The screening and expression of polysaccharide deacetylase from Caulobacter crescentus and its function analysis.

The bacterium Caulobacter crescentus secretes an adhesive polysaccharide called holdfast, which is the known strongest underwater adhesive in nature. The deacetylase encoded by hfs (holdfast synthesis) H gene is a key factor affecting the adhesion of holdfast. Its structure and function are not yet clear, and whether other polysaccharide deacetylases exist in C. crescentus is still unknown. The screening of both HfsH and its structural analogue as well as their purification from the artificial expression products of Escherichia coli is the first step to clarify these questions. Here, we determined the conserved domains of HfsH via sequence alignment among carbohydrate esterase family 4 enzymes and screened out its structural analogue (CC_2574) in C. crescentus. The recombinant HfsH and CC_2574 were effectively expressed in E. coli. Both of them were purified by chromatography from their corresponding productions in E. coli and were then functionally analyzed. The results indicated that a high deacetylase activity (61.8 U/mg) was observed in recombinant HfsH but not in CC_2574, which suggesting that HfsH might be the irreplaceable gene mediating adhesion of holdfast in C. crescentus. Moreover, the divalent metal ions Zn2+ , Mg2+ , and Mn2+ could promote the activity of recombinant HfsH at the concentration from 0.05 to 1 mM, but inhibit its activity when the concentration exceeds 1 mM. In sum, our study first realized the artificial production of polysaccharide deacetylase HfsH and its structural analogue, and further explored their functions, both of which laid the foundation for the development of new adhesive materials.

Interrelationships between amphiregulin, kisspeptin, FSH and FSH receptor in promotion of human ovarian cell functions.

The aim of this study was to investigate: (1) the ability of granulosa cells to produce amphiregulin (AREG), kisspeptin (KISS) and FSH receptor (FSHR); (2) the role of AREG and KISS in the control of ovarian functions; (3) the effect of FSH and KISS on AREG; and (4) the ability of KISS to affect FSHR and to modify FSH action on AREG output by human ovarian granulosa cells. We examined: (1) time-dependent accumulation of AREG; (2) effects of AREG (0, 1, 10, 100ng/mL) and KISS (0, 1, 10, 100ng/mL) on granulosa cell functions; and (3) the effects of KISS (0, 1, 10, 100ng/mL), FSH (0, 1, 10, 100ng/mL), and their combinations on AREG release. Viability, markers of proliferation [accumulation ofproliferating cell nuclear antigen (PCNA) cyclin B1 and sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro)benzene sulfonic acid hydrate (XTT formazan)] and apoptosis (accumulation of bax, caspase 3 and terminal deoxynucleotidyl transferase dUTP nick-end labelling), accumulation of KISS, FSHR and steroid hormones, and AREG release were analysed by Trypan blue exclusion test, quantitative immunocytochemistry, XTT, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays and enzyme-linked immunosorbent assay. AREG promoted cell viability, proliferation and steroid hormone output, and inhibited apoptosis. KISS (1 and 10ng/mL) stimulated viability, proliferation, steroid hormone release and occurrence of FSHR and suppressed apoptosis and AREG output; KISS (100ng/mL) had the opposite effect. FSH stimulated AREG release, whilst addition of KISS reversed this FSH effect. FSH mimicked and promoted the inhibitory effect of KISS on AREG release. These results suggest an intra-ovarian production and a functional interrelationship between AREG, KISS, FSH and FSHR in direct regulation of basic ovarian cell functions.

Role of FSH and FSH receptor on HUVECs migration.

