The Determination and Evaluation of the Biological Activities for the Commercialization of Recombinant Follicle-Stimulating Hormone in Vitro

Follicle-stimulating hormone (FSH) plays a central role in mammals reproduction, with the actions of FSH mediated by follicle-stimulating hormone receptors (FSHRs) on the surface of target cells. The purposes of this study were to determine and evaluate the biological activities for the commercialization of recombinant follicle-stimulating hormone (rFSH) in vitro through the cellular internalization using cloned 293T-FSHR cell lines as target. Using imaging approaches we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus by endocytosis. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization, but the rFSH have promoted the internalization of the fluorescent, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.

Follicle-stimulating Hormone(FSH) Induced Internalization of Porcine FSH Receptor in Cultured Porcine Granulosa Cells and Chinese Hamster Ovary Cells Transfected with Recombinant Porcine FSH Receptor cDNA

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125 I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

Role of follicle-stimulating hormone and estradiol benzoate in recovering spermatogenesis in tamoxifen-injured rats

Objective:To evaluate the effects of follicle-stimulating hormone (FSH) and estradiol benzoate (EB) on the recovery of spermatogenesis, histology, sexual hormones levels and testicular gene expression in testes of tamoxifen-injured rats.Methods:Forty adult rats were divided into eight groups in a factorial arrangement of tamoxifen and hormonal treatments. Half of the groups orally received 0.6 mg/kg tamoxifen, and 30 d later tamoxifen and no-tamoxifen groups (controls) were paired and assigned into four hormonal treatments with daily intramuscular injections for 10 consecutive days: 1 mL saline (control);7.5 IU FSH;12 μg/kg EB;and 7.5 IU FSH+12 μg/kg EB. One day after the last treatment, spermatozoa were recovered from epididymis, blood was processed for sex hormones concentration (testosterone, FSH and luteinizing hormone) and testes were processed for histology and RNA extraction for expression of genes related to apoptosis [caspase 3, inducible nitric oxide synthase (iNOS) and B-cell lymphoma-2 (Bcl-2)].Results: Control groups did not show significant changes in most parameters, but hormonal treatments decreased caspase 3 and iNOS and increased Bcl-2 expression. Tamoxifen significantly decreased counts, motility and viability of spermatozoa, Bcl-2 expression and sex hormones. It increased intertubular space, caspase 3 and iNOS expression, and induced seminiferous tubular atrophy. The hormonal treatments reverted spermatogenesis, hormonal levels and histology compared with controls, however not attaining the same sperm quality as controls.Conclusions:Tamoxifen is clearly detrimental to spermatogenesis and overall testicular structure and function, whereas hormonal therapy with FSH and EB can improve testicular function and revert tamoxifen-induced azoospermia.

Follicle-stimulating hormone(FSH)levels prior to prostatectomy are not related to long-term oncologic or cardiovascular outcomes for men with prostate cancer

Prior research suggests a link between circulating levels of follicle-stimulating hormone(FSH)and prostate cancer outcomes.FSH levels may also explain some of the observed differences in cardiovascular events among men treated with gonadotropin-releasing hormone(GnRH)antagonists compared to GnRH agonists.This study evaluates the association between preoperative FSH and long-term cardiovascular and oncologic outcomes in a cohort of men with long follow-up after radical prostatectomy.We performed a cohort study utilizing an institutional biobank with annotated clinical data.FSH levels were measured from cryopreserved plasma and compared with sex steroids previously measured from the same samples.Differences in oncologic outcomes between tertiles of FSH levels were compared using adjusted cox regression models.Major adverse cardiovascular events(MACE)were similarly assessed using hospital admission diagnostic codes.A total of 492 patients were included,with a median follow-up of 13.1(interquartile range:8.9–15.9)years.Dehydroepiandrosterone sulfate(DHEA-S)levels,but not other androgens,negatively correlated with FSH levels on linear regression analysis(P=0.03).There was no association between FSH tertile and outcomes of biochemical recurrence,time to castrate-resistant prostate cancer,or time to metastasis.MACEs were identified in 50 patients(10.2%),with a mean time to first event of 8.8 years.No association with FSH tertile and occurrence of MACE was identified.Our results do not suggest that preoperative FSH levels are significantly associated with oncologic outcomes among prostate cancer patients treated with radical prostatectomy,nor do these levels appear to be predictors of long-term cardiovascular risk.

LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells

Long noncoding RNAs(lncRNAs)are expressed in different species and different tissues,and perform different functions,but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone(FSH).In general,we have revealed lnc RNA-micro RNA(mi RNA)-messenger RNA(m RNA)interactions that may play important roles in rat primary pituitary cells.In this study,a new lncRNA was identified for the first time.First,we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization,which indicated that this lncRNA was distributed mainly in the cytoplasm.Next,we used RT-qPCR and enzyme-linked immunosorbent assay(ELISA)to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion.In addition,mothers against decapentaplegic homolog 2(Smad2)was highly expressed in our sequencing results.We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2.We used RNA immunoprecipitation-qPCR(RIP-qPCR)and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2.Fluorescence in situ hybridization(FISH)showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm.Finally,we determined the relationship among lncRNA-m18as1,miR-18a-5p,and the Smad2/3 pathway.Overall,we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.

Effect of Progestin-Primed Ovarian Stimulation Protocol in Infertile Women with Basal Follicle-Stimulating Hormone Levels ≥15 IU/L: A Retrospective Analysis

Objective:To evaluate the efficacy of progestin-primed ovarian stimulation(PPOS)protocol in infertile women with high basal follicle-stimulating hormone(FSH)levels≥15 IU/L.Methods:Patients with high basal FSH levels≥15 IU/L with autologous oocytes from September 2016 to March 2019 were reviewed.Either medroxyprogesterone acetate 4 mg/d or clomiphene citrate(CC)50 mg/d was administered daily from day 3 to the trigger day.When serum FSH levels decreased to≤15.0 IU/L,a low dose of human menopausal gonadotropin(hMG)75/150 IU/d was administered to promote late follicular development.Results:Two hundred and twenty women were retrospectively analyzed in this study.Among them,139 patients were administered with PPOS protocol as the study group,and 81 patients were administered with CC protocol as the control group.The numbers of received oocytes and viable embryos were higher in the study group than those in the control group(1.5±1.2 vs.1.2±0.8 and 0.8±0.8 vs.0.5±0.6,respectively,P<0.05).However,hMG duration and dosage were significantly higher in the study group than those in the control group(4.2±2.7 d vs.1.1±2.3 d and 609.1±424.5 IU vs.140.7±231.3 IU,respectively,P<0.01).Incidence of luteinizing hormone surge and cycle cancellation rate were lower in the study group than those in the control group with statistical difference(2.88%vs.16.05%and 36.50%vs.50.63%,respectively,P<0.05).Conclusions:PPOS protocol can effectively downregulate the endogenous FSH levels.Compared with CC protocol,treatment with PPOS protocol in patients with high basal FSH levels≥15 IU/L could receive more oocytes and more viable embryos.

Auricular acupoint application combined with Fujing prescription treatment for patients with premature ovarian failure

Background:To study the efficacy of Fujing prescription combined with auricular acupoint application in premature ovarian failure and its effect on hormone levels.Methods:From March 2018 to March 2020,100 patients with premature ovarian failure treated in our outpatient clinic were selected and randomly divided into a control group of 50 cases and a treatment group of 50 cases.The control group was treated with Femoston for 28 consecutive days,and the treatment group was treated with not only Femoston,but also Fujing prescription and auricular acupoint application.The patients in the two groups were treated with a 3-month course of treatment.The clinical efficacy,traditional Chinese medicine integral,serum hormone level,immune index and the occurrence of adverse reactions were compared between the two groups.Results:After treatment,the indexes of IgG,IgM and IgA in the treatment group were all increased(P<0.05 for all);compared with the control group,the incidence of adverse reactions in the treatment group was lower(P<0.05).Conclusion:The combination of Fujing prescription and auricular acupoint application in the treatment of premature ovarian failure can improve patients'clinical symptoms significantly,resulting in a decrease in follicle-stimulating hormone levels and an increase in estrogen levels.The combination of Fujing prescription and acupoint application has provided a clinical basis for the TCM treatment of premature ovarian failure.

