The SRR according to NOA etiologies were: Y chromosome azoospermia factor c microdeletions (26/46, 56.5%), Klinefelter syndrome (KS), 36/85, 42.4%), idiopathic (110/398, 27.6%), cryptorchidism (20/29, 69.0%), chromosome anomalies (7/13, 53.9%), orchitis (45/50, 90.0%), and cancer (3/6, 50.0%). The SRR were different for spermatogonia arrest (26/96, 27.1%), maturation arrest (76/177, 42.9%), and SCOS (30/80, 37.5%) according to histological examinations. The clinical pregnancy rate was similar among the NOA etiologies. The high-quality embryo rate differed between successful (54.7%) and unsuccessful (40.9%) pregnancies. Moreover, the successfully pregnant women (28.99 years) were younger than the unsuccessfully pregnant ones (30.92 years); (4) Conclusions: The SRR from patients with NOA was associated with the etiology and histological categories, while the clinical outcome was associated with the high-quality embryo rate and the female partner's age.

P-137 A deep learning model using time-lapse imaging data to predict successful clinical pregnancy and embryo euploidy

Abstract Study question Are the deep learning models helpful in selecting embryos with a high probability of clinical pregnancy and euploidy from time-lapse imaging during embryo growth? Summary answer Our deep learning models using time-lapse imaging data predicted the probabilities of successful clinical pregnancy and euploidy of embryos with high accuracy. What is known already Recent studies have attempted to select the most viable embryo using deep learning of time-lapse imaging during incubation. However, most studies included embryos that were not transferred due to low evaluation by the embryologist as embryos with unsuccessful pregnancies, or they mixed single and multiple embryo transfer cases in the analyses. Some studies’ algorithms rely on humans’ subjective interpretation of morphological and morphokinetic features, leading to embryologist-to-embryologist and within-embryologist variabilities. Some reports, including the study predicting embryo aneuploidy, used a snapshot image for prediction. These approaches have restricted the potential of deep learning in embryo selections. Study design, size, duration Retrospective analyses of time-lapse videos of 2,192 embryos with clinical pregnancy outcomes, defined as confirmed gestational sacs in utero, and 1,467 embryos with karyotype outcomes examined by preimplantation genetic testing for aneuploidy (PGT-A). The data were obtained from two different in-vitro fertilization clinics between November 2017 and December 2021. All transfers were performed as a single embryo transfer. Participants/materials, setting, methods The deep learning model was trained using time-lapse videos with known clinical pregnancy outcomes for predicting the probability of clinical pregnancy with given time-lapse video sequences and patient ages (CP model). Another deep learning model was trained using time-lapse videos with known karyotype outcomes by PGT-A for predicting the probability of euploidy (AP model). The models’ predictive powers were measured using the average area under the curve (AUC) of the receiver operating characteristic curve. Main results and the role of chance The CP model predicted clinical pregnancy with an AUC of 0.693. The AP model predicted embryo aneuploidy with an AUC of 0.702. Limitations, reasons for caution The CP model is based on the embryos that were transferred because the embryologist judged them as having a high probability of pregnancy. Clinical pregnancy may be associated with maternal factors besides embryo factors. Wider implications of the findings Our two deep learning models, the CP and AP models, will be helpful for selecting the embryos to be transferred. The CP model may increase pregnancy rates in patients undergoing assisted reproductive technology. The AP model may reduce the miscarriage caused by embryo aneuploidy. Trial registration number not applicable

A Biallelic Mutation in CCDC103 Impairs Sperm Motility due to the Absence of Dynein Arms

Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by destructive respiratory disease and laterality abnormalities due to randomized left–right body asymmetry. In men, PCD is also often associated with infertility due to immotile sperm owing to a malfunction of the sperm flagella. Pathogenic mutations have been found in more than 50 genes. Nonetheless, not all patients with PCD experience infertility. Therefore, to better understand the impact of PCD-associated mutations on male fertility, it is necessary to clarify the role of these genes in spermatogenesis. The CCDC103 p.His154Pro mutation has a high prevalence in PCD. Here, we present the identification and functional analysis of a biallelic mutation in CCDC103 identified in a familial case of PCD associated with male infertility. The biallelic CCDC103 mutations, NM_213607:c.161_162del(p.His55Serfs ∗9) and NM_213607:c.461A > C (p.His154Pro), were identified by whole-exome sequencing. Sanger sequencing validation was performed on all available family members, and the mutation was recessively separated with an infertility phenotype. The c.161_162del mutation breaks the reading frame of the protein and, therefore, is predicted to produce a nonfunctional protein. The tertiary structure of CCDC103-mutated protein indicated a significant conformational change that likely affected protein function. Transmission electron microscopy of spermatozoa showed that both the mid and principal regions of the flagellum lacked dynein arms, which was confirmed via immunofluorescence staining. Using the method of laser-assisted immotile sperm selection combined with intracytoplasmic sperm injection, the patient’s wife has a successful clinical pregnancy. These results extend the phenotype spectrum of the CCDC103 mutation in PCD.