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein that regulates follicle maturation through its binding to follicle-stimulating hormone receptor (FSHR). Endothelial cells express FSHR, but its exact role in endothelial cells remains unclear. Here we show that FSHR expression was detectable in human umbilical vein endothelial cells (HUVECs). FSH stimulation promoted HUVECs migration but not proliferation. Because FSHR is a GPCR, FSH treatment triggers the activation of cAMP-PKA signaling pathways, and the JAK-STAT, PI3K-AKT, and JNK-MAPK pathways. RNAi of FSHR dramatically attenuated the activation effect of FSH on HUVECs migration, as well as the related signaling pathways. Treatment of FSH in HUVECs also transcriptionally upregulated the expression of VAV3 and LAMA2, suppression either of VAV3 or LAMA2 by RNAi attenuated the FSH's effect on HUVECs migration. All of these results indicated a functional role of FSH in the regulation of endothelial cells.

Can improvement in hormonal and energy balance reverse cardiovascular risk factors in athletes with amenorrhea?

Female athletes display a high prevalence of hypothalamic amenorrhea as a result of energy imbalance. In these athletes with amenorrhea, decreased luteinizing hormone/follicule-stimulating hormone secretion leads to deficiency in endogenous estrogen. The severe estrogen deficiency in these athletes may increase cardiovascular risk similar to that in postmenopausal women. This review discusses the potential cardiovascular risk factors in athletes with amenorrhea as a result of hypoestrogenism, which include endothelial dysfunction and unfavorable lipid profiles. We also consider the potential to reverse the cardiovascular risk by restoring energy or hormonal imbalance along the reproductive axis in athletes with amenorrhea.

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography.

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C-termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

Amplification of FSH signalling by CFTR and nuclear soluble adenylyl cyclase in the ovary.

The cAMP/PKA pathway is one of the most important signalling pathways widely distributed in most eukaryotic cells. The activation of the canonical cAMP/PKA pathway depends on transmembrane adenylyl cyclase (tmAC). Recently, soluble adenylyl cyclase (sAC), which is activated by HCO3- or Ca2+ , emerges to provide an alternative way to activate cAMP/PKA pathway with the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- /HCO3- -conducting anion channel, as a key player. This review summarizes new progress in the investigation of the CFTR/HCO3- -dependent sAC signalling and its essential role in various reproductive processes, particularly in ovarian functions. We present the evidence for a CFTR/HCO3- -dependent nuclear sAC signalling cascade that amplifies the FSH-stimulated cAMP/PKA pathway, traditionally thought to involve tmAC, in granulosa for the regulation of oestrogen production and granulosa cell proliferation. The implication of the CFTR/HCO3- /sAC pathway in amplifying other receptor-activated cAMP/PKA signalling in a wide variety of cell types and pathophysiological processes, including aging, is also discussed.

FSH-FSHR3-stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer.

Despite extensive research, genetic basis of premature ovarian failure (POF) and ovarian cancer still remains elusive. It is indeed paradoxical that scientists searched for mutations in FSH receptor (FSHR) expressed on granulosa cells, whereas more than 90% of cancers arise in ovary surface epithelium (OSE). Two distinct populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) exist in OSE, are responsible for neo-oogenesis and primordial follicle assembly in adult life, and are modulated by FSH via its alternatively spliced receptor variant FSHR3 (growth factor type 1 receptor acting via calcium signaling and the ERK/MAPK pathway). Any defect in FSH-FSHR3-stem cell interaction in OSE may affect folliculogenesis and thus result in POF. Ovarian aging is associated with a compromised microenvironment that does not support stem cell differentiation into oocytes and further folliculogenesis. FSH exerts a mitogenic effect on OSE and elevated FSH levels associated with advanced age may provide a continuous trigger for stem cells to proliferate resulting in cancer, thus supporting gonadotropin theory for ovarian cancer. Present review is an attempt to put adult ovarian biology, POF, aging, and cancer in the perspective of FSH-FSHR3-stem cell network that functions in OSE. This hypothesis is further supported by the recent understanding that: i) cancer is a stem cell disease and OSE is the niche for ovarian cancer stem cells; ii) ovarian OCT4-positive stem cells are regulated by FSH; and iii) OCT4 along with LIN28 and BMP4 are highly expressed in ovarian cancers.