Homeostatic relevance of vitamin D in maintaining male fertility in human: Down-regulation of oxidative stress and up-regulation of anti-oxidative defense and steroidal hormones

Objective:To evaluate correlation between the levels of vitamin D and male infertility as well as to determine the efficacy of vitamin D in improving the male fertility by up-regulating the levels of testosterone and spermatogenesis.Methods: In the present study, 130 male patients (aged 25-70 years) having fertility defects were screened and 145 healthy individuals were taken as control. All human subjects were screened for 4-hydroxynonenal, isoprostane-F2α, 8-hydroxy-2′-deoxyguanosine, vitamin D, luteinizing hormone, follicle stimulating hormone, testosterones, malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, and nitric oxide.Results: The screening analysis revealed that the levels of luteinizing hormone, follicle stimulating hormone, and testosterone were lower in male infertile subjects compared to healthy subjects. Similarly, the levels of vitamin D [(17.17 ± 2.30) ng/mL] and calcium[(6.29 ± 0.31) mg/dL] were significantly lower in infertile groups compared to the normal healthy groups. Moreover, the study revealed that the levels of superoxide dismutase, catalase, and glutathione peroxidase were significantly higher in healthy subjects compared to the infertile subjects.Conclusions:Vitamin D exhibits strong relevance to male fertility by maintaining the levels of sex hormones (luteinizing hormone, follicle stimulating hormone, and testosterone), up-regulating the antioxidant defense (superoxide dismutase, catalase, and glutathione peroxidase), and down-regulating the oxidative stress (malondialdehyde, nitric oxide, and inducible nitric oxide synthase species).

An examination of predictive markers for successful sperm extraction procedures:a linear model and systematic review

The authors performed a comprehensive review of current literature to create a model comparing commonly evaluated variables in male factor infertility,for example,follicle-stimulating hormone(FSH),testicular volume(TV),and testosterone(T),to better predict sperm retrieval rate(SRR).Twenty-nine studies were included,9 with data on conventional testicular sperm extraction(cTESE)for a total of 1227 patients and 20 studies including data on microdissection testicular sperm extraction(mTESE)for a total of 4760 patients.A weighted-means value of SRR,FSH,T,and TV was created,and a weighted linear regression was then used to describe associations among SRR,type of procedure,FSH,T,and TV.In this study,weighted-means values demonstrated mTESE to be superior to cTESE with an SRR of 51.9%vs 40.1%.Multiple weighted linear regressions were created to describe associations among SRR,procedure type,FSH,T,and TV.The models showed that for every 1.19 mIU ml^(−1)increase in FSH,there would be a significant decrease in SRR by 1.0%.Seeking to create a more clinically relevant model,FSH values were then divided into normal,moderate elevation,and significant elevation categories(FSH<10 mIU ml^(−1),10–19 mIU ml^(−1),and>20 mIU ml^(−1),respectively).For an index patient undergoing cTESE,the retrieval rates would be 57.1%,44.3%,and 31.2%for values normal,moderately elevated,and significantly elevated,respectively.In conclusion,in a large meta-analysis,mTESE was shown to be more successful than cTESE for sperm retrievals.FSH has an inverse relationship to SRR in retrieval techniques and can alone be predictive of cTESE SRR.

