Demographic distributions and clinical results of assisted reproduction techniques in Turkey in 2019: a descriptive survey.

Objective: The aim of this study was to describe characteristics and outcomes of assisted reproductive technology (ART) cycles performed in 2019 in Turkey. Material and Methods: One-hundred and sixty-five ART centers in Turkey were invited to submit data. The survey was sent to center directors via e-mail with anonymous links by Qualtrics™. The survey involved questions about their patient characteristics, clinical practices, and outcomes. Results: Forty-one (24.8%) centers responded to e-mails, and data gathered from 25 centers was included in the analyses. In 25 centers, 18,127 fresh or frozen transfers were carried out during the study period, of which 7796 (43.0%) were fresh and the rest were either frozen (45.2%) or embryo transfers (ET) with preimplantation genetic testing (PGT) (11.8%). The live birth rate per ET was as 30.6%, 40.1%, and 50.7% in fresh, frozen and PGT cycles, respectively. A single embryo was transferred in 65.3% of all transfers and singleton live births comprised 86.1% of all deliveries. For cycles with intrauterine insemination, 1407 were started in 2019, and 195 clinical pregnancies, 150 live births with 19 multiple pregnancies occurred. A total of 1513 ART cycles were initiated for foreign patients. Russia (29.6%), Germany (7.4%), Iraq (4.6%), Uzbekistan (3.1%), and Syria (1.4%) were the top five countries with most patients coming to Turkey for ART. Conclusion: The survey results are in parallel with the reports of international institutions and organizations. With repeated editions, the data collected with annual surveys can be used to inform ART practices in the coming years.

Comparing performance between clinics of an embryo evaluation algorithm based on time-lapse images and machine learning.

PURPOSE: This article aims to assess how differences in maternal age distributions between IVF clinics affect the performance of an artificial intelligence model for embryo viability prediction and proposes a method to account for such differences. METHODS: Using retrospectively collected data from 4805 fresh and frozen single blastocyst transfers of embryos incubated for 5 to 6 days, the discriminative performance was assessed based on fetal heartbeat outcomes. The data was collected from 4 clinics, and the discrimination was measured in terms of the area under ROC curves (AUC) for each clinic. To account for the different age distributions between clinics, a method for age-standardizing the AUCs was developed in which the clinic-specific AUCs were standardized using weights for each embryo according to the relative frequency of the maternal age in the relevant clinic compared to the age distribution in a common reference population. RESULTS: There was substantial variation in the clinic-specific AUCs with estimates ranging from 0.58 to 0.69 before standardization. The age-standardization of the AUCs reduced the between-clinic variance by 16%. Most notably, three of the clinics had quite similar AUCs after standardization, while the last clinic had a markedly lower AUC both with and without standardization. CONCLUSION: The method of using age-standardization of the AUCs that is proposed in this article mitigates some of the variability between clinics. This enables a comparison of clinic-specific AUCs where the difference in age distributions is accounted for.

An annotated human blastocyst dataset to benchmark deep learning architectures for in vitro fertilization.

Medical Assisted Reproduction proved its efficacy to treat the vast majority forms of infertility. One of the key procedures in this treatment is the selection and transfer of the embryo with the highest developmental potential. To assess this potential, clinical embryologists routinely work with static images (morphological assessment) or short video sequences (time-lapse annotation). Recently, Artificial Intelligence models were utilized to support the embryo selection procedure. Even though they have proven their great potential in different in vitro fertilization settings, there is still considerable room for improvement. To support the advancement of algorithms in this research field, we built a dataset consisting of static blastocyst images and additional annotations. As such, Gardner criteria annotations, depicting a morphological blastocyst rating scheme, and collected clinical parameters are provided. The presented dataset is intended to be used to train deep learning models on static morphological images to predict Gardner's criteria and clinical outcomes such as live birth. A benchmark of human expert's performance in annotating Gardner criteria is provided.

