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.

Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

Aim： To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone （FSH） or both agents given together. Methods： From postnatal day （PND） 5 to 15 male rats were daily injected with 12.5 μg of 1713-estradiol benzoate （EB） or 7.5 IU of human purified FSH （hFSH） or EB ＋ hFSH or solvents （control）. On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUN-EL method. Results： Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB ＋ hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB ＋ hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB ＋ hFSH. Conclusion： At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.

Influence of leptin on luteinizing hormone and follicle stimulating hormone secreted from cultured rat anterior pituitary cells

BACKGROUND： Leptin may regulate reproductive function via release of hypothalamic neuropeptide Y. However, it is unknown whether this regulatory effect is limited to the hypothalamus. OBJECTIVE： To detect the effect of different dosages of leptin on luteinizing hormone （LH） and follicle stimulating hormone （FSH） secretion from in vitro cultured rat anterior pituitary cells. DESIGN： Contrast study based on cells. SETTING： This study was performed in the Basic Institute of Chengde Medical College, Chengde City, Hebei Province, China from March to June 2007. MATERIALS： Eighteen female Wistar rats of three months of age, weighing 200-220 g, and of clean grade were used. Leptin was provided by Peprotech Company, DMEM culture medium by Invitrogen Company, and the radioimmunological kit by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. METHODS： Three glandular organs were regarded as one group for culture of anterior pituitary cells. In the control group, saline was added to the culture medium instead of leptin. In the leptin group, leptin was prepared into different concentrations of 1×10^-12, 1×10^-11, 1×10^-9, 1×10^-7, and 1×10^-6 mol/L for stimulation of cultured cells. The culture supernatant was obtained at three hours after additional of saline/leptin. MAIN OUTCOME MEASURES： Contents of LH and FSH were detected by radioimmunology. RESULTS： Following leptin stimulation, LH release increased with increasing concentrations of leptin up to 1×10^-9 mol/L, where LH release peaked. LH release then progressively decreased with increasing leptin concentrations （P 〈 0.01）. LH release in the leptin （1×10^-12, 1×10^-11, 1×10^-7, and 1×10^-6 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. FSH content in the leptin （1×10^-11, 1×10^-9, and 1×10^-7 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. CONCLUSION： Leptin can directly affect pituitary tissue to promote the secretion of LH and FSH in a dose-dependent manner.

Urinary follicle stimulating hormone can be used as a biomarker to assess male reproductive function

Aim: To develop an algorithm for use in population-based studies to assess testicular function by measurements of totalurinary follicle stimulating hormone (FSH). Methods: Total concentrations of urinary FSH were measured in a groupof 44 men at the University of California, Davis (UCD) and were compared to FSH measurements in serum. On thebasis of these and other published data, a urinary FSH value of >2 ng/mg creatinine (Cr) was selected as the cutoffpoint to identify men with elevated serum FSH ( > 12 IU/L) or low sperm counts ( < 20 million/mL). Results: Thesensitivity and specificity of this algorithm for detecting elevated serum FSH in a group of 58 agricultural workers in thePeople's Republic of China were 100 % and 50 %, respectively. The sensitivity and specificity of this algorithm fordetecting low sperm counts in a population of 105 infertility patients at UCD were 58 % and 76 %, respectively.Conclusion: This test may have particular value in identifying populations with no evidence of testicular toxicity, andin which labor-intensive semen studies may not be feasible.

Fluoride Exposure,Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women

The effects of fluoride exposure on thefunctions of reproductive and endocrine systemshave attracted widespread attention in academiccircle nowadays. However, it is unclear whether thegene-environment interaction may modify thesecretion and activity of hypothalamus-pituitary-ovarian （HPO） axis hormones. Thus, the aim of thisstudy was to explore the influence of fluorideexposure and follicle stimulating hormone receptor（FSHR） gene polymorphism on reproductivehormones in Chinese women. A cross sectionalstudy was conducted in seven villages of HenanProvince, China during 2010-2011. A total of 679women aged 18-48 years were recruited throughcluster sampling and divided into three groups, i.e.endemic fluorosis group （EFG）, defluoridationproject group （DFPG）, and control group （CG） basedon the local fluoride concentration in drinkingwater. The serum levels of gonadotropin releasinghormone （GnRH）, follicle stimulating hormone（FSH）, luteinizing hormone （LH）, and estradiol （E2）were determined respectively and the FSHRpolymorphism was detected by real time PCR assay.The results provided the preliminary evidenceindicating the gene-environment interaction onHPO axishormones in women.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Association between Two Polymorphisms of Follicle Stimulating Hormone Receptor Gene and Susceptibility to Polycystic Ovary Syndrome: a Meta-analysis