Atypical pituitary hormone–target tissue axis

A long-held belief is that pituitary hormones bind to their cognate receptors in classical target glands to actuate their manifold functions.However,a number of studies have shown that multiple types of pituitary hormone receptors are widely expressed in non-classical target organs.Each pituitary gland-derived hormone exhibits a wide range of nonconventional biological effects in these non-classical target organs.Herein,the extra biological functions of pituitary hormones,thyroid-stimulating hormone,follicle-stimulating hormone,luteinizing hormone,adrenocorticotrophic hormone,and prolactin when they act on non-classical organs were summarized,defined by the novel concept of an“atypical pituitary hormone–target tissue axis.”This novel proposal explains the pathomechanisms of abnormal glucose and lipid metabolism,obesity,hypertension,fatty liver,and atherosclerosis while offering a more comprehensive and systematic insights into the coordinated regulation of environmental factors,genetic factors,and neuroendocrine hormones on human biological functions.The continued exploration of the physiology of the“atypical pituitary hormone–target tissue axis”could enable the identification of novel therapeutic targets for metabolic diseases.

Regulation of anti-Mullerian hormone(AMH)in males and the associations of serum AMH with the disorders of male fertility

Anti-Mullerian hormone(AMH)is a functional marker of fetal Sertoli cells.The germ cell number in adults depends on the number of Sertoli cells produced duri ng peri natal development.Rece ntly,AMH has received increasi ng attenti on in research of disorders related to male fertility.This paper reviews and summarizes the articles on the regulation of AMH in males and the serum levels of AMH in male fertility-related disorders.We have determined that follicle-stimulating hormone(FSH)promotes AMH transcription in the absenee of androgen signaling.Testosterone inhibits the transcriptional activation of AMH.The undetectable levels of serum AMH and testosterone levels indicate a lack of functional testicular tissue,for example,that in patients with anorchia or severe Klinefelter syndrome suffering from impaired spermatogenesis.The normal serum testosterone level and undetectable AMH are highly suggestive of persistent Mullerian duct syndrome(PMDS),combined with clinical manifestations.The levels of both AMH and testosterone are always subnormal in patients with mixed disorders of sex development(DSD).Mixed DSD is an early-onset complete type of disorder with fetal hypogonadism resulting from the dysfunction of both Leydig and Sertoli cells.Serum AMH levels are varying in patients with male fertility-related disorders,in cludi ng pubertal delay,severe con genital hypog on adotropic hypogonadism,nonobstructive azoospermia,Klinefelter syndrome,varicocele,McCune-Albright syndrome,and male senescence.

Predictive significance of serum inhibin B on testicular haploid gamete retrieval outcomes in nonobstructive azoospermic men

The purpose of this study was to determine the diagnostic accuracy of serum inhibin B(INHB)as a predictor of the retrieval outcome of testicular haploid gametes(spermatids and testicular spermatozoa)in nonobstructive azoospermic men.Serum hormone levels,testicular volume,and histological evaluation were performed in 403 Chin ese non obstructive azoospermic men.Testicular haploid gamete was successfully retrieved in 213 of 403 patients(52.85%).The haploid gamete group always had higher INHB levels than the non-haploid gamete group.According to the receiver operating characteristic(ROC)curve analysis,INHB was a good predictor of testicular haploid gamete retrieval outcome in all patients(sensitivity:77.93%and specificity:91.58%)and patients with normal follicle-stimulating hormone(FSH;sensitivity:88.52%and specificity:70.83%).The area under the ROC curve(AUC)of INHB was similar to that of FSH in all patients or patients with normal FSH.In patients with elevated FSH,INHB was superior to FSH in predicting the presence of haploid gamete(AUC:0.73 vs 0.55,P<0.05),with a sensitivity of 60.00%and a specificity of 80.28%.It con eluded that serum INHB as an effective marker for spermatoge nesis was a sign ificant predictor of testicular haploid gamete retrieval outcomes in nonobstructive azoospermic men.Especially,INHB is superior to FSH in predicting the presenee of haploid gamete in the patients with elevated FSH.