Dynamic view of assisted reproduction in Turkey from 1996 to 2020.

RESEARCH QUESTION: What are the trends and changes in patient demographics and practices in treatment with assisted reproductive technology (ART) in Turkey over 25 years? DESIGN: Data on patient demographics, cycle characteristics and clinical outcome of 29,541 cycles in 22,867 women who underwent treatment with ART between 1996 and 2020 were analysed according to calendar years. Regression and interrupted time series analysis were used to assess changes in patient characteristics, trends and effects of interventions on outcome. RESULTS: From 1996 to 2020, the average age of women undergoing treatment with ART increased from 32.1 to 36.0 years (r = 0.96), the proportion of women over 40 more than tripled (9% versus 28.7%; r = 0.97) and the average duration of infertility at presentation dropped from 8.4 to 4 years (r = -0.98) (P < 0.0001, for all). Diminished ovarian reserve became the major indication by 2015. Gonadotrophin-releasing hormone antagonists dominated ovarian stimulation by 2009. The average number of oocytes retrieved decreased from 11.5 to 7.8 (r = -0.86, P < 0.0001). Blastocyst-stage transfers gradually increased, comprising 51% of all transfers in 2020 (r = 0.86, P < 0.0001). The mean number of embryos transferred decreased from 3.9 to 1.5. Clinical pregnancy rates (CPR) per embryo transfer remained stable for fresh transfers (range: 31.6-43.9%) but increased from 13% to 30.3% in cryopreserved transfers. The estimate of effect of blastocyst vitrification was significant (P = 0.001). The multiple birth rate declined from 30.4% to 7.1%. CONCLUSIONS: Remarkable changes were seen in patient demographics, treatment indications, and clinical and laboratory practices. Increased use of single embryo transfer and improvements in cryopreservation techniques helped maintain high CPR while reducing multiple births.

What is the optimum number of follicular flushes in mono-follicular in-vitro fertilization cycles in a poor responder population?

OBJECTIVE: Assessment of the optimal number of follicular flushes on retrieval rate and quality of oocytes in mono-follicular in-vitro fertilization (IVF) cycles. MATERIAL AND METHODS: A retrospective analysis of 246 oocyte pick-up procedures in mono-follicular IVF cycles of 226 poor responder women was performed. The primary endpoint was oocyte retrieval rate in the initial aspirate versus subsequent flushing episodes. The secondary endpoints were oocyte maturity, fertilization rates and embryo cleavage. RESULTS: The procedure was successful in 187 cycles (76%), of which 160 metaphase-II oocytes were retrieved. Retrieval rates were similar for natural and modified natural cycles (p=0.595). The initial aspirate provided 54% of the total yield and the rest was obtained from up to four episodes of flushing. Follicular flushing increased oocyte recovery rate from 41.1% to 76%. None of the oocytes retrieved after three flushes fertilized. Oocyte maturity, fertilization and embryo cleavage rates were comparable for oocytes from the initial aspirate and one or two episodes of flushing. Oocytes obtained after the third flushing episode developed into poor quality embryos. CONCLUSION: Flushing confers a benefit for oocyte recover rates in mono-follicular IVF cycles in poor responder women. However, more than three attempts at flushing were not associated with good outcome.

Platelet-rich plasma another add-on treatment getting out of hand? How can clinicians preserve the best interest of their patients?

Add-on treatments in IVF are utilized to a great extent but without sufficient evidence showing their effectiveness. Since the offered treatments are usually costly and may be associated with yet unknown risks, this practice is not in the best interest of couples that may go to great lengths to conceive and have an offspring carrying their own genetic make-up. A recent addition to this armamentarium is the administration of platelet-rich plasma (PRP) in women with diminished ovarian reserve, implantation failures, and a thin endometrium. The only evidence for PRP comes from small scale and mostly before and after studies with clinically irrelevant end points. PRP has not been subjected to a rigorous clinical trial. It is a typical example of an add-on gaining widespread popularity based on biological plausibility and mind-bending theoretical presumptions. We should be extremely cautious prior to implementing PRP on a widescale and await the results of well-designed studies.

Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen.

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml-1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

Luteal granulosa cells from natural cycles are more capable of maintaining their viability, steroidogenic activity and LH receptor expression than those of stimulated IVF cycles.

STUDY QUESTION: Are there any differences in the molecular characteristics of the luteal granulosa cells (GC) obtained from stimulated versus non-stimulated (natural) IVF cycles that may help explain the defective luteal phase in the former? SUMMARY ANSWER: Luteal GC of stimulated IVF cycles, particularly those of agonist-triggered antagonist cycles, are less viable ex vivo, express LH receptor and anti-apoptotic genes at lower levels, undergo apoptosis earlier and fail to maintain their estradiol (E2) and progesterone (P4) production in comparison to natural cycle GC. WHAT IS KNOWN ALREADY: Luteal function is defective in stimulated IVF cycles, which necessitates P4 and/or hCG administration (known as luteal phase support) in order to improve clinical pregnancy rates and prevent miscarriage. The luteal phase becomes shorter and menstruation begins earlier than a natural cycle if a pregnancy cannot be achieved, indicative of early demise of corpus luteum (premature luteolysis). Supra-physiological levels of steroids produced by multiple corpora luteae in the stimulated IVF cycles are believed to inhibit LH release directly via negative feedback actions on the hypothalamic-pituitary-ovarian axis resulting in low circulating levels of LH and a defective luteal phase. We hypothesized that some defects in the viability and steroidogenic activity of the luteal GC of the stimulated IVF cycles might contribute to this defective luteal phase in comparison to natural cycle GC. This issue has not been studied in human before. STUDY DESIGN, SIZE, DURATION: A comparative translational research study of ex vivo and in vitro models of luteal GC recovered from IVF patients undergoing natural versus stimulated IVF cycles was carried out. Luteinized GC were obtained from 154 IVF patients undergoing either natural (n = 22) or stimulated IVF cycles with recombinant FSH and GnRH agonist (long) (n = 44), or antagonist protocol triggered conventionally either with recombinant hCG (n = 46) or with a GnRH agonist (n = 42). GC were maintained in vitro for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cellular viability (YO-PRO-1 staining), the expression of the steroidogenic enzymes, pro-apoptotic genes [Bcl-2-associated death promoter (BAD), Bcl-2-associated X protein (BAX) and Caspase-3 (CASP3)], anti-apoptotic genes [RAC-alpha serine/threonine-protein kinase (AKT-1) and Bcl-2-like protein 2 (BCL2-L2)], LH receptor, vascular endothelial growth factor (VEGF) (using real-time quantitative PCR at mRNA level and western blot immunoprecipitation assay at protein level) and in vitro E2 and P4 production (electrochemiluminescence immunoassay) were compared in GC among the groups. MAIN RESULTS AND THE ROLE OF CHANCE: Natural cycle GC were significantly more viable ex vivo (88%) compared to their counterparts of the stimulated IVF cycles (66, 64 and 37% for agonist and antagonist cycles triggered with hCG and GnRH agonist respectively, P < 0.01). They were also more capable of maintaining their vitality in culture compared to their counterparts from the stimulated IVF cycles: at the end of the 6-day culture period, 74% of the cells were still viable whereas only 48, 43 and 22% of the cells from the agonist and antagonist cycles triggered with hCG and agonist respectively, were viable (P < 0.01). The mRNA expression of anti-apoptotic genes (AKT-1 and BCL2-L2) was significantly lower, while that of pro-apoptotic genes (BAD, BAX and CASP3) was significantly higher in the stimulated cycles, particularly in the agonist-triggered antagonist cycles, compared to natural cycle GC (P < 0.01 for long protocol and antagonist hCG trigger, P < 0.001 for agonist trigger). The expression of steroidogenic enzymes (stAR, SCC, 3ß-HSD and aromatase) and VEGF was significantly higher in the agonist and hCG-triggered antagonist cycles compared to natural cycle GC. Therefore, in vitro E2 and P4 production in cells from the stimulated IVF cycles was significantly higher than their counterparts obtained from the natural cycles in the first 2 days of culture. However, after Day 2, their viability and hormone production began to decline very rapidly with the most drastic decrease being observed in the agonist-triggered cycles. By contrast, natural cycle GC maintained their viability and produced E2 and P4 in increasing amounts in culture up to 6 days. In vitro P production and the mRNA and protein expression of LH receptor, VEGF and 3ß-HSD were most defective in the agonist-triggered antagonist cycles compared to natural and agonist and hCG-triggered antagonist cycles. In vitro hCG treatment of a subset of the cells from the agonist-triggered cycles improved their viability, increased E2 and P4 production in vitro and up-regulated the mRNA expression of anti-apoptotic gene BCL-L2 together with steroidogenic enzymes stAR, SCC, 3B-HSD, LH receptor and VEGF. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The limitations include analysis of luteinized GC only might not reflect the in vivo mechanisms involved in survival and function of the whole corpus luteum; GC recovered during oocyte retrieval belong to a very early stage of the luteal phase and might not be representative; effects of ovulation triggered with hCG may not equate to the endogenous LH trigger; the clinical characteristics of the patients may vary among the different groups and it was not possible to correlate stimulation-related molecular alterations in luteal GC with the clinical outcome, as no oocytes have been utilized yet. Therefore, our findings do not conclusively rule out the possibility that some other mechanisms in vivo may also account for defective luteal function observed in stimulated IVF cycles. WIDER IMPLICATIONS OF THE FINDINGS: Ovarian stimulation is associated with significant alterations in the viability and steroidogenic activity of luteal GC depending on the stimulation protocol and mode of ovulation trigger. Reduced survival and down-regulated expression of 3B-HSD, LH receptor and VEGF leading to compromised steroid production in stimulated cycles, and particularly in the agonist-triggered cycles, may at least in part help explain why the luteal phase is defective and requires exogenous support in these cycles. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koç University Research Center for Translational Medicine (KUTTAM), equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest.