Competent blastocyst and receptivity endometrium improved clinical pregnancy in fresh embryo transfer cycles: a retrospective cohort study

BackgroundEmbryo quality is usually regarded as a key predictor of successful implantation and clinical pregnancy potential. The identification of embryos that have the capacity to implant and result in a healthy pregnancy is a crucial part of in vitro fertilization (IVF). Usually, morphologically high-quality embryos are chosen for embryo transfer in IVF treatment. The aim of this study was to assess the association between the available blastocyst formation rate and the clinical pregnancy outcome following the first fresh embryo transfer cycle and provide systematic individual treatment to adjust endometrial receptivity for the next transfer cycle.MethodsThis retrospective, single-center study included 512 fresh embryo transfers conducted between 11/2019 and 08/2021, which consisted of 385 cleavage-stage (Day 3) and 127 blastocyst-stage (Day 5) embryo transfers. The two groups were divided into a clinical pregnancy group and a nonclinical pregnancy group for comparison. The association between the available blastocyst formation rate and the clinical pregnancy rate in the Day 3 and Day 5 transfer groups were considered.ResultsIn the Day 3 group, there were 275 clinical pregnancies, and the clinical pregnancy rate was 71.43%. Although the two pronuclei (2PN) oocyte rate and available embryo rate at Day 3 were significantly higher in the clinical pregnancy group than the nonclinical pregnancy group (P < 0.05), the blastocyst formation rate and the available blastocyst formation rate were not significantly different between the clinical pregnancy group and the nonclinical pregnancy group (P > 0.05). In the Day 5 group, there were 81 clinical pregnancies, and the clinical pregnancy rate was 63.78%. No baseline characteristics showed any obvious differences between the clinical pregnancy group and nonclinical pregnancy group (P > 0.05). The blastocyst formation rate in the nonclinical pregnancy group was higher than that in the clinical pregnancy group, but the difference was not statistically significant (81.06% vs. 77.03%, P = 0.083). Interestingly, the available blastocyst formation rate and the Day 5 available blastocyst formation rate were significantly higher in the nonclinical pregnancy group than the clinical pregnancy group (66.19% vs. 60.79%, P = 0.014; 54.58% vs. 46.98%, P = 0.007).ConclusionsIn fresh cycles, the available blastocyst formation rate was not associated with the clinical pregnancy outcome for Day 3 embryo transfers, and the available blastocyst formation rate was not positively correlated with the clinical pregnancy outcome for Day 5 embryo transfers.

Oncologic and reproductive outcomes after fertility-sparing surgery for bilateral borderline ovarian tumors: A retrospective study

ObjectivesTo investigate the oncological safety and fertility outcomes of different fertility-sparing surgery procedures for bilateral borderline ovarian tumors (BOTs) and to identify the safest and most effective approach to help patients conceive with minimal risk. Study DesignA retrospective study of 144 patients (≤40 years) with pathologically confirmed bilateral BOTs were included in the study.The effects of surgery type on fertility outcome and recurrence were compared. Cox regression analysis was employed to determine potential prognostic factors. Survival analysis utilized the Kaplan–Meier method. ResultsThree therapeutic modalities were applied in our study, including bilateral ovarian cystectomy (BOC; n = 29), unilateral adnexectomy + contralateral cystectomy (UAC; n = 4) and radical surgery (n = 61). Totally 33 cases (22.9 %) relapsed during the follow-up period. In 37 % of cases administered conservative surgery, relapses were diagnosed in the first 2 years. Only conservative surgery and adjuvant chemotherapy were risk factors for recurrence. Meanwhile, a pregnancy rate of 55.4 % was obtained in patients with bilateral BOTs. The pregnancy rate was slightly higher but no significant (P = 0.539) difference in patients treated with BOC (n = 17, 63 %) compared with UAC (n = 29, 55.8 %) group. GnRHa treatment significantly improved the clinical pregnancy rate in this study(P = 0.029). ConclusionsSatisfactory pregnancy rate can be achieved after conservative surgery in patients with bilateral BOTs. BOC is worth recommending for bilateral borderline ovarian tumors and a critical factor in fertility is the preservation of maximum healthy ovarian tissue. Patients should make a pregnancy plan in 2 years after the first surgery. GnRHa increase the rate of successful clinical pregnancies.

