Combination Of Luteinizing Hormone Research Articles

An Observational Retrospective Cohort Trial on 4,828 IVF Cycles Evaluating Different Low Prognosis Patients Following the POSEIDON Criteria.

Objective: To study the actual controlled ovarian stimulation (COS) management in women with suboptimal response, comparing clinical outcomes to the gonadotropins consume, considering potential role of luteinizing hormone (LH) addition to follicle-stimulating hormone (FSH).Design: Monocentric, observational, retrospective, real-world, clinical trial on fresh intra-cytoplasmic sperm injection (ICSI) cycles retrieving from 1 to 9 oocytes, performed at Humanitas Fertility Center from January 1st, 2012 to December 31st, 2015.Methods: COS protocols provided gonadotropin releasing-hormone (GnRH) agonist long, flare-up, short and antagonist. Both recombinant and urinary FSH were used for COS and LH was added according to the clinical practice. ICSI outcomes considered were: gonadotropins dosages; total, mature, injected and frozen oocytes; cumulative, transferred and frozen embryos; implantation rate; pregnancy, delivery and miscarriage rates. Outcomes were compared according to the gonadotropin regimen used during COS.Results: Our cohort showed 20.8% of low responders, defined as 1–3 oocytes retrieved and 79.2% of “suboptimal” responders, defined as 4–9 oocytes retrieved. According to recent POSEIDON stratification, cycles were divided in group 1 (6.9%), 2 (19.8%), 3 (11.7%), and 4 (61.5%). The cohort was divided in 3 groups, according to the gonadotropin's regimen. Women treated with FSH plus LH showed worst prognostic factors, in terms of age, basal FSH, AMH, and AFC. This difference was evident in suboptimal responders, whereas only AMH and AFC were different among treatment groups in low responders. Although a different result, in terms of oocytes and embryos detected, major ICSI outcomes (i.e., pregnancy and delivery rates) were similar among groups of COS treatment. Outcomes were significantly different among Poseidon groups. Implantation, pregnancy and delivery rates were significantly higher in Poseidon group 1 and progressively declined in other POSEIDON groups, reaching the worst percentage in group 4.Conclusions: In clinical practice, women with worst prognosis factors are generally treated with a combination of LH and FSH. Despite low prognosis women showed a reduced number of oocytes retrieved, the final ICSI outcome, in terms of pregnancy, is similarly among treatment group. This result suggests that the LH addition to FSH during COS could improve the quality of oocytes retrieved, balancing those differences that are evident at baseline.Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03290911

Insulin and Insulin-Like Growth Factors (IGFs) promote Vascular Endothelial Growth Factor (VEGF-A) secretion by human luteinizing granulosa cells following Controlled Ovarian Stimulation (COS)