Joint modeling of cross-sectional health outcomes and longitudinal predictors via mixtures of means and variances.

Joint modeling methods have become popular tools to link important features extracted from longitudinal data to a primary event. While most modeling strategies have focused on the association between the longitudinal mean trajectories and risk of an event, we consider joint models that incorporate information from both long-term trends and short-term variability in a longitudinal submodel. We also consider both shared random effect and latent class (LC) approaches in the primary-outcome model to predict a binary outcome of interest. We develop simulation studies to compare and contrast these two modeling strategies; in particular, we study in detail the effects of the primary-outcome model misspecification. Among other findings, we note that when we analyze data from a shared random-effect using a LC model while the information from the longitudinal data is weak, the LC approach is more sensitive to such a model misspecification. Under this setting, the LC model has a superior performance in within-sample prediction that cannot be duplicated when predicting new samples. This is a unique feature of the LC approach that is new as far as we know to the existing literature. Finally, we use the proposed models to study how follicle stimulating hormone (FSH) trajectories are related to the risk of developing severe hot flashes for participating women in the Penn Ovarian Aging Study.

Human steroidogenesis: implications for controlled ovarian stimulation with exogenous gonadotropins.

In the menstrual cycle, the mid-cycle surge of gonadotropins (both luteinising hormone [LH] and follicle-stimulating hormone [FSH]) signals the initiation of the periovulatory interval, during which the follicle augments progesterone production and begins to luteinise, ultimately leading to the rupture of the follicle wall and the release of an oocyte. The administration of gonadotropins in controlled ovarian stimulation (COS) leads to supraphysiological steroid concentrations of a very different profile compared with those seen during natural cycles. It has been suggested that these high steroid concentrations cause alterations in endometrial development, affecting oocyte viability in assisted reproductive technology. Furthermore, it has been proposed that elevated progesterone levels have a negative effect on the reproductive outcome of COS. This may arise from an asynchrony between embryo stage and endometrium status at the window of implantation. The regulation of progesterone production by the developing follicles during COS is a complicated interplay of hormonal systems involving the theca and granulosa cells, and the effect of the actions of both LH and FSH. The present paper reviews current knowledge of the regulation of progesterone in the human ovary during the follicular phase and highlights areas where knowledge remains limited. In this review, we provide in-depth information outlining the regulation and function of gonadotropins in the complicated area of steroidogenesis. Based on current evidence, it is not clear whether the high levels of progesterone produced during COS have detrimental effects on fertility.

Age-specific reference values for serum FSH and estradiol levels throughout the reproductive period.

High serum day 3 FSH levels are associated with poor ovarian reserve and reduced fertility, but the interpretation of FSH values according to age is still not univocal. The purpose of this study was to determine age-dependent reference values in women with regular menstrual cycles and FSH as a guide for specialists. The study was performed at the Department of Mother-Infant of a University-based tertiary care centre. One-hundred ninety-two healthy normal menstruating women were recruited for the study. All patients attended the department on menstrual cycle day 3 for a blood sample for FSH and estradiol determination. A linear relationship between FSH or estradiol serum levels and age was observed. The FSH level increased by 0.11 IU for every year of age (1 IU for every 9 years of age). The values of FSH and estradiol corresponding to the 5th, 25th, 50th, 75th, 95th centiles for any specific age have been calculated. Serum FSH levels need to be interpreted according to age-dependent reference values. Serum FSH levels on 95th centile for any age may represent a warning sign for reduced ovarian reserve.

The follicle-stimulating hormone triggers rapid changes in mitochondrial structure and function in porcine cumulus cells.