Mosaic embryo transfer versus additional IVF with PGT-A Cycle: a decision model comparing live birth rate and cost.

To evaluate the relative live birth rate and net cost difference between mosaic embryo transfer and an additional cycle of IVF with PGT-A for patients whose only remaining embryos are non-euploid. A decision analytic model was designed with model parameters varying based on discrete age cutoffs (<35, 35-37, 38-39, 40-42, 43-44, >44). Model inputs included probabilities of successful IVF, clinical pregnancy, and live birth as well as costs of IVF with PGT-A, embryo transfer, live birth, amniocentesis, and dilation and curettage. All costs were modeled from the healthcare system perspective and adjusted for inflation to 2023 $USD. Model outcomes were sub-stratified by degree and type of mosaicism. For patients younger than 43, an additional cycle of IVF with PGT-A resulted in a higher relative live birth rate (<35, +20%; 35-37, +15%; 38-39, +17%; 40-42, +6%; average, +14.5%) compared to mosaic embryo transfer with an average additional cost of $16,633. For patients older than 42, mosaic embryo transfer resulted in a higher live birth rate (43-44, +5%; >44, +3%; average, +4%) while on average costing $9572 less than an additional cycle of IVF with PGT-A. Mosaic embryo transfers are a superior alternative to an additional cycle of IVF with PGT-A for patients older than 42 whose only remaining embryos are non-euploid. Mosaic embryo transfers also should be considered for patients younger than 42 who are unable to pursue additional autologous IVF cycles. Counseling and care should be personalized to individual patients and embryos.

Molecular Mechanisms Determining Mammalian Oocyte Quality with the Treatment of Cancer Therapy.

Cancer is a global public health issue and remains one of the leading causes of death in the United States (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). It is estimated in the US in 2022, about 935,000 new cases of cancer will be diagnosed in women, and the probability of developing invasive cancer is 5.8% for females younger than 50years old (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). However, advances in screening programs, diagnostic methods, and therapeutic options have greatly increased the five-year survival rate in reproductive-age women with a variety of cancers. Given the clinical consequences of gonadotoxic cancer therapies, young, female cancer survivors may face compromised fertility, premature ovarian insufficiency, early-onset menopause, and endocrine dysregulation (Bedoschi et al. Future Oncol. 12:2333-44, 2016). Gonadotoxic side effects may include decreased oocyte quality within surviving follicles, loss of ovarian follicles, and impaired ovarian function. In reproductive-age women, oocyte quality is an important element for successful clinical pregnancies and healthy offspring as poor-quality oocytes may be a cause of infertility (McClam et al. Biol Reprod. 106:328-37, 2022; Marteil et al. Reprod Biol. 9:203-24, 2009; Krisher. J Anim Sci. 82: E14-E23, 2004). Thus, it is critical to determine the quantity and quality of surviving follicles in the ovary after cancer treatment and to assess oocyte quality within those surviving follicles as these are markers for determining the capacity for ovarian function restoration and future fertility, especially for young cancer survivors (Xu et al. Nat Med. 17:1562-3, 2011). The long-term effects of cancer therapeutics on oocyte quality are influenced by factors including, but not limited to, individual patient characteristics (e.g. age, health history, comorbidities, etc.), disease type, or treatment regimen (Marci et al. Reprod Biol Endocrinol. 16:1-112, 2018). These effects may translate clinically into an impaired production of viable oocytes and compromised fertility (Garutti et al. ESMO Open. 6:100276, 2021).

Associations between Urinary Concentrations of Disinfection Byproducts and in Vitro Fertilization Outcomes: A Prospective Cohort Study in China.

Experimental studies show that disinfection byproducts (DBPs) can inhibit oocyte maturation, decrease fertilization capacity, and impair embryo development, but human evidence is lacking. We aimed to evaluate the associations between exposure to drinking water DBPs and in vitro fertilization (IVF) outcomes. The study included 1,048 women undergoing assisted reproductive technology (ART) treatment between December 2018 and January 2020 from a prospective cohort study, the Tongji Reproductive and Environmental study in Wuhan, China. Exposure to DBPs was assessed by dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in up to four urine samples, which were collected on the day of both enrollment and oocyte retrieval. Multivariable generalized linear mixed models, accounting for multiple IVF cycles per woman, were applied to evaluate the associations between urinary biomarkers of DBP exposures and IVF outcomes. Stratified analyses were used to explore the potential effect modifiers. The included 1,048 women underwent 1,136 IVF cycles, with 960 (91.6%), 84 (8.0%), and 4 (0.4%) women contributing one cycle, two cycles, and three cycles, respectively. We found that elevated quartiles of urinary DCAA and TCAA concentrations were associated with reduced numbers of total oocytes and metaphase II oocytes and that urinary DCAA concentrations with a lower proportion of best-quality embryos (all p for ). Moreover, elevated quartiles of urinary DCAA concentrations were associated with decreased proportions of successful implantation, clinical pregnancy, and live birth (14%, 15%, and 15% decreases in adjusted means comparing the extreme quartiles, respectively; all p for ). Stratification analyses showed that the inverse associations of urinary TCAA concentrations with multiple IVF outcomes were stronger among women y of age (p for ). Exposure to drinking water DBPs was inversely associated with some IVF outcomes among women undergoing ART treatment. Further study is necessary to confirm our findings. https://doi.org/10.1289/EHP12447.