Objective To investigate the association between two polymorphisms of follicle stimulating hormone receptor(FSHR) gene and polycystic ovary syndrome(PCOS) susceptibility. Methods Case-control studies on relationship of Thr307 Ala and Asn680 Ser polymorphisms in FSHR gene and PCOS susceptibility were searched from Pub Med, ISI web of knowledge, EBSCO, and China National Knowledge Infrastructure(CNKI) databases up to March 21, 2013. The pooled odds ratio(OR) and 95% confidence interval(CI) were calculated using fixed- or random-effect model based on heterogeneity test in 5 genotype models analyses. Results A total of 11 studies were included in the Meta-analysis. The random-effect analysis showed Asn680 Ser was significantly associated with the reduced susceptibility to PCOS with dominant model(Asn/Asn+Asn/Ser vs. Ser/Ser, OR=0.83, 95% CI: 0.69-1.00), recessive model(Asn/Asn vs. Asn/Ser+ Ser/Ser, OR=0.84, 95% CI: 0.72-0.98), homozygote comparison(Asn/Asn vs. Ser/Ser, OR=0.79, 95% CI: 0.63-0.98), and the allele contrast(Asn vs. Ser, OR=0.87, 95% CI: 0.79-0.97) respectively(P=0.02, I2=56.0%), being protective factors for PCOS. However, no significant associations were found between Thr307 Ala and PCOS. Conclusion There might be a significant association between Asn680 Ser polymorphism and PCOS.

Follicle-stimulating hormone autoantibody is involved in idiopathic spermatogenic dysfunction

Aim： To detect the anti-follicle-stimulating hormone （FSH） antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction. Methods： The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay （ELISA）. The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling （HOS） test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy （TEM）. Results： The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa （swollen） was 45.1% ±3.5% in the FSH antibody-positive group and 59.1% ± 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti- FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group. Conclusion： These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.

Cloning and Transcriptional Activity of Follicle-Stimulating Hormone Receptor Promoter in the Jintang Black Goat

[ Objective] To clone follicle-stimulating hormone receptor （FSHR） promoter in the Jintang black goat, study its transcriptional activity, and provide a basis for alternative splicing of FSHR gene. [Method] The total DNA were extracted from the womb of Jintang black goat, and one pair of primers were designed for amplification of FSHR promoter fragments, then the sequences and homology were analyzed. The FSHR promoter fragment was inserted into the pcFSHRB1 expression vector to substitute the CMV promoter and construct the pcFSHRB2 expression vector. The pcFSHRB1 and pcFSHRB2 expression vectors were transformed into HEK293 cells, respectively. Then these cells were collected after 24 and 48 h treatment with 2 mlU/ml follicle-stimulating hormone （FSH）, and the cAMP levels were detected. [Result] The FSHR promoter sequence of Jin- tang black goat had 34.2% homology to that of chicken and 41.6% to that of rat, respectively. The transcription initial site of FSHR was at -576 bp and its upstream sequences contained two TATA-boxes, four CAAT-boxes, one E-box and one Wl-box. After treating for 24 and 48 h, the cAMP levels of pcFSHRB2 were respectively 299.581 3 and 125.528 1 pmol/L; and that of pcFSHRB1 were respectively 120.057 1 and 109.940 7 pmoVL. [Conclusion] The FSHR promoter of Jintang black goat is a typical type 2 eukaryotic promoter, and it is also a strong promoter.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200~ implanted heifers

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

In order to assess the impact of mRNA degradation on steady state levels of follicle stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time dependent changes in FSHR mRNA content were determined by nuclease protection solution hybridization assay (NPA) or by qualitative reverse transcription competitive polymerase chain reaction (RT PCR) in cultured hFSHR YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of uridine into total RNA, by 90 % within 1 h in hFSHR Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time dependent decrease in FSHR mRNA content in hFSHR Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Effect of Follicle-Stimulating Hormone Dosage, Seasons and Treatment Frequency on Super-Ovulation in Goats

[ Objective] To explore the effects of FSH （ Follicle-stimulating hormone）, seasons and treatment frequency on super-ovulation in donor goats and to provide necessary data for research about embryo transfer and embryo biotechnology in Yangzhou area. [ Method] Two FSH dosages （240 and 300 I U）, two seasons （April- June and October- December）, and two treatment frequencies （one or two times） were used to induce super-ovulation in goats. [ Result] Both average ovulation point and number of transferable embryos were significantly different between the goats given 240 IU FSH and those given 300 lU FSH （average ovulation point, 10.12 vs 15.55; number of transferable embryos, 8.82 vs 13.15） at the 0.05 level. Both average ovulation point and number of transferable embryos were also significantly different between April -June and October- December （ average ovulation point, 9.05 vs 15.55; number of transferable embryos, 7.05 vs 13.15） at the 0.05 level. Super-ovulation effect was not significantly different between the two treatment frequencies. [ Conduslonl The FSH dosages and seasons have significant impact on super-ovu- lation, but repeat super-ovulation does not have the same impact.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

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.