Oocyte maturation is a key process during which the female germ cell undergoes resumption of meiosis and completes its preparation for embryonic development including cytoplasmic and epigenetic maturation. The cumulus cells directly surrounding the oocyte are involved in this process by transferring essential metabolites, such as pyruvate, to the oocyte. This process is controlled by cyclic adenosine monophosphate (cAMP)-dependent mechanisms recruited downstream of follicle-stimulating hormone (FSH) signaling in cumulus cells. As mitochondria have a critical but poorly understood contribution to this process, we defined the effects of FSH and high cAMP concentrations on mitochondrial dynamics and function in porcine cumulus cells. During in vitro maturation (IVM) of cumulus-oocyte complexes (COCs), we observed an FSH-dependent mitochondrial elongation shortly after stimulation that led to mitochondrial fragmentation 24 h later. Importantly, mitochondrial elongation was accompanied by decreased mitochondrial activity and a switch to glycolysis. During a pre-IVM culture step increasing intracellular cAMP, mitochondrial fragmentation was prevented. Altogether, the results demonstrate that FSH triggers rapid changes in mitochondrial structure and function in COCs involving cAMP.

Role of follicle-stimulating hormone on biliary cyst growth in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the progressive development of renal and hepatic cysts. Follicle-stimulating hormone (FSH) has been demonstrated to be a trophic factor for biliary cells in normal rats and experimental cholestasis induced by bile duct ligation (BDL). AIMS: To assess the effect of FSH on cholangiocyte proliferation during ADPKD using both in vivo and in vitro models. METHODS: Evaluation of FSH receptor (FSHR), FSH, phospho-extracellular-regulated kinase (pERK) and c-myc expression in liver fragments from normal patients and patients with ADPKD. In vitro, we studied proliferating cell nuclear antigen (PCNA) and cAMP levels in a human immortalized, non-malignant cholangiocyte cell line (H69) and in an immortalized cell line obtained from the epithelium lining the hepatic cysts from the patients with ADPKD (LCDE) with or without transient silencing of the FSH gene. RESULTS: Follicle-stimulating hormone is linked to the active proliferation of the cystic wall and to the localization of p-ERK and c-myc. This hormone sustains the biliary growth by activation of the cAMP/ERK signalling pathway. CONCLUSION: These results showed that FSH has an important function in cystic growth acting on the cAMP pathway, demonstrating that it provides a target for medical therapy of hepatic cysts during ADPKD.

High water temperature impairs ovarian activity and gene expression in the brain-pituitary-gonadal axis in female red seabream during the spawning season.

Temperature plays a pivotal role in the control of seasonal reproduction in temperate fish species. It is well known that temperatures that exceed a certain threshold impair gonadal development, maturation, and spawning. However, the endocrine mechanisms that underlie these effects are poorly understood. We evaluated the effect of high water temperature on the brain-pituitary-gonadal (B-P-G) axis of a perciform fish, red seabream, Pagrus (Chrysophrys) major during its spawning season (April-May). Fish were reared at either high (24 °C: H-group) or optimal/control (17 °C: C-group) temperatures for 5 or 10 d. After 5 d, the transcript abundance of gonadotropin-releasing hormone-1 (GnRH1) in the brain and GnRH receptor (GnRH-R) and FSH-ß in the pituitary were significantly lower in H-group than in C-group. Conversely, there was no difference in pituitary LH-ß mRNA levels, serum concentrations of estradiol-17ß (E2), or the gonadosomatic index (GSIs) between H- and C-groups on Day 5. After 10 d, the ovaries of all H-group fish had completely regressed and were filled with only perinucleolar stage primary oocytes and atretic oocytes. The brain GnRH1 expression, pituitary GnRH-R and pituitary LH-ß expression, serum E2 concentrations, and the GSI were significantly lower in the H-group on Day 10. Our results suggest that high water temperature is the proximate driver of the termination of the spawning season of female red seabream. The effect appears to be mediated by suppression of gene expression in the B-P-G axis.

Clinical features and analysis in 1385 Chinese patients with pituitary adenomas.