Bologna criteria are predictive for ovarian response and live birth in subsequent ovarian stimulation cycles.

PURPOSE: The ESHRE Working Group on Poor Ovarian Response defined a set of variables to define poor responders, named as the Bologna Criteria, but several concerns have been raised regarding their applicability and prognostic significance. In order to evaluate the clinical relevance of the criteria, we retrospectively analyzed the ovarian response and live birth rates in women who had consecutive IVF attempts, according to their fulfillment of the criteria. METHODS: The study group comprised 1153 and 288 women who had two and three consecutive ovarian stimulation (OS) cycles between May 2010 and January 2017, respectively. We compared the ovarian response and live birth rates in subsequent IVF attempts of Bologna criteria-defined poor responder women and women who did not fulfill the Bologna criteria. RESULTS: Women who fulfilled the criteria achieved higher rates of poor ovarian response (76.2% vs 14.3% and 60.3% vs 13.4%) and lower live birth rates (14.6% vs 33.3% and 12.9% vs 34.3%) in their second and third OS cycles, respectively (both p < 0.001) compared to women who did not fulfill the criteria. The former group also had lower number of oocytes and lower likelihood of having embryo transfer in their subsequent OS cycles. The criteria were able to predict both ovarian response and clinical outcome in the subsequent cycle in < 40-year-old women, whereas they were predictive only for the ovarian response but not for the clinical outcome in women over 40 years of age, who exhibited very low live birth rates regardless of the fulfillment of the criteria. CONCLUSIONS: The results of this study show that the Bologna criteria are clinically relevant in terms of prediction of ovarian response and clinical outcome in subsequent OS cycles.

