Patients with gynecological malignancies are similar to other IVF patients without cancer for clinical and molecular reproductive parameters and DNA damage response pattern.

This study intended to investigate if gynecological cancers compromise ovarian function and reduce the success of assisted reproduction techniques (ART). No clinical and molecular data together is available on this issue for gynecological or other organ cancers. Steroidogenic pathways and DNA damage response characteristics of the granulosa cells retrieved from the 39 gynecological cancer patients were analyzed together with their clinical ART characteristics in comparison to 31 control ART patients. Patients with gynecological malignancies were similar to the control IVF patients for the number of mature oocytes retrieved, fertilization rates and embryo development competency. Molecular analyses of the granulosa cells retrieved from these cancer patients did not detect any perturbations in gonadotropin receptor expression and response, sex steroid production, cholesterol utilization/storage and, DNA damage response pattern in comparison to control IVF patients without cancer. This study provides the first reassuring clinical and molecular combined data set that the presence of gynecological malignancy does not appear to have any detrimental effect on clinical IVF cycle characteristics and ovarian functioning at molecular level.

Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

Real-Time Visualization of Cholesterol Trafficking in Human Granulosa Cells Using Confocal Live Cell Microscopy as a Tool to Study the Novel Role of Autophagy in Sex Steroid Synthesis.

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. We have very recently demonstrated that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LDs) with lysosomes to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis in human ovary and testis. In this chapter, we describe live cell confocal microscopy technique that allows us to monitor real-time cholesterol trafficking and the association of cholesterol-laden LDs with lysosome (lipophagy) in human granulosa cells.

Progesterone signaling in the regulation of luteal steroidogenesis.

The corpus luteum is the major source of progesterone, the essential hormone for female reproductive function. While progesterone activity has been the subject of extensive research for decades, characterization of non-canonical progesterone receptor/signaling pathways provided a new perspective for understanding the complex signal transduction mechanisms exploited by the progesterone hormone. Deciphering these mechanisms has significant implications in the management of luteal phase disorders and early pregnancy complications. The purpose of this review is to highlight the complex mechanisms through which progesterone-induced signaling mediates luteal granulosa cell activity in the corpus luteum. Here, we review the literature and discuss the up-to-date evidence on how paracrine and autocrine effects of progesterone regulate luteal steroidogenic activity. We also review the limitations of the published data and highlight future research priorities.

Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis.

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. Previous studies documented the role of autophagy in sex steroid hormone biosynthesis in different animal models and human testis. Here we demonstrate in this study that sex steroid hormones estrogen and progesterone are produced through the same autophagy-mediated mechanism in the human ovary in addition to the human testis. In brief, pharmacological inhibition and genetic interruption of autophagy through silencing of autophagy genes (Beclin1 and ATG5) via siRNA and shRNA technologies significantly reduced basal and gonadotropin-stimulated estradiol (E2), progesterone (P4) and testosterone (T) production in the ex vivo explant tissue culture of ovary and testis and primary and immortalized granulosa cells. Consistent with the findings of the previous works, we observed that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LD)s with lysosome to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis. Gonadotropin hormones are likely to augment the production of sex steroid hormones by upregulating the expression of autophagy genes, accelerating autophagic flux and promoting the association of LDs with autophagosome and lysosome. Moreover, we detected some aberrations at different steps of lipophagy-mediated P4 production in the luteinized GCs of women with defective ovarian luteal function. The progression of autophagy and the fusion of the LDs with lysosome are markedly defective, along with reduced P4 production in these patients. Our data, together with the findings of the previous works, may have significant clinical implications by opening a new avenue in understanding and treatment of a wide range of diseases, from reproductive disorders to sex steroid-producing neoplasms, sex steroid-dependent malignancies (breast, endometrium, prostate) and benign disorders (endometriosis).

Breast cancer treatment and ovarian function.

The aim of this study was to provide an update on ovarian function and the mechanisms of gonadal damage after exposure to chemotherapy in breast cancer survivors. The alkylating agents are toxic to both primordial and growing follicles. However, anti-metabolite drugs are more likely to destroy preantral and antral follicles. Younger patients are more likely to have a higher ovarian reserve, and therefore, more likely to retain some residual ovarian function after exposure to gonadotoxic regimens. However, there can be significant variability in ovarian reserve among patients of the same age. Furthermore, patients with critically diminished ovarian reserve may continue to menstruate regularly. Therefore age and menstrual status are not reliable indicators of good ovarian reserve and might give a false sense of security and result in an adverse outcome if the patient is consulted without considering more reliable quantitative markers of ovarian reserve (antral follicle count and anti-Müllerian hormone) and fertility preservation is not pursued. In contrast to well-documented ovarian toxicity of older chemotherapy regimens, data for newer taxane-containing protocols have only accumulated in the last decade and data are still very limited regarding the impact of targeted therapies on ovarian function.