AIM: The prevalence of pituitary adenomas (PAs) is increasing as the development of imaging techniques. Few studies systematically documented the profile of these tumors in Chinese population. Our study is aimed to investigate the clinical features of PAs including the clinicopathologies, manifestations, and tumor recurrence. METHODS: A retrospective analysis of clinic records of patients (median age, 39 years) with PAs was performed. A total of 1385 patients diagnosed and treated surgically at Tongji hospital, Hubei Province, China during the years 1987 through 2009 were identified that met our inclusion criteria and formed the study group. RESULTS: The distribution of each PA subtype was nonfunctioning pituitary adenomas (NFPA) occupying 40.0% of the total subjects, pure prolactin-secreting (PRL+) adenomas 18.6%, mixed adenomas 14.4%, growth hormone-secreting (GH+) adenomas 6.0%, follicle-stimulating hormone/luteinizing hormone-secreting (FSH/LH+) adenomas 5.9%, adrenocorticotropic hormone-secreting (ACTH+) adenomas 4.6%, and thyroid-stimulating hormone-secreting (TSH+) adenomas 0.6%. The most common initial symptoms were visual disturbances (N.=664), endocrine disturbances (N.=645), and headaches (N.=532). Patients who complained of endocrine disturbances mostly presented menstrual dysfunction in females whereas acromegaly in males. A total of 45 cases developed to recurrence, most of which occurred within 3 years after surgery. PRL+ adenoma showed the lowest frequency of recurrence (0.8%). CONCLUSION: Most adenomas were secretory PAs, with prolactinomas being the most common subtype. Visual defects, endocrine disorders, and headaches were the most common symptoms. Patients with PRL+ adenoma had the lowest chance to undertake recurrence. More clinical care and research activities are needed to improve the outcome of these patients.

Spontaneous ovarian hyperstimulation syndrome.

Spontaneous forms of the ovarian hyperstimulation syndrome (sOHSS) are nearly always reported between 8 and 14 weeks of pregnancy and also with follicle-stimulating hormone (FSH) producing pituitary adenoma. The syndrome has been previously reported in rare instances of increased production of human chorionic gonadotrophin (hCG) such as multiple pregnancies, hydatiforme mole, polycystic ovary disease and elevated concentrations of thyroid-stimulating hormone (TSH) in hypothyreoidism. High levels of these hormones are able to stimulate by natural promiscuous activation the wild-type FSHr, resulting in sporadic presentations of the syndrome. Since 2003, only six different activating FSHr gene mutations have been reported in cases of familial or habitual sOHSS. In addition to five mutations which have been found in the transmembrane helices (Asp567Asn, Asp567Gly, Thr449Ile, Thr449Ala, Ile545Thr), the first germline mutation (c.383C > A, p. Ser 128 Tyr) in the extracelullar domain was identified. All five mutants were abnormally activated by TSH and normal levels of hCG while displaying constitutive activity. In contrast to these mutations, the p.Ser128Tyr mutant displayed an increase in sensitivity only toward hCG. Accordingly, the mutated FSHrs, may be hyperstimulated by the pregnancy-derived hCG or TSH, inducing the occurrence of the syndrome. In the differential diagnosis, malignancy, pregnancy luteoma and hyperreactio luteinalis would have to be excluded. In almost all of the cases the disease regresses spontaneously and could be managed expectantly or conservatively, but with termination of pregnancy or surgery in cases of complications.

[The molecular design and drug development of recombinant long-acting follicle stimulating hormone].

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone.

BACKGROUND: Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. METHODS: To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. RESULTS: Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED(50) values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. CONCLUSION: High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin.

Structure of human follicle-stimulating hormone in complex with its receptor.