Spontaneous and in vitro fertilization pregnancies have comparable first trimester screening profiles for Down syndrome

Objective: We aimed to compare the first trimester screening profiles of spontaneous (n=972) and in in vitro fertilization (IVF) pregnancies (n=339) in a population of patients who had uncomplicated singleton pregnancies comparable for maternal age, gestation, body mass index, and ethnicity. Material and Methods: A non-interventional analysis of retrospective cohort data and review of the literature. Results: All IVF pregnancies were achieved via intracytoplasmic sperm injection using the same ovarian stimulation protocol with recombinant follicle-stimulating hormone and a gonadotropin-releasing hormone antagonist, cetrorelix acetate. The means of the multiple of median (MoM) of pregnancy-associated plasma protein-A (PAPP-A) were slightly lower in the fresh (1.19±0.6 vs 1.33±0.7, respectively; p=0.056) and frozen embryo transfer (1.03±0.5 vs 1.33±0.7, respectively; p=0.036) IVF pregnancies compared with natural conceptions. However, when the medians of the MoMs of PAPP-A and beta-human chorionic gonadotrophin (ß-hCG), and their distributions were compared across the mode of conception, there were no differences between IVF pregnancies spontaneous pregnancies. Furthermore, the scatterplot diagram and curve fitting regression analyses revealed no difference in the temporal relations of ß-hCG and PAPP-A with each other and gestational age between spontaneous and IVF pregnancies. Conclusion: These results support the notion that uncomplicated singleton IVF pregnancies have similar first trimester screening profiles to spontaneous conceptions.

High responders are not exempt from detrimental effects of prematurely rising progesterone levels in fresh embryo transfer cycles.

RESEARCH QUESTION: Are high-responder IVF patients protected from the deleterious effect of prematurely elevated serum progesterone level on the probability of pregnancy? DESIGN: In this retrospective cohort study, 2971 autologous fresh embryo transfer IVF cycles with gonadotrophin-releasing hormone agonist long protocol were analysed to investigate whether the detrimental effect of prematurely rising progesterone levels on clinical pregnancy rate (CPR) varies depending on the magnitude of ovarian response. Nine different evenly spaced intervals were constructed for serum progesterone level on the human chorionic gonadotrophin day (<0.5/0.5-0.9/1-1.4/1.5-1.9/2-2.4/2.5-2.9/3-3.4/3.5-3.9/>4 ng/ml). Then, IVF cycles in each of these intervals were further divided into low (≤3 oocytes), normal (4-15 oocytes) and high responders (≥16 oocytes). RESULTS: The progressive rise of serum progesterone from the <0.5 to the >4 ng/ml interval caused a gradual and continuous decline in the CPR of all three types of ovarian response. The absolute difference in the CPR between the lowest and the highest progesterone groups was not related to the magnitude of ovarian response (-26.6%, -37.7% and -40.7% for the low, normal and high responders, respectively). On multivariate logistic regression analysis, the detrimental effect of progesterone started at 1.5-1.9 ng/ml, 3.0-3.4 ng/ml and 4.0-4.4 ng/ml intervals for the low, normal and high responders, respectively. CONCLUSION: High responders are not exempt from the detrimental effects of prematurely rising serum progesterone levels but the threshold interval where the detrimental effect begins is higher in the high responders compared with the low and normal responders.

FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization.