Embryo selection through artificial intelligence versus embryologists: a systematic review.

What is the present performance of artificial intelligence (AI) decision support during embryo selection compared to the standard embryo selection by embryologists? AI consistently outperformed the clinical teams in all the studies focused on embryo morphology and clinical outcome prediction during embryo selection assessment. The ART success rate is ∼30%, with a worrying trend of increasing female age correlating with considerably worse results. As such, there have been ongoing efforts to address this low success rate through the development of new technologies. With the advent of AI, there is potential for machine learning to be applied in such a manner that areas limited by human subjectivity, such as embryo selection, can be enhanced through increased objectivity. Given the potential of AI to improve IVF success rates, it remains crucial to review the performance between AI and embryologists during embryo selection. The search was done across PubMed, EMBASE, Ovid Medline, and IEEE Xplore from 1 June 2005 up to and including 7 January 2022. Included articles were also restricted to those written in English. Search terms utilized across all databases for the study were: ('Artificial intelligence' OR 'Machine Learning' OR 'Deep learning' OR 'Neural network') AND ('IVF' OR 'in vitro fertili*' OR 'assisted reproductive techn*' OR 'embryo'), where the character '*' refers the search engine to include any auto completion of the search term. A literature search was conducted for literature relating to AI applications to IVF. Primary outcomes of interest were accuracy, sensitivity, and specificity of the embryo morphology grade assessments and the likelihood of clinical outcomes, such as clinical pregnancy after IVF treatments. Risk of bias was assessed using the Modified Down and Black Checklist. Twenty articles were included in this review. There was no specific embryo assessment day across the studies-Day 1 until Day 5/6 of embryo development was investigated. The types of input for training AI algorithms were images and time-lapse (10/20), clinical information (6/20), and both images and clinical information (4/20). Each AI model demonstrated promise when compared to an embryologist's visual assessment. On average, the models predicted the likelihood of successful clinical pregnancy with greater accuracy than clinical embryologists, signifying greater reliability when compared to human prediction. The AI models performed at a median accuracy of 75.5% (range 59-94%) on predicting embryo morphology grade. The correct prediction (Ground Truth) was defined through the use of embryo images according to post embryologists' assessment following local respective guidelines. Using blind test datasets, the embryologists' accuracy prediction was 65.4% (range 47-75%) with the same ground truth provided by the original local respective assessment. Similarly, AI models had a median accuracy of 77.8% (range 68-90%) in predicting clinical pregnancy through the use of patient clinical treatment information compared to 64% (range 58-76%) when performed by embryologists. When both images/time-lapse and clinical information inputs were combined, the median accuracy by the AI models was higher at 81.5% (range 67-98%), while clinical embryologists had a median accuracy of 51% (range 43-59%). The findings of this review are based on studies that have not been prospectively evaluated in a clinical setting. Additionally, a fair comparison of all the studies were deemed unfeasible owing to the heterogeneity of the studies, development of the AI models, database employed and the study design and quality. AI provides considerable promise to the IVF field and embryo selection. However, there needs to be a shift in developers' perception of the clinical outcome from successful implantation towards ongoing pregnancy or live birth. Additionally, existing models focus on locally generated databases and many lack external validation. This study was funded by Monash Data Future Institute. All authors have no conflicts of interest to declare. CRD42021256333.

Decreased CD44v3 expression impairs endometrial stromal cell proliferation and decidualization in women with recurrent implantation failure

BackgroundThe precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF.MethodsMid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation.ResultsThe expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth.ConclusionThe reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.

Testicular Sperm Extraction and Intracytoplasmic Sperm Injection in Management of Obstructive Azoospermia: A Two-Year Multicenter Review in Ghana.