Polymorphism of Follicle-Stimulating Hormone Receptor Gene in Ningxia Tan Sheep

[ Objective] To study the polymorphism of follicle-stimulating hormone receptor （FSHR） gene in Ningxia Tan sheep and thus to provide a theoretical basis for breeding. I Methodl Genotypes of 111 healthy Ningxia Tan sheep were examined by polymerase chain reaction and restriction fragment length polymorphism （PCR-RFLP）. [ResultS] A 306-bp fragment was amplified. The PCR products digested with restriction enzyme Alu I showed polymorphism with three genotypes, L e., GG, CG and CC. The genotypic frequencies of GG, CG and CC were 0.135 1 （ 15 individuals）, 0.666 7 （74 individuals） and 0.198 2 （22 individuals）, respectively. The allele frequencies of G and C were 0.468 5 and 0.531 5, respectively.[ Conclusion] FSHR aene is Dolvmomhic in Ninaxia Tan Sheeo.

Anti-Müllerian hormone and antral follicle count predict ovarian response in women less than 45 years following GnRH antagonist multiple-dose protocol

Objective:To speculate which of the following parameters:antral follicle count(AFC),anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH)and age can be used as a predictor of ovarian response to gonadotropin-releasing hormone(GnRH)antagonist stimulation multiple-dose protocol in women under 45 years,and to determine the cutoff value of these parameters and their correlations for predicting low and high ovarian response.Methods:This prospective study included 462 women with the mean age of(29.3±6.5)years.All women were subjected to the GnRH antagonist stimulation multiple-dose protocol.On the second day of the menstrual cycle,ultrasonography was conducted to determine AFC in both ovaries.Peripheral blood samples were collected to evaluate the level of estradiol,FSH,luteinizing hormone,prolactin,thyroid-stimulating hormone,and AMH.The women were divided into three groups:low response(AHH<1 ng/mL,n=173),normal response(AMH=1.0-3.5 ng/mL,n=175),and high response(AMH>3.5 ng/mL,n=114).Results:A significant decrease was found in the age and FSH level in the high response group compared to other groups(P<0.001).Conversely,a significant increase was shown in AMH,estradiol on human chorionic gonadotropin(hCG)day,AFC,mature oocytes,fertilized oocytes,and embryos transferred in the high response group compared to the other two groups(P<0.001).The receiver operating characteristic(ROC)curves demonstrated that AFC and AMH had the highest accuracy,followed by basal FSH level and age in the prediction of low ovarian reserves(P<0.001)with cutoff values of≤4.50 and≤0.95 for AFC and AMH,respectively.Moreover,the ROC analysis showed that AFC had the highest accuracy,followed by AMH level and age in the prediction of high ovarian reserves with a cutoff value of≥14.50,≥3.63,and≤27.50 years,respectively(P<0.01).A significant decrease was observed in women's age,estradiol level,and oocyte fertilization rate in pregnant women compared to non-pregnant women(P<0.001).Additionally,significant negative correlations were found between the AFC,the number of mature oocytes,fertilized oocytes,embryos transferred,and the age of pregnant women(P<0.001).Conclusions:AFC and AMH predict low and high ovarian response to GnRH antagonist stimulation multiple-dose protocol in women under 45 years.

关元命门序贯针刺激活FSHR/cAMP/PKA通路促进早发性卵巢功能不全模型大鼠颗粒细胞增殖的机制研究

【目的】观察关元命门序贯针刺方案对早发性卵巢功能不全(POI)模型大鼠的治疗作用及机制。【方法】将雌性SD大鼠分为空白组、模型组、蛋白激酶A(PKA)抑制剂(H89)+针刺组、针刺组各12只。除空白组,其他3组大鼠采用雷公藤多苷片灌胃制备POI模型。模型成功建立后,空白组和模型组每日捆绑一次;针刺组大鼠在动情间期取关元穴针刺,在动情前期取命门穴针刺;H89+针刺组按照针刺组针刺方案干预,在每次针刺前30 min内腹腔注射H89。连续干预20 d。各组大鼠分别在干预后第1个动情间期和动情前期取材。酶联免疫吸附分析(ELISA)检测动情间期促卵泡激素(FSH)、雌二醇(E2)水平,Western Blot法检测动情间期促卵泡激素受体(FSHR)、芳香化酶P450(P450arom)蛋白表达,细胞计数试剂盒8(CCK-8)法检测动情间期和动情前期颗粒细胞活性,免疫组织化学法检测动情前期增殖细胞核抗原(PCNA)蛋白表达水平。【结果】(1)与空白组比较,模型组和H89+针刺组血清FSH水平显著升高(P<0.01),E2水平显著降低(P<0.001);H89+针刺组FSH水平与模型组无差异(P>0.05),E2水平低于模型组(P<0.05);针刺组FSH水平低于模型组和H89+针刺组(P<0.05),与空白组无差异(P>0.05),E2水平显著高于模型组和H89+针刺组(P<0.01),仍低于空白组(P<0.05)。(2)模型组和H89+针刺组FSHR、P450arom蛋白表达均低于空白组(P<0.01);H89+针刺组FSHR蛋白表达水平与模型组无差异(P>0.05),P450arom蛋白表达水平低于模型组(P<0.05);针刺组FSHR、P450arom蛋白表达水平均高于模型组和H89+针刺组(P<0.05),但仍低于空白组(P<0.05)。(3)模型组和H89+针刺组GCs活性和PCNA平均光密度值均低于空白组(P<0.05);H89+针刺组GCs活性和PCNA平均光密度值均低于模型组(P<0.05);针刺组的GCs活性和PCNA平均光密度值显著高于模型组和H89+针刺组(P<0.05或P<0.01)。【结论】关元命门序贯针刺方案可通过上调促卵泡激素受体(FSHR)/环磷酸腺苷(cAMP)/蛋白激酶A(PKA)通路FSHR、P450arom蛋白的表达,调控性激素水平,提高GCs活性和促进GCs细胞增殖,从而改善POI。