STUDY QUESTION: Can granulosa cells produce progesterone (P) in response to FSH stimulation? SUMMARY ANSWER: FSH actively promotes P synthesis and output from granulosa cells without luteinization by up-regulating the expression and increasing enzymatic activity of 3ß-hydroxysteriod dehydrogenoase (3ß-HSD), which converts pregnenolone to P. WHAT IS KNOWN ALREADY: Serum P level may rise prematurely prior to ovulation trigger in stimulated IVF cycles and adversely affect implantation and clinical pregnancy rates by impairing endometrial receptivity. STUDY DESIGN, SIZE, DURATION: A translational research study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian cortical samples (n = 15) and non-luteinizing FSH-responsive human mitotic granulosa cell line (HGrC1) were stimulated with rec-FSH at 12.5, 25 and 50 mIU/ml concentrations for 24 and 48 h. FSH receptor expression was knocked-down and up-regulated in the granulosa cells using short hairpin RNA (shRNA) technology and activin-A administration, respectively. The expressions of the steroidogenic enzymes were analyzed at mRNA level by real-time quantitative RT-PCR, and protein level by western blot and immunoprecipitation assay. The enzymatic activity of 3ß-HSD was measured using a spectrophotometric method. In vitro estradiol (E2) and P productions of the cells before and after FSH stimulation were measured by electro-chemiluminescence immunoassay method. MAIN RESULTS AND THE ROLE OF CHANCE: Stimulation of the HGrC1 cells with FSH resulted in a dose-dependent increase in the mRNA and protein level of 3ß-HSD. Overall, when all time points and FSH doses were analyzed collectively, FSH significantly up-regulated the mRNA expression of its own receptor (3.73 ± 0.06-fold, P < 0.001), steroidogenic acute regulatory protein (stAR, 1.7 ± 0.03-fold, P < 0.01), side-chain cleavage enzyme (SCC, 1.75 ± 0.03-fold, P < 0.01), aromatase (4.49 ± 0.08-fold, P < 0.001), 3ß-HSD (1.68 ± 0.02-fold, P < 0.01) and 17ß-hydroxy steroid dehydrogenase (17ß-HSD, 2.16 ± 0.02-fold, P < 0.01) in the granulosa cells. Expression of 17α-hydroxylase (17α-OH, 1.03 ± 0.01-fold P > 0.05) did not significantly change. Similar changes were observed in the protein expression analysis of these enzymes on western blotting after FSH stimulation. FSH significantly increased 3ß-HSD, 17ß-HSD and aromatase in a dose-dependent manner but did not affect 17α-OH. Protein expression of P was increased along with 3ß-HSD after FSH stimulation, which was further evidenced by immunoprecipitation assay. Enzymatic activity of 3ß-HSD was significantly enhanced by FSH administration in the HGrC1 cells in a dose-dependent manner. In line with these findings P output (1.05 ± 0.3 vs. 0.2 ± 0.1 ng/ml, respectively, P < 0.001) from the samples stimulated with FSH were significantly increased along with E2 (1918 ± 203 vs. 932 ± 102 pg/ml, respectively, P < 0.001) compared to unstimulated controls. FSH-induced increase in 3ß-HSD expression was amplified and reversed in the HGrC1 cells when FSH receptor expression was up-regulated by activin-A and down-regulated with shRNA, respectively. LIMITATIONS AND REASONS FOR CAUTION: As only the effect of FSH was studied we cannot extrapolate our findings to the potential effects of HMG and recombinant LH. WIDER IMPLICATIONS OF THE FINDINGS: This data provides a molecular explanation for the largely unexplained phenomenon of P rise during the follicular phase of gonadotropin stimulated IVF cycles. Our findings may progress the research to uncover potential mechanisms for preventing premature P rise that appears to be associated with inferior outcomes in women undergoing IVF. STUDY FUNDING/COMPETING INTEREST(S): Funded by the School of Medicine and the Graduate School of Health Sciences of Koc University. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: None.

6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for transforming growth factor ß1-enhanced invasion of Panc1 cells in vitro.