Objective: To investigate the role of insulin-related growth factors and luteinizing hormone (LH) in the physiological regulation of VEGF-A production by luteinized granulosa cells (LGC) from preovulatory follicles in humans. Design: Prospective, comparative, experimental study. Materials and Methods: LGC were obtained from women (n=6), with IRB approval, by follicular aspiration after undergoing COS for in-vitro fertilization. Subjects (33.5 ± 1.2 years, mean age ± SEM) had regular menstrual cycles with no evidence of PCOS or other endocrinopathies. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 μg/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin and 25 μg/ml human LDL. Cells were incubated in the presence and absence of 100 ng/ml recombinant human (r-h) LH (Ares Serono) and 0, 1, 10, 100 ng/ml of r-h IGF-1, IGF-II or insulin (Sigma). Media were collected daily and assayed for free VEGF-A (Quantikine VEGF ELISA, R&D Systems). After 3 days (d), cells were fixed, Crystal Violet stained and analyzed for DNA content to estimate cell numbers. Data were analyzed by two-way repeated ANOVA, using a randomized block design. Results: In the absence of LH or insulin-related growth factors, LGCs secreted low but detectable levels of VEGF-A through d 3 of culture (179–304 pg/ml on d 3). However, exposure to IGF-I or -II resulted in a dose-related increase in VEGF-A levels by d 1 (e.g., 568 vs 183 pg/ml or 453 vs 278 pg/ml,100 ng/ml IGF-I or-II vs controls, respectively; p<0.05), which was sustained through d 3 (1001 vs 179 pg/ml or 701 vs 255 pg/ml, p<0.05). Conversely, the addition of insulin (100 ng/ml) did not increase VEGF-A levels until d 2 (737 vs 436 pg/ml, p<0.05). Although LH alone did not increase VEGF-A levels above controls on d 1–3, the combination of LH and insulin-related growth factors increased VEGF-A levels to a greater extent than IGFs alone at all doses (1181 vs 717 or 800 vs 567 pg/ml, LH + 10 ng/ml IGF-I or -II vs IGF-I or -II alone, respectively, on d 2; p<0.05). The synergistic effect of LH with IGF-I/-II was evident earlier on d 2 and at lower doses (10 ng/ml), compared to insulin and LH, which was not significant until d 3. There was no change in DNA content following incubation for 3 days in the presence of IGF-I, -II, insulin or LH when compared to controls. However, DNA content decreased (p<0.05) in the presence of LH + IGF-II at the maximum dose. Conclusion: Exposure to both insulin-related growth factors and LH is required for optimal VEGF-A secretion by human LGC. Thus, local (IGFs) as well as endocrine (LH, insulin) factors may combine to regulate VEGF-A production, and thus angiogenesis and vascular permeability, in the primate ovulatory follicle and corpus luteum. Evidence of insulin/IGFs, as well as LH, regulation of VEGF-A production supports further studies on their role in regulating angiogenic factors during COS cycles in women with endocrinopathies, such as PCOS. Supported by: NICHD U54 HD18185, RR 00163.

Effects of gonadotropins on bovine oocytes matured in TCM-199

The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during in vitro maturation of bovine oocytes in TCM-199 without serum were evaluated. Bovine oocytes with compact cumulus cells were collected from slaughterhouse-derived ovaries and cultured in Hepes-buffered TCM-199 supplemented with 5 mg/mL BSA, 1 μg/mL estradiol-17β, FSH (0, 0.015, 0.05, 0.15, 1.5 or 15 ng/mL; Experiment 1), LH (0, 0.14, 1, 7 or 49 μg/mL; Experiment 2) and combinations of 1 or 10 ng/mL FSH and 1 or 10 μg/mL LH (Experiment 3) at 39°C in 5% CO2 in air. After 22 h of maturation, cumulus expansion was estimated by scoring from 0 (no expansion) to 4 (full expansion of cumulus mass). In vitro fertilization was done with Percoll (45/90%) separated bull sperm at 1 × 106 sperm/mL in tert-TALP with 5 U/mL heparin. At 18 to 20 h post-insemination, presumptive zygotes were transferred to a chemically defined medium (CDM-1) supplemented with 0.5% BSA and nonessential amino acids for 72 h and then moved to CDM-2, additionally supplemented with essential amino acids. Zygotes were cultured at 39°C in 5% CO2, 5% O2 and 90% N2 for 8 days. During Experiments 1 and 2, cumulus expansion increased in proportion to concentrations of FSH and LH. Cleavage rates and development to blastocysts were not significantly different among FSH and LH treatments. In Experiment 3, cumulus expansion of bovine oocytes was maximal when 1 ng/mL FSH and 1 μg/mL LH were added to IVM medium, but cumulus expansion again was not related to developmental ability, although cleavage rates were improved slightly (P<0.05) by the combination of LH and FSH. Blastocyst quality, estimated by the size of inner cell mass, was not different between combinations of FSH and LH, and the numbers of nuclei were not different. Although expansion of cumulus cells surrounding bovine oocytes was altered in response to FSH and/or LH in semi-defined medium, cumulus expansion was not related to rates of cleavage or subsequent embryonic development in vitro. The effects of LH on cumulus expansion can be explained by as little as 1 part per 10,000 contamination with FSH.

Effect of prostaglandins on luteal function during early pregnancy in pigs.