IVF characteristics and the molecular luteal features of random start IVF cycles are not different from conventional cycles in cancer patients.

STUDY QUESTION: Are the IVF parameters and the steroidogenic luteal characteristics of random-start IVF cycles different from conventional cycles in cancer patients? SUMMARY ANSWER: No; controlled ovarian stimulation cycles randomly started at late follicular phase (LFP) and luteal phase (LP) are totally comparable to those conventional IVF cycles started at early follicular phase (EFP) in terms of the expression of the enzymes involved in cholesterol utilization and steroid hormone biosynthesis pathways, gonadotropin receptor expression and, estradiol (E2) and progesterone (P4) production in addition to the similarities in ovarian response to gonadotropin stimulation, oocyte yield, fertilization rate and embryo development competency in cancer patients. WHAT IS KNOWN ALREADY: Random start ovarian stimulation protocols are commonly employed for oocyte and embryo freezing for fertility preservation in cancer patients with time constraints who do not have sufficient time to undergo ovarian stimulation initiated conventionally at EFP of the next cycle. No data is available regarding the molecular steroidogenic features of these cycles analyzed together with the clinical IVF characteristics in cancer patients. We aimed to address this question in this study to help understand how similar the random start cycles are to the conventional start ones. STUDY DESIGN, SIZE, DURATION: A clinical translational research study conducted in 62 cancer patients undergoing IVF for fertility preservation between the years 2017 and 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixty-two patients who were diagnosed with different types of cancer and underwent ovarian stimulation for oocyte (n = 41) and embryo (n = 21) cryopreservation using GnRH antagonist protocol and human menopausal gonadotropins before receiving cancer treatment/surgery were enrolled in the study. For patients with breast cancer and endometrial cancer the aromatase inhibitor letrozole was used with gonadotropin stimulation. Ovarian stimulation was initiated conventionally at EFP in 22 patients and served as control while it was started at LFP in 20, and mid-LP in the other 20 patients. The luteinized granulosa cells (GCs) were recovered from follicular aspirates during oocyte retrieval procedure and used for the experiments separately for each individual patient. The expression of the enzymes involved in sex steroid biosynthesis (StAR, 3ß-HSD, Aromatase) and cholesterol synthesis (3-hydroxy 3-methylglutaryl Co-A reductase (HMG-Co-A reductase)), utilization (hormone sensitive lipase (HSL)), and storage (Acetyl-Coenzyme A acetyltransferase 1 (ACAT-1)), and gonadotropin receptor expression status were analyzed using immunoblotting and RT-PCR methods. Laser confocal immunofluorescence imaging was applied to analyze and compare the expression patterns of the steroidogenic enzymes and their relation with mitochondria. In vitro E2 and P4 production by the cells were compared among the groups. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline demographic and IVF characteristics of the patients undergoing the conventional start and random start IVF cycles were similar. Duration of gonadotropin stimulation was significantly longer in LFP and LP start cycles in comparison to the conventional ones. Ovarian response to gonadotropin stimulation, mature and total oocyte yield, fertilization and Day 5 blastulation rates of the embryos were comparable between the conventional versus random start cycles. When the luteal GCs of these random start cycles were analyzed we could not find any gross differences between these cycles in terms of the viability index and gross light microscopic morphologic features. More detailed analysis of the molecular luteal characteristics of the cells using RT-PCR, immunoblotting methods revealed that the expression profiles of the gonadotropin receptors, and the enzymes involved in sex steroid biosynthesis and cholesterol synthesis/utilization, and the steroidogenic activity of the luteal GCs of the random start cycles are almost identical to those of the conventional start cycles. Confocal image analysis demonstrated similar patterns in the signal expression profiles of the steroidogenic enzymes and their co-localization within mitochondria. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Caution should be exercised when interpreting our data and counseling cancer patients seeking fertility preservation because it is still unclear if previous exposure to cancer drugs, different ovarian pathologies or infertility etiologies, previous ovarian surgery and/or any other underlying diseases that are concomitantly present with cancer may cause a difference between conventional and random start stimulation protocols in terms of IVF parameters, luteal function and reproductive outcome. Relatively low number of patients in each stimulation protocol and pooling of luteal GCs for each patient rather than individual analysis of each follicle and oocyte are additional limitations of our study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide reassurance that random start protocol offers cancer patients an equally good prospect of fertility preservation as conventional IVF. STUDY FUNDING/COMPETING INTEREST(S): 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. TRIAL REGISTRATION NUMBER: N/A.