Transforming growth factor ß1 (TGFß1) is a well-established inducer of the epithelial-mesenchymal transition (EMT) that is essential for the acquisition of malignant properties, such as invasion, in tumor cells. Although recent studies suggest that the EMT in tumor cells is associated with reprogramming of energy metabolism and TGFß1 has been shown to stimulate glycolysis in multiple primary cell lines, little is known about TGFß1's effect on glycolysis and glycolytic regulators in transformed cells. Given the known regulatory role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) in glycolysis and association of glycolytic activity with malignant features such as invasion, we sought to investigate whether TGFß1 regulates PFKFB3 expression and if PFKFB3 is involved in the TGFß1-mediated increase in the invasive ability of the Panc1 cell cline-a well-established model of TGFß1-initiated EMT. Herein we demonstrate that TGFß1 induces PFKFB3 expression and stimulates glycolysis in Panc1 cells. We also show that siRNA silencing of PFKFB3 prevents the stimulation of glycolysis and in vitro invasive ability of Panc1 cells by TGFß1. Furthermore, PFKFB3 silencing suppresses the TGFß1-mediated induction of the Snail protein, suggesting that PFKFB3 is required for the regulation of Snail expression by TGFß1. Taken together, our study identifies PFKFB3 as a key TGFß1 effector protein that mediates TGFß1's effect on Snail expression, invasion, and glycolysis.

Vaginal Progesterone Gel for Luteal Support After Cleavage Stage Embryo Transfer: Once or Twice a Day?

OBJECTIVE: To determine whether twice daily dosing of progesterone vaginal gel (PVG) is better for luteal phase support (LPS) than once daily dosing. STUDY DESIGN: Retrospective study including 456 women aged ≤42 years who underwent assisted reproductive technology with long GnRH agonist protocol. Blastocyst transfers and difficult embryo transfers were excluded. LPS was started with 90 mg PVG once daily on the evening of oocyte retrieval and continued until negative pregnancy test or 10th week of pregnancy in both groups. PVG dosage was doubled on the day of embryo transfer in the twice-daily group. RESULTS: Age, duration of infertility, and number of oocytes collected were similar. Numbers of embryos transferred were 2.9 and 2.8 in the once-daily and twice-daily groups, respectively (p=0.04). Embryo implantation (23.96% vs. 27.95%) and clinical pregnancy (50.9% vs. 56.5%) rates favored twice-daily dosage; however, differences were statistically nonsignificant, and the study had 20% power to demonstrate significance. When our results were pooled with a prior trial comparing once and twice daily dosing, twice daily dosing seemed to significantly increase clinical pregnancy rate (rate ratio: 1.18, 95% CI 1.01-1.38). CONCLUSION: Trends favoring twice daily dosing are encouraging findings and require further investigation.

Assessment of human embryo development using morphological criteria in an era of time-lapse, algorithms and 'OMICS': is looking good still important?

With the worldwide move towards single embryo transfer there has been a renewed focus on the requirement for reliable means of assessing embryo viability. In an era of 'OMICS' technologies, and algorithms created through the use of time-lapse microscopy, the actual appearance of the human embryo as it progresses through each successive developmental stage to the blastocyst appears to have been somewhat neglected in recent years. Here we review the key features of the human preimplantation embryo and consider the relationship between morphological characteristics and developmental potential. Further, the impact of the culture environment on morphological traits, how key morphological qualities reflect aspects of embryo physiology, and how computer-assisted analysis of embryo morphology may facilitate a more quantitative approach to selection are discussed. The clinical introduction of time-lapse systems has reopened our eyes and given us a new vantage point from which to view the beauty of the initial stages of human life. Rather than a future in which the morphology of the embryo is deemed irrelevant, we propose that key features, such as multinucleation, cell size and blastocyst differentiation should be included in future iterations of selection/deselection algorithms.

Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

STUDY QUESTION: Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER: The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY: Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION: This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE: The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION: Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS: GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells.