Functioning gonadotroph adenoma

To the Editor:Pituitary gonadotroph adenoma is a type of non-functioning pituitary adenoma of which approximately 35%ecrete biologically active luteinizing hormone(LH)or follicle-stimulating hormone(FSH),and these adenomas are named functioning gonadotroph adenoma(FGA).We present three cases of FSH-secreting adenomas who were admitted to our hospital between 2014 and 2016.Case 1:A 37-year-old woman was admitted due to menstrual irregularity and galactorrhea for 2 years.The laboratory tests showed a significantly elevated estradiol 2(E2)level,and multiple cyst fusions could be observed by transvaginal ultrasonography.The pituitary magnetic resonance imaging(MRI)revealed an upper-right wing distension and detected a mass with a size of approximately 7.5 mm×10.7 mm in the saddle area,and a pituitarymacroadenoma was considered.The patient underwent a transsphenoidal microsurgical resection of the pituitary adenoma.The pathologic examination showed a positive result for FSH,LH,and SF-1,and a negative result for growth hormone,insulin-like growth factor 1,TPIT,and PIT1.

Endocrine aberrations of human nonobstructive azoospermia

Nonobstructive azoospermia(NOA)refers to the failure of spermatogenesis,which affects approximately 1%of the male population and contributes to 10%of male infertility.NOA has an underlying basis of endocrine imbalances since proper human spermatogenesis relies on complex regulation and cooperation of multiple hormones.A better understanding of subtle hormonal disturbances in NOA would help design and improve hormone therapies with reduced risk in human fertility clinics.The purpose of this review is to summarize the research on the endocrinological aspects of NOA,especially the hormones involved in hypothalamic–pituitary–testis axis(HPTA),including gonadotropin-releasing hormone,follicle-stimulating hormone,luteinizing hormone,prolactin,testosterone,estradiol,sex hormone binding globulin,inhibin B,anti-Müllerian hormone,and leptin.For the NOA men associated with primary testicular failure,the quality of currently available evidence has not been sufficient enough to recommend any general hormone optimization therapy.Some other NOA patients,especially those with hypogonadotropic hypogonadism,could be treated with hormonal replacement.Although these approaches have succeeded in resuming the fertility in many NOA patients,the prudent strategies should be applied in individuals according to specific NOA etiology by balancing fertility benefits and potential risks.This review also discusses how NOA can be induced by immunization against hormones.

Effect of varicocele repair on sperm retrieval rate and testicular histopathological patterns in men with nonobstructive azoospermia

Varicocele adversely affects semen parameters.However,the effect of varicocele repair on the sperm retrieval rate and testicular histopathological patterns in men with nonobstructive azoospermia has not been widely reported.We retrospectively assessed the sperm retrieval rates and testicular histopathological patterns in men with nonobstructive azoospermia who were referred to the Urology Clinic in Dr.Cipto Mangunkusumo Hospital(Jakarta,Indonesia)and Bunda General Hospital(Jakarta,Indonesia)between January 2009 and December 2019.We compared patients who had undergone a surgical sperm retrieval procedure for assisted reproductive technology no earlier than three months after varicocele repair and those who had not undergone varicocele repair.The study included 104 patients(age range:26–54 years),42 of whom had undergone varicocele repair before the sperm retrieval procedure and 62 who had not.Motile spermatozoa were found in 29(69.1%)and 17(27.4%)patients who had undergone varicocele repair before the sperm retrieval procedure and those who had not undergone the repair,respectively(relative risk:2.51;95%confidence interval:1.60–3.96;P<0.001).A predicted probabilities graph showed consistently higher sperm retrieval rates for patients with varicocele repair,regardless of their follicle-stimulating hormone levels.Patients who underwent varicocele repair showed higher testicular histopathological patterns(P=0.001).In conclusion,men with nonobstructive azoospermia and clinical varicocele who underwent varicocele repair before the sperm retrieval procedure had higher sperm retrieval rates compared to those who did not undergo varicocele repair.