Cholesterol uptake or trafficking, steroid biosynthesis, and gonadotropin responsiveness are defective in young poor responders.

OBJECTIVE: To investigate whether poor ovarian response in young patients undergoing in vitro fertilization simply involves lesser follicle growth due to diminished ovarian reserve or whether there are intrinsic perturbations in the ovary. DESIGN: A translational research study. SETTING: University Hospital Translational Research Center. PATIENT(S): A total of 40 patients undergoing in vitro fertilization (20 normal and 20 poor responders) with ovarian stimulation using a gonadotropin-releasing hormone antagonist and recombinant follicle-stimulating hormone were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Luteal granulosa cells obtained during oocyte retrieval procedures were used for the experiments. Cell culture, quantitative real-time polymerase chain reaction, immunoblotting, confocal time-lapse live-cell imaging, and hormone assays were used. RESULT(S): We tracked the steroidogenic pathway starting from the very initial step of cholesterol uptake to the final step of estradiol and progesterone production in luteal granulosa cells and identified some previously unknown intrinsic defects in the poor responders. Most notably, the expression of low-density lipoprotein receptors was significantly down-regulated and the uptake of cholesterol and its cytoplasmic accumulation and transportation to mitochondria were substantially delayed and reduced in the poor responders. Further, the expression of the steroidogenic enzymes steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase, and aromatase as well as gonadotropin receptors was defective, and the response of the cells to exogenous follicle-stimulating hormone and human chorionic gonadotropin was blunted, leading to compromised basal and gonadotropin-stimulated estradiol and progesterone production in the poor responders. CONCLUSION(S): This study demonstrates that poor ovarian response in young individuals should not simply be regarded as lesser follicle growth due to diminished ovarian reserve because the underlying pathogenetic mechanisms appear to be much more complex.

Terminal differentiation of human granulosa cells as luteinization is reversed by activin-A through silencing of Jnk pathway.

Molecular mechanisms underlying luteinization (terminal differentiation of granulosa and theca cells after ovulation) and luteolysis (demise of corpus luteum) are poorly understood in human ovary. Here we report that activin-A, after binding to its cognate receptors induces a functional luteolytic state and reverses luteinization phenotype by downregulating the expression of the steroidogenic enzymes, LH receptor and VEGF and reducing estradiol (E2) progesterone (P4) production and upregulating FSH receptor and cyclin D1 expression in human primary luteinized granulosa cells. Further, this action of activin-A involves downregulation of JNK signaling pathway and is opposite to that of human chorionic gonadotropin (hCG), which acts as a luteotropic hormone and improves luteal function through the activation of JNK pathway in the same cell type. Reversal of luteinization phenotype in luteal granulosa cells by activin-A potentially makes this hormone an attractive candidate for use under certain clinical situations, where induction of luteolysis and rapid reduction of endogenous sex steroid levels are beneficial such as ovarian hyperstimulation syndrome (OHSS), in which the ovaries hyper-respond to gonadotropin stimulation by producing too many growing follicles along with development of ascites, pleural effusion, and hemo-concentrations as a result of increased vascular permeability and leakage of intravascular volume into third spaces. Our work unveils a previously undefined role for activin-A and JNK signaling pathway in human corpus luteum biology, that might have a direct clinical impact in assisted reproductive technologies.

There is a cycle to cycle variation in ovarian response and pre-hCG serum progesterone level: an analysis of 244 consecutive IVF cycles.

We aimed to answer one key question, that was not previously addressed as to whether serum progesterone (P4-hCG day) and its co-variates (estradiol (E2-hCG day) and the number of retrieved oocytes) of a given cycle can be predictive of the subsequent cycle when both cycles are consecutive and comparable for the stimulation protocol, gonadotropin dose and duration of stimulation. We analyzed such 244 consecutive (< 6 months) IVF cycles in 122 patients with GnRH agonist long protocol and found that P4, E2 and the number of retrieved oocytes significantly vary between the two cycles. Although P4 increased (ranging from 4.7 to 266.7%) in the 2nd cycle in 61 patients, E2 and the number of retrieved oocytes, which are normally positively correlated with P4 paradoxically decreased in the 41% and 37.7% respectively, of these same 61 patients. When a similar analysis was done in the 54 out of 122 patients (44.3%) in whom serum P4 was decreased in the 2nd cycle, the mean decrease in P4 was - 34.1 ± 23.3% ranging from - 5.26 to - 90.1%. E2 and the number of retrieved oocytes paradoxically increased in the 42.3% and 40.7% of these 54 patients respectively. P4 remained the same only in the 7 (5.7%) of these 122 patients. These findings indicate that late follicular phase serum P4 may change unpredictably in the subsequent IVF cycle. The changes are not always necessarily proportional with ovarian response of previous cycle suggesting that growth characteristics and steroidogenic activities of antral cohorts may exhibit considerable cycle to cycle variations.