The objective of this study was to evaluate treatment outcomes and assess predictors of clinical pregnancy in obstructive azoospermia cases treated with testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) in Ghana. This study was a retrospective study conducted on 67 men seeking treatment for obstructive azoospermia at two study sites in Ghana from January 2018 to December 2019. First, archived data were reviewed and treatment outcomes of cases of obstructive azoospermia from the hospital records were evaluated. Infertile men who met the inclusion criteria were recruited. Descriptive data were expressed in the form of frequencies and percentages. The dependent and independent variables were analyzed using multiple logistic regression and reported as odds ratios (ORs). The confidence interval (CI) was set at 95% and a p-value <0.05 was considered significant. The mean age of male participants was 42.43±9.11 years (mean±SD) while the mean age of their partners was 32.89±5.73 years (mean±SD). The average duration of infertility before intervention was 5.01±3.60 years (mean±SD). Successful pregnancy was observed in 52.2% (35/67) of the participants. After adjusting for confounders, the rate of a successful clinical pregnancy was 0.07 lower for every additional year increase in the male's age [AOR=0.93 (95%CI=0.87-0.99), p=0.02]. Overall the rate of clinical pregnancy following TESE/ICSI from our study was 52.2%. A man's age was a strong predictor of successful clinical pregnancy among couples treated with TESE-ICSI for obstructive azoospermia in Ghana.

P-388 Isolation and characterization of human uterine fluid lavage-derived extracellular vesicles by three different methods for optimal embryo transfer timing discovery

Abstract Study question What is the suitable method for isolating extracellular vesicles (EVs) from a small volume of Uterine Fluid Lavage (UFL)? Summary answer It seems that the qEV column can help in faster, precise, and gentle isolation of EVs from small amounts of UFL. What is known already Several studies have discovered that existing EVs in the UFL, which are informative carriers, reflect cytological and molecular conditions of the uterine environment. The relative amounts of the EV components are different in various physiological situations. So, they are the ideal source for precise molecular analysis of the uterine condition to identify the Window of Implantation (WOI) for appropriate embryo transfer (ET) timing. There is limited information describing the most efficient method for EV isolation from a low-volume sample of biological fluids. In particular, EV separation from some biological fluids such as UFL is more complicated because of the viscosity. Study design, size, duration The study was conducted in two steps; first, the pre-implantation UFL sampling effect was studied on the implantation and clinical pregnancy rates. UFLs were obtained from 20 IVF Frozen Embryo Transfer (FET) patients as the case group. The control group consisted of 20 FET patients without lavage. In the second step, UFLs (n = 30) were collected and pooled. The EVs were extracted by ultracentrifugation (UC), Sucrose cushion (Suc), and Size-exclusion chromatography (by the qEV column) methods. Participants/materials, setting, methods First, UFL was collected two days before frozen blastocyst ET from the case group using an intrauterine insemination catheter attached to a syringe. Then, patients were compared with control at the seventh week. Second, EVs were extracted from pooled UFL samples and characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western Blot (WB). Albumin contamination was detected by SDS-PAGE. Total RNA was extracted, and Real-time PCR was performed for six housekeeping genes. Main results and the role of chance A comparison between case and control groups indicated no significant difference for implantation and clinical pregnancy rates. Moreover, we have confirmed the presence of EVs in UFL that were successfully isolated, by the UC, Suc, the qEV methods. The SEM images showed that the collected vesicles were morphologically spherical. The DLS analysis revealed that the intensity peak of isolated particles with the UC and Suc methods were 281 nm and 216 nm, respectively. Extracted EVs using the qEV technique were smaller and homogenized in size, with an intensity peak of 67.7 nm. SDS-PAGE of EVs showed a weaker albumin band in the qEV column (with a 100 kDa filter) method than the UC, Suc, and qEV column (with a 30 kDa filter) methods. Although, their quantification showed no significant differences. WB analysis indicated common EV markers (D81, CD9, CD63, and TSG101) showed better expression using the qEV extraction technique. But CD9 and TSG101 had lower expression levels in EVs isolated using Suc and UC, respectively. Calnexin was not detected in the isolated EVs. The GAPDH, 18SrRNA, ACTIN, GUSB, HMBS, and YWHAZ genes were detected by real-time PCR in all three EV extraction methods. The 18SrRNA gene showed better CT values. Limitations, reasons for caution Uterine fluid samples collection from qualified study groups and the patient’s follow-up for ensuring successful implantation and clinical pregnancy are time-consuming. The low volume of uterine fluid lavage sample and consequently the low number of extracellular vesicles inside it was also the limitation of this study. Wider implications of the findings Endometrial receptivity assay is difficult to study non-invasively. Herein, endometrial secretome EVs as a minimally invasive source for uterine environment molecular assessment is presented. High-throughput analysis of EV components can offer biomarkers for individual determination of ET timing, providing an additional method to use with transvaginal-ultrasonography for assessing endometrial receptivity. Trial registration number not applicable