FSHR基因多态性、BMI及性激素与多囊卵巢综合征发病风险的相关性分析

目的探讨体重指数(BMI)、性激素及卵泡刺激素受体(FSHR)基因rs2268361和rs2349415位点的单核苷酸多态性(SNP)与多囊卵巢综合征(PCOS)发病风险的相关性。方法收集2021年3-8月山西医科大学第一医院生殖医学科门诊就诊的213例PCOS患者(PCOS组)与207名健康对照者(对照组)的外周血,随机收集PCOS组与对照组各32例卵泡液。计算PCOS组与对照组的BMI;采用免疫化学发光法检测两组外周血卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_(2))、睾酮(T)、孕酮(P)和泌乳素(PRL)水平;采用聚合酶链式反应(PCR)和高分辨熔解曲线(HRM)分析两组FSHR基因rs2268361和rs2349415位点多态性;采用实时定量PCR检测两组外周血及卵巢颗粒细胞FSHR mRNA表达水平。结果LH与LH/FSH呈明显正相关(r=0.88,P<0.05);PCOS组BMI、E2、LH、LH/FSH、T水平明显高于对照组(P<0.05),FSH水平明显低于对照组(P<0.001);HRM分析显示,PCOS组rs2349415位点CC、CT、TT基因型频率分别为55.9%、34.3%和9.8%,对照组分别为68.6%、23.2%和8.2%,PCOS组C、T等位基因频率分别为73.0%、27.0%,对照组分别为80.2%、19.8%,两组基因型频率及等位基因频率比较差异均有统计学意义(P<0.05);PCOS组卵巢颗粒细胞中FSHR mRNA表达水平明显高于对照组(P=0.004),其中rs2349415 TT基因型FSHR mRNA表达水平高于CC(P=0.002)和CT基因型(P=0.035)。结论高水平的BMI、LH、E2及rs2349415位点T等位基因可增加PCOS的发病风险。

来曲唑、FSH促排卵方案及HRT方案在PCOS患者冻融单囊胚胎移植内膜准备中的应用

目的探讨来曲唑、卵泡刺激素(FSH)促排卵方案及激素替代治疗(HRT)方案在多囊卵巢综合征(PCOS)患者冻融单囊胚胎移植内膜准备中的应用效果。方法选择2021年1月至2022年12月厦门市妇幼保健院206例接受冻融单囊胚移植的PCOS患者作为研究对象,按随机数表法分为A组69例、B组69例和C组68例。三组患者分别采用不同内膜准备方案,其中A组使用来曲唑方案,B组使用FSH促排卵方案,C组使用HRT方案。比较三组患者的治疗情况、妊娠情况及母婴结局。结果三组患者的胚胎移植个数比较差异无统计学意义(P>0.05);三组患者的生化妊娠、临床妊娠、异位妊娠比较差异均无统计学意义(P>0.05),而A组患者的持续妊娠、活产率分别为60.87%、57.97%,B组分别为59.42%、55.07%,均高于C组的41.18%、36.76%,差异均有统计学意义(P<0.05),但A组和B组患者的持续妊娠、活产率比较差异无统计学意义(P>0.05);三组患者的妊娠期糖尿病、妊娠期高血压、前置胎盘、剖宫产、产后出血发生率及分娩孕周、新生儿体质量和早产比较差异均无统计学意义(P>0.05)。结论与HRT方案相比,使用来曲唑、FSH促排卵方案进行内膜准备有助于提高PCOS患者冻融单囊胚胎移植后的持续妊娠率及活产率。