In-vitro AMH production of ovarian tissue samples in culture correlates with their primordial follicle pool.

OBJECTIVE: We aimed to investigate if there is a correlation between in-vitro AMH production and primordial follicle reserve of the ovarian cortical samples in culture. METHODS: Seven patients undergoing laparoscopic excision of ovarian dermoid cysts were included in the study. 0.5 × 0.5 cm of ovarian cortical samples embedded within the cyst wall were removed and cultured for one day. Then, the cultured cortical pieces were fixed, paraffin-embedded and serially sectioned for histormorphometric analysis. AMH and estradiol (E2) production of the samples after one-day culture period were measured in the spent culture media. Primordial follicle density was expressed as the number of primordial follicles per mm2. Pearson correlation and linear regression analyses were applied. RESULTS: The mean age of the patients was 29.2 ± 6.8 (ranging from 18 to 36). There was a negative correlation between age and PF density (r=-0.92, %95CI: -0.99 to -0.76, p < 0.001). In-vitro AMH level of the cortical samples was significantly associated with age (R2 = 0.67, p = 0.023), primordial follicle density (R2 = 0.71, p = 0.015). There was a borderline significance between in-vitro levels of AMH and E2 level (R2 = 0.55, p = 0.058). A similar comparison could not be made for secondary follicles (preantral and small antral follicles) because of their rarity in the histological sections analyzed. CONCLUSIONS: This histomorphometric study provides evidence that in-vitro AMH production of the ovarian cortical samples reflects primordial follicle pool of the samples.

Anogenital distance in newborn infants conceived by assisted reproduction and natural conception.

RESEARCH QUESTION: Does anogenital distance (AGD) differ in newborn infants conceived through assisted reproduction technology (ART) compared with those conceived naturally? DESIGN: This case-control study looked at anthropometric and anogenital measurements in 247 male and 200 female newborns born after ART (n = 121) or natural conception (n = 326), within 24 h of birth. Anogenital measurements included distance from the centre of the anus to the anterior clitoris (AGDAC) and to the posterior fourchette (AGDAF) in female infants, and from the centre of the anus to the posterior base of the scrotum (AGDAS) and to the anterior base of the penis (AGDAP) in male infants. RESULTS: ART mothers were older, more likely to be nulliparous and delivered by Caesarean section at an earlier gestational week. AGDAS of male infants was approximately twice the AGDAF of female infants (17.6 ± 5.0 versus 9.1 ± 3.6 mm). AGDAF in female infants conceived by ART compared with those conceived naturally was not significantly different (8.8 ± 3.6 versus 9.3 ± 3.6 mm; P = 0.404). AGDAC were also comparable for both groups (27.4 ± 6.3 versus 27.7 ± 7.1 mm; P = 0.770). In male infants, no significant difference was seen between ART and natural conception groups in terms of AGDAS (17.4 ± 4.6 versus 17.7 ± 5.2 mm, P = 0.742) and AGDAP (37.5 ± 6.6 versus 38.0 ± 6.7 mm, P = 0.589). When adjusted for gestational age, weight, length and head circumference, mode of conception was not associated with differences in any of the anogenital measurements. CONCLUSIONS: AGD measurements in infants conceived by ART are no different from those of infants conceived naturally.

A comparative molecular analysis of DNA damage response, cell cycle progression, viability and apoptosis of malignant granulosa cells exposed to gemcitabine and cisplatin.