The effect of intrauterine hCG injection before embryo transfer on pregnancy rate in frozen embryo transfer cycles

ObjectiveHCG (human chorionic gonadotropin), which is secreted by cytotrophoblast cells, plays an important role in improving pregnancy outcomes among patients with infertility or related problems. In this study, we evaluate the effect of intrauterine hCG injection prior to frozen embryo transfer on pregnancy outcomes. MethodsIn this clinical trial study, among women with infertility problems referred to (XXX) and those with frozen embryos were included in the study. 155 patients in the intervention group received 500 units of hCG while 157 in control group received saline prior to embryo transfer. Along with demographic data, successful in vitro fertilization and clinical pregnancy, loss of pregnancy, successful transplantation, and biochemical parameters were compared among the two groups. ResultsThe mean age of the patients included in the study was 32.97 ± 3.31 years. The level of anti-Mullerian hormone, follicle stimulating hormone and the grade of frozen embryos were not significantly different between the two groups (P > 0.05). The rate of laboratory pregnancy in the intervention group was significantly higher than in the control group (51% vs 35%), p = 0.006. The rate of successful implantation and clinical pregnancy in the intervention group was also significantly higher, p = 0.01 and p = 0.006, respectively. Overall loss of pregnancy in intervention group was 78.1% and 86.0% in control group which was not significantly different, p = 0.068. ConclusionThe outcomes of our study showed that 500 IU of hCG prior to embryo transfer improves the rate of clinical and laboratory pregnancy. However, it does not reduce the rate of loss of pregnancy. Further studies are therefore required in this area.

Falsely elevated serum estradiol in woman of reproductive age led to unnecessary intervention and delayed fertility opportunity: a case report and literature review

BackgroundThe optimal management of patients in reproductive endocrinology relies on the accuracy and validity of sex hormone assays. Endogenous or exogenous substances can compete with the analyte. This competition can result in interfering errors and falsely indicate elevated serum levels. Obvious interference in estradiol assays appears to occur rarely. Consequently, clinicians who are not familiar with the potential of interference could be misled. In addition to unnecessary investigations and interventions and severe mental stress, falsely elevated estradiol results can result in missed or delayed fertility opportunities.CaseA 28-year-old female with pregnancy demand was diagnosed with polycystic ovary syndrome, Hashimoto’s thyroiditis and subclinical hypothyroidism. She was found to have persistently elevated levels of serum estradiol in the early follicular phase (between 527 and 642 pg/mL). Screening workup was performed for nearly 11 months to find the causes. Serum tumor biomarkers were normal. Abdominal and pelvic computed tomography were negative for adrenal or adnexal masses. A left mesosalpinx cyst and benign pathological results were achieved by laparoscopic surgery. Hormonal substances and dietary supplements were absent, as determined by dietary records. Ultrasound confirmed follicles could grow slowly and eventually ovulate. Falsely elevated estradiol levels were suspected due to the discrepancy among high estradiol levels, follicle growth and normal gonadotropin levels. Immunological interference by heterophile antibody was finally verified by two competitive chemiluminescent immunoassay platforms (estradiol levels in the early follicle phase: 619 pg/mL, Siemens ADVIA CENTAUR and 60 pg/mL, Beckman, DxI 800). Successful clinical pregnancy was eventually achieved by combining induced ovulation, ultrasound monitoring and intercourse guidance.ConclusionsAnalytical interference and laboratory error should be suspicious at first when the clinical characteristics contradict the laboratory results of serum hormones. Measuring serum estradiol with another immunoassay platform is an easy and non-time-consuming method to exclude the heterophile interfering.

Clinical outcome of intrauterine administration of peripheral mononuclear cells or human chorionic gonadotropin in unexplained implantation failure.