Follicle-stimulating hormone (FSH) is central to reproduction in mammals. It acts through a G-protein-coupled receptor on the surface of target cells to stimulate testicular and ovarian functions. We present here the 2.9-A-resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular hormone-binding domain of its receptor (FSHR(HB)). The hormone is bound in a hand-clasp fashion to an elongated, curved receptor. The buried interface of the complex is large (2,600 A2) and has a high charge density. Our analysis suggests that all glycoprotein hormones bind to their receptors in this mode and that binding specificity is mediated by key interaction sites involving both the common alpha- and hormone-specific beta-subunits. On binding, FSH undergoes a concerted conformational change that affects protruding loops implicated in receptor activation. The FSH-FSHR(HB) complexes form dimers in the crystal and at high concentrations in solution. Such dimers may participate in transmembrane signal transduction.

Follicular-Stimulating Hormone, Luteinizing Hormone, and Prolactin Serum Level in Patients with Oral Lichen Planus in Comparison to Healthy Population.

MATERIALS AND METHODS: In this cross-sectional case control study, the serum level of LH, FSH, and prolactin of 40 women with lichen planus who have been referred to Shiraz Dental Faculty, Oral and Maxillofacial Disease Department during 2018-2019 has been evaluated in comparison to 40 healthy controls. Data were analyzed by SPSS version 18. Two-way ANOVA and Mann-Whitney test were used for data analysis. RESULTS: The mean serum level of FSH and LH was significantly higher in OLP patients while this difference was not reported for prolactin. Only FSH mean serum level was significantly higher in nonmenopausal OLP patients. The distribution of prolactin and FSH hormones' serum level was in normal range. CONCLUSIONS: The high serum level of FSH and LH can affect OLP pathogenesis by estrogen and progesterone modulation.

Optimising Follicular Development, Pituitary Suppression, Triggering and Luteal Phase Support During Assisted Reproductive Technology: A Delphi Consensus.

Background: A Delphi consensus was conducted to evaluate global expert opinions on key aspects of assisted reproductive technology (ART) treatment. Methods: Ten experts plus the Scientific Coordinator discussed and amended statements plus supporting references proposed by the Scientific Coordinator. The statements were distributed via an online survey to 35 experts, who voted on their level of agreement or disagreement with each statement. Consensus was reached if the proportion of participants agreeing or disagreeing with a statement was >66%. Results: Eighteen statements were developed. All statements reached consensus and the most relevant are summarised here. (1) Follicular development and stimulation with gonadotropins (n = 9 statements): Recombinant human follicle stimulating hormone (r-hFSH) alone is sufficient for follicular development in normogonadotropic patients aged <35 years. Oocyte number and live birth rate are strongly correlated; there is a positive linear correlation with cumulative live birth rate. Different r-hFSH preparations have identical polypeptide chains but different glycosylation patterns, affecting the biospecific activity of r-hFSH. r-hFSH plus recombinant human LH (r-hFSH:r-hLH) demonstrates improved pregnancy rates and cost efficacy versus human menopausal gonadotropin (hMG) in patients with severe FSH and LH deficiency. (2) Pituitary suppression (n = 2 statements): Gonadotropin releasing hormone (GnRH) antagonists are associated with lower rates of any grade ovarian hyperstimulation syndrome (OHSS) and cycle cancellation versus GnRH agonists. (3) Final oocyte maturation triggering (n=4 statements): Human chorionic gonadotropin (hCG) represents the gold standard in fresh cycles. The efficacy of hCG triggering for frozen transfers in modified natural cycles is controversial compared with LH peak monitoring. Current evidence supports significantly higher pregnancy rates with hCG + GnRH agonist versus hCG alone, but further evidence is needed. GnRH agonist trigger, in GnRH antagonist protocol, is recommended for final oocyte maturation in women at risk of OHSS. (4) Luteal-phase support (n = 3 statements): Vaginal progesterone therapy represents the gold standard for luteal-phase support. Conclusions: This Delphi consensus provides a real-world clinical perspective on the specific approaches during the key steps of ART treatment from a diverse group of international experts. Additional guidance from clinicians on ART strategies could complement guidelines and policies, and may help to further improve treatment outcomes.