STUDY QUESTION: Do different chemotherapy drugs exert the same magnitude of cytotoxicity on dormant primordial follicles and the growing follicle fraction in the ovary in vivo and on mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro? SUMMARY ANSWER: Cyclophosphamide (alkylating agent) and cisplatin (alkylating like) impacted both primordial and pre-antral/antral follicles and both mitotic and non-mitotic granulosa cells, whereas the anti-metabolite cancer drug gemcitabine was detrimental only to pre-antral/antral follicles and mitotic non-luteinized granulosa cells. WHAT IS KNOWN ALREADY: It is already known that anti-metabolite cancer drugs are less detrimental to the ovary than alkylating and alkylating like agents, such as cyclophosphamide and cisplatin. This assumption is largely based on the results of clinical reports showing lower rates of amenorrhea in women receiving anti-metabolite agent-based regimens compared with those treated with the protocols containing an alkylating drug or a platinum compound. But a quantitative comparison of gonadotoxicity with a histomorphometric proof of evidence has not been available for many chemotherapy drugs. Therefore, we combined in this study in vivo and in vitro models of human and rat origin that allows a comparative analysis of the impact of different chemotherapy agents on the ovary and granulosa cells using real-time quantitative cell indices, histomorphometry, steroidogenesis assays, and DNA damage and cell death/viability markers. We also aimed to investigate if there is a difference between mitotic and non-mitotic granulosa cells in terms of their sensitivity to the cytotoxic actions of chemotherapy drugs with different mechanisms of action. This issue has not been addressed previously. STUDY DESIGN, SIZE, DURATION: This translational research study involved in vivo analyses of ovaries in rats and in vitro analyses of granulosa cells of human and rat origin. PARTICIPANTS/MATERIALS, SETTING, METHODS: For the in vivo assays, 54 4- to 6-week old Sprague-Dawley young female rats were randomly allocated into four groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. LIMITATIONS, REASONS FOR CAUTION: This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells.

Laboratory procedures for human in vitro fertilization.

Successful and consistent outcomes in human in vitro fertilization (IVF) can be readily achieved by all IVF clinics through consideration and optimization of each procedure associated with the collection and processing of gametes, culminating in the resultant culture and transfer of healthy embryos. Furthermore, understanding the interactions between the individual components of the IVF cycle will assist when trouble-shooting possible problems in a laboratory which could have an adverse effect on cycle outcome. This article will review handling of oocytes and embryo culture, preparation of gametes for insemination and microinjection, selection of the most viable gametes and embryos, cryopreservation, and successful embryo transfer from the laboratory perspective.

Association between blastocyst morphology and outcome of single-blastocyst transfer.

The aim of this study was to assess the ability of three individual blastocyst morphology parameters - expansion and hatching (EH) stage, inner cell mass (ICM) grade and trophectoderm grade - to predict outcome of a cycle with single-blastocyst transfer. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly (P<0.005 for each) associated with positive human chorionic gonadotrophin, clinical and ongoing pregnancy rates and live birth rates, while only the ICM grade was significantly (P=0.033) associated with early pregnancy loss rate. Blastocyst EH stage was the only significant predictor of live birth (P=0.002) in the multiple logistic regression. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the EH stage. Transfer of a blastocyst with ICM grade A may reduce the risk of early pregnancy loss. Choosing the embryo(s) with the best implantation potential is essential for securing each couple the highest chance of achieving pregnancy after assisted reproduction. The selection of embryo(s) for transfer at the blastocyst stage is based on morphology parameters of expansion and hatching stage, inner cell mass grade and trophectoderm grade. The aim of this study was to assess the relative impact of each parameter in predicting the probability of a successful outcome. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 patients who underwent single-blastocyst transfer on day 5 were included. Statistical analysis showed that all three blastocyst morphology parameters were significantly associated with positive human chorionic gonadotrophin (ßHCG), clinical and ongoing pregnancy rates and live birth rates. Only the inner cell mass grade was significantly associated with early pregnancy loss between the positive ßHCG test and confirmation of ongoing pregnancy 10-11weeks after transfer. The expansion and hatching stage was the only significant predictor of live birth in the multiple logistic regression analysis. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the expansion and hatching stage. Transfer of a blastocyst with inner cell mass grade A may reduce the risk of early pregnancy loss.