We aimed to provide a comparative characterization of DNA damage response elements, survival/apoptosis and cell cycle progression of the malignant granulosa cells exposed to gemcitabine and cisplatin. Malignant granulosa tumor cell lines COV434 and KGN were used for the experiments. Cell viability, proliferation, DNA damage response and apoptosis were investigated. Cell cycle progression was assessed. In vitro estradiol (E2) and AMH productions of the cells were measured. Exposure of asynchronous malignant granulosa cells to gemcitabine caused growth arrest, induced DNA damage and activated cellular stress pathways, cell cycle checkpoint sensors and triggered apoptosis as evidenced by increased expression of phospho-p38, γ-histone H2AX, phospho-Chk-1/phospho-Chk-2, and cleaved forms of PARP and caspase-3 in a dose dependent manner. In vitro E2 and AMH productions of the cells were decreased along with reduction in viable cell mass. Cisplatin treatment produced a similar response but it was associated with JNK activation rather than p38. When the cells were synchronized and treated with gemcitabine at G2/M transition, the degradation of cyclin B1 and dephosphorylation of cdc-2 at Tyr 15 residue did not occur, resulting in cycle arrest. Similar effects on cell cycle progression was also observed in cisplatin. However, it was associated with JNK activation and higher expression of γ-histone H2AX and cleaved forms of caspase-3 and PARP, indicative of more extensive DNA damage and apoptosis in the cells. This descriptive study provides evidence that gemcitabine exerts cytotoxic effects and causes perturbations in cell cycle progression of malignant granulosa cells.

hCG Improves Luteal Function and Promotes Progesterone Output through the Activation of JNK Pathway in the Luteal Granulosa Cells of the Stimulated IVF Cycles†.

Human chorionic gonadotropin (hCG) is a luteotropic hormone that promotes the survival and steroidogenic activity of corpus luteum (CL) by acting through luteinizing hormone receptors (LHRs) expressed on luteinized theca and granulosa cells (GCs). Therefore, it is used to support luteal phase in in vitro fertilization (IVF) cycles to improve clinical pregnancy rates and prevent miscarriage. However, the molecular mechanism underlying this action of hCG is not well characterized. To address this question, we designed an in vitro translational research study on the luteal GCs obtained from 58 IVF patients. hCG treatment at different concentrations and time points activated c-Jun N-terminal kinase (JNK) pathway and significantly increased its endogenous kinase activity along with upregulated expression of steroidogenic enzymes (steroidogenic acute regulatory protein (stAR), 3ß-Hydroxysteroid dehydrogenase (3ß-HSD)) in a dose-dependent manner in the luteal GCs. As a result, in vitro P production of the cells was significantly enhanced after hCG. When JNK pathway was inhibited pharmacologically or knocked-down with small interfering RNA luteal function was compromised, P4 production was declined along with the expression of stAR and 3ß-HSD in the cells. Further, hCG treatment after JNK inhibition failed to correct the luteal defect and promote P4 output. Similar to hCG, luteinizing hormone (LH) treatment improved luteal function as well and this action of LH was associated with JNK activation in the luteal GCs. These findings could be important from the perspective of CL biology and luteal phase in human because we for the first time identify a critical role for JNK signaling pathway downstream LHR activation by hCG/LH in luteal GCs. SUMMARY SENTENCE: JNK signaling pathway plays a central role in the upregulated expression of the steroidogenic enzymes StAR and 3b-HSD and augmented progesterone production by hCG/LH in human luteal granulosa cells.

Intrauterine administration of peripheral mononuclear cells in recurrent implantation failure: a systematic review and meta-analysis.

It has been proposed that intrauterine administration of peripheral blood mononuclear cells (PBMCs) modulates maternal immune response through a cascade of cytokines, chemokines and growth factors to favor implantation. We conducted a meta-analysis to verify the effect of intrauterine PBMC administration on the outcome of embryo transfer in women with recurrent implantation failure (RIF). All relevant trials published in PubMed, Web of Science and Cochrane library databases were searched. Two randomized controlled trials and three cohort studies (1173 patients in total) matched the inclusion criteria. No differences in live birth rates were seen between the PBMC-treated patients and controls (OR: 1.65, 95% CI: 0.84-3.25; p = 0.14; I2: 66.3%). The clinical pregnancy rate was significantly higher in women who received intrauterine PBMCs before embryo transfer compared with those who did not (OR: 1.65, 95% CI: 1.30-2.10; p = 0.001, heterogeneity; I2: 60.6%). Subgroup analyses revealed a significant increase in clinical pregnancy rates with the administration of PBMCs in women with ≥3 previous failures compared with controls (OR: 2.69, 95% CI: 1.53-4.72; p = 0.001, I2: 38.3%). In summary, the data did not demonstrate an association between the administration of PBMCs into the uterine cavity before fresh or frozen-thawed embryo transfer and live birth rates in women with RIF. Whether intrauterine PBMC administration significantly changes live birth and miscarriage rates requires further investigation.

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.

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.