Intrauterine administration of autologous peripheral blood mononuclear cells (PBMCs) or human chorionic gonadotropin (hCG) has been proposed to facilitate embryo implantation, while its effect on clinical outcome of women with previous implantation failure (RIF) in frozen/thawed embryo transfer (FET) cycles is still unclear. A total 523 patients having not experienced successful clinical pregnancy were enrolled in our study, including 207 repeat implantation failure (RIF) patients, and 316 patients with previous implantation failures but failed to meet the diagnostic criteria for RIF (non-RIF). Autologous PBMCs were cultured with hCG for 4h in the hCG-activated PBMC-treated group (n=73 in RIF patients, n=112 in non-RIF patients), and then intrauterine administered 2 days before FET. In the hCG-treated group (n=67 in RIF patients, n=100 in non-RIF patients), recombinant hCG was administered 2 days before FET. The control group (n=67 in RIF patients, n=104 in non-RIF patients) underwent FET without intrauterine administration. In RIF patients, the clinical pregnancy rate of the above three groups are 56.16%, 53.73%, and 43.28%, respectively (p=.276). The implantation rate and live birth rate showed no significant differences (p>.05). For non-RIF patients, higher clinical pregnancy rate was also seen in PBMC intrauterine group (57.15%) and hCG intrauterine group (58.00%) than controls (50.96%) but without statistical significance. There were no significant differences of implantation rate and live birth rate (p>.05). Intrauterine administration of hCG-activated PBMC and hCG did not improve clinical outcomes for both RIF and non-RIF patients before FET embryo transfer.

The associations of serum metals concentrations with the intermediate and pregnancy outcomes in women undergoing in vitro fertilization (IVF)

BackgroundToxic and essential trace elements are reported to have impact on female fertility. However, studies on the potential synergistic or antagonistic effects of metal mixtures on IVF outcomes remain limited. ObjectiveTo evaluate whether serum concentrations of metals, individually and as mixtures, are associated with pregnancy outcomes in women undergoing IVF. MethodsIn a prospective birth cohort study about IVF from the First Affiliated Hospital of Anhui Medical University (n = 1184), we measured the concentrations of serum metals by ICP-MS according to a previously established method. Oocyte/embryo development indicators and follow-up results were also collected. The individual and joint effects of metals were estimated using logistic regressions and Bayesian kernel machine regressions (BKMR). ResultsAt embryonic stage, we found negative associations between the serum lead (Pb) (β = −0.14, 95%CI: −0.32, −0.04) and cadmium (Cd) (β = −0.24, 95%CI: −0.39, −0.09) concentrations and the high-quality embryos rate; and positive associations between the serum cobalt (Co) (β = 0.18, 95%CI: 0.05, 0.31) and selenium (Se) (β = 0.17, 95%CI: 0.06, 0.41) concentrations and the MII rate. Regarding to the pregnancy outcomes, the serum Pb was negatively related with successful implantation (OR=0.85, 95%CI: 0.77, 0.94) and clinical pregnancy (OR=0.95, 95%CI: 0.91, 0.99); and positively associated with spontaneous abortion (OR=1.39, 95% CI: 1.02, 1.91). The BKMR analysis showed linear or parabolic associations between the metal mixtures and pregnancy outcomes, with Pb showing the highest posterior inclusion probabilities. ConclusionsThe toxic (Pb, Cd) and essential (Co, Se) metals could be incorporated as simultaneous predictors of IVF outcomes including potential antagonistic effects, in which Pb exhibits major contributions.

Predıctıve value of pregnancy of follıcular fluıd fetuın-A and -B levels ın infertıle women after intra-cytoplasmic sperm injection.

We aimed to investigate the value of follicular fluid fetuins-A and -B to predict successful IVF and pregnancy outcomes in infertile women with poor, normal, and high ovarian reserve. The follicular fluid of 96 infertile women who underwent intra-cytoplasmic sperm injection (ICSI) procedure was analyzed. Fetuins-A and -B levels were examined and compared in those who could achieve pregnancy and those who could not. Receiver operating characteristic curve analyzes were used to determine cut-off and statistically significant associations for fetuins-A and -B. Follicular fluid fetuin-A levels were higher in cases with weak ovarian reserve (OR) (p < 0.05) and higher in patients who did not achieve clinical pregnancy (p < 0.05). Conversely, the follicular fluid fetuin-B levels were lower in cases with poor OR (p < 0.05) and were lower in patients who did not achieve a clinical pregnancy (p < 0.05). A follicular fluid fetuin-A concentration ≤ 19.12 ng/mL had a sensitivity and specificity of 94.74% and 93.1%, respectively, at predicting clinical pregnancy. While the follicular fluid fetuin-B concentration >24.7ng/mL had sensitivity and specificity of 71.1% and 51.7%, respectively, for clinical pregnancy prediction. Overall, high levels of follicular fluid fetuin-A may be independently associated with unsuccessful IVF irrespective of OR grouping. A low level of follicular fetuin-B was also associated with failed IVF. The sensitivity and specificity were found to be higher for fetuin-A in predicting clinical pregnancy. Therefore, the follicular fluid fetuin-A may be more predictive for successful IVF and clinical pregnancy outcomes than follicular fluid fetuin-B.

Success rate and ART outcome of microsurgical sperm extraction in non obstructive azoospermia: A retrospective study.

BackgroundThe management of non-obstructive azoospermia (NOA) disease relies on microdissection testicular sperm extraction (micro-TESE). Few studies have assessed the role of micro-TESE in men with NOA in our country.Objective The aim of the current study was to investigate the success rate of micro-TESE.Materials and MethodsThis retrospective descriptive-analytical study was conducted on 463 men with NOA in Yazd Reproductive Sciences Institute during September 2017 through September 2019. Sperm were retrieved and frozen according to the rapid sperm freezing protocol. After preparing the oocyte of the male partner's spouse, sperms were thawed and then entered the intracytoplasmic sperm injection process. The clinical pregnancy of individuals was confirmed via ultrasound. Demographic data were extracted from medical records.ResultsThe success rate of micro-TESE was 38% and successful fertilization, biochemical pregnancy, clinical pregnancy, and live birth were observed in 111 (85.4%), 29 (22.3%), 29 (22.3%) and 14 (10.7%) men, respectively. A significant difference was seen between the two groups, regarding age (p = 0.01). In addition, the mean follicle-stimulating hormone in men with positive micro-TESE was significantly lower than in men with negative micro-TESE (p = 0.02).Conclusion The success of pregnancy in couples with NOA managed via micro-TESE was significant. The study found that the success rate of micro-TESE was higher in older men and in those with lower follicle-stimulating hormone levels.

P–694 Natural cycle vs Accumulation of oocytes. Which strategy is better for women with the poor ovarian response (POR)?

Abstract Study question The aim of this study is to determine whether repetitive natural cycles (Strategy A) or oocyte accumulation (Strategy B) is a more effective strategy. Summary answer There was no statistical difference between the strategies however, the number of attempts for successful outcome with strategy A was lower than in strategy B. What is known already For populations with poor ovarian response natural cycles (NC) or modified natural cycles (mNC) with minimal stimulation have been implemented as a preferable option as opposed to conventional ovarian stimulation. Due to the development of advanced vitrification techniques, the accumulation of oocytes has become available. Previous studies (2011, 2013, 2019) suggest that accumulation of oocytes could be a successful alternative to repetitive natural cycles for poor responders aged ≥35 years, showing higher clinical pregnancy rates. Moreover, the embryo-transfer cancellation and miscarriage rate were significantly lower in the oocyte accumulation strategy. Study design, size, duration Present retrospective cohort study included a selection of patients with POR treated from 2019–2020, which were divided into 2 strategies. Strategy A included 324 natural cycles or modified natural cycles with successful oocyte retrievals (Female mean age: 36.3 years). The strategy B consisted of 46 cycles with thawed oocytes that were accumulated through cryopreservation in several attempts (average n = 2,3) prior in NC or mNC (Female mean age: 37.6 years). Participants/materials, setting, methods POR was defined by following criteria: 1) advanced maternal age (≥35 years) or; 2) previous POR (≤3 oocytes with a conventional ovarian stimulation protocol); 3) abnormal ovarian reserve test; The vitrification of the oocytes was performed using Kuwayama method (Cryotech®, Japan), while thawing was performed using Cryotop method (Kitazato®, Japan). Prior to fertilization, oocytes were cultured for 2 hours in IVF medium (Origio®, Denmark). The unsuccessful transvaginal oocyte retrievals (TVOR) were excluded from both strategies. Main results and the role of chance The clinical pregnancy rate per started cycle and per transfer in Strategy A vs Strategy B were 15,4% vs 15,2% and 28.08% vs 20%, respectively. Strategy A seemed to achieve higher rates; however, the difference was not statistically significant (Student’s t-test, p &amp;lt; 0.05). Throughout all TVORs the rate of successful retrieval was 71.7%. The embryo-transfer cancellation rates in Strategy A vs Strategy B were 38,2% vs 8,69%, respectively. With strategy A the average amount of attempts for successful clinical pregnancy was 1.86. With strategy B the average amount of cryopreservations for oocyte accumulation was 2.3. Limitations, reasons for caution The number of patients in strategy B was significantly lower than in strategy A. Larger study with an increased number of samples is necessary to confirm the results. In addition calculation of cost-effectiveness in each strategy. The unsuccessful TVORs were excluded from both strategies, which significantly affects the statistical rates. Wider implications of the findings: This study might help in developing and selecting more appropriate strategies for women with POR. The findings might help to determine the amount of time and attempts required for a successful outcome for patients aged ≥35. It can also be helpful in regulating the financial part of artificial reproductive technology. Trial registration number N/A