Luteal cells were obtained by digestion of luteal tissue of cyclic (day 12) and early pregnant (days 12, 20 and 30) pigs. Suspensions of the dispersed luteal cells (5 x 10(4) cells ml-1) were incubated for 2 h in minimum essential medium (MEM) alone (control) and MEM with different concentrations of prostaglandin F2 alpha (PGF2 alpha) and PGE2 (0.01, 0.1, 1, 10, 100 and 1000 ng ml-1) and luteinizing hormone (LH) 100 and 1000 ng ml-1, or with combinations of LH + PGF2 alpha and LH + PGE2. Net progesterone production was measured in the incubation media by direct radioimmunoassay. The overall response pattern of the luteal cells to exogenous hormones on day 12 of the oestrous cycle and pregnancy differed (P < 0.05) from treatment on day 20 and 30 of pregnancy. In general progesterone production was higher (P < 0.05) and the response to PGF2 alpha and PGE2 treatment was most obvious on day 12 of the oestrous cycle and pregnancy. Overall, PGF2 alpha stimulated progesterone production in a dose-dependent manner (P < 0.05). The response to PGE2 was of a quadratic nature (P < 0.05) in which the lowest and the highest doses of PGE2 were associated with a greater production of progesterone than were the intermediate doses. Treatment of luteal cells with PGF2 alpha + LH or PGE2 + LH caused overall inhibition (P < 0.05) of progesterone production compared with treatment with each hormone alone. This interaction was not affected by the dose of LH used. These findings indicate that PGF2 alpha and PGE2 are involved in the autocrine control of corpus luteum function.

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradkinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5μM) on: 1) prostaglandin biosynthesis (PGE 2, PGF 2α and 6-keto-PGF 1α), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1μg/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE 2, and PGF 2α, (p ≤ 0.01) but not on 6-keto-PGF 1α synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE 2, PGE 2α and 6-keto-PGF 1α. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast ti LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.

Regulation of Leydig Cell Function by Prolactin, Growth Hormone and Luteinizing Hormone

The effects of prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) on testicular LH receptor concentration and on testosterone synthesis in response to LH (testicular responsiveness) was studied in mature intact and hypophysectomized rats. Hypophysectomy reduced LH receptor concentration by 80% and testicular responsiveness to LH by 70%, 7 days after surgery. Daily treatment with LH initiated immediately following surgery resulted in a further dose‐dependent decrease in LH receptors and a dose‐dependent increase in testicular responsiveness. Loss of LH receptors was not due to occupancy of the receptor by exogenous LH. PRL (150 μg/day) or GH (150 μg/day) partially prevented the loss of LH receptors in hypophysectomized saline‐treated rats. The effect of PRL plus GH on LH receptor concentration was additive. The combination of LH (5 μg/day), PRL and GH prevented any loss of LH receptors after hypophysectomy. A positive effect of LH on its receptor occurred in the presence of PRL. Treatment of hypophysectomized rats with 5 μg LH + 150 μg PRL enhanced the effect observed with PRL alone on maintenance of LH receptors. PRL, administered together with higher doses of LH (25 or 50 μg/day) prevented LH from exerting a negative effect on LH receptor concentration. Similar treatment with GH plus LH neither allowed a positive effect of LH nor prevented higher doses of LH from exerting a negative effect on LH receptor concentration. Loss in testicular LH receptors was also demonstrated in intact rats which received a single administration of LH. Administration of 150 μg PRL for 3 days partially prevented the LH‐induced loss in LH receptors, while treatment with 150 μg GH had no effect on LH‐induced loss of testicular LH receptors.Despite the ability of PRL to increase LH receptor concentration in hypophysectomized rats, PRL treatments did not enhance testicular responsiveness to LH. Only when LH was administered together with either PKL or G H was testicular responsiveness to LH maintained at intact control values. These data indicate that 1) maintenance of testicular LH receptor concentration in adult hypophysectomized rats is dependent on the combined effects of PKL, GH and LH; 2) PRL not only prevents LH from exerting a negative effect, but allows LH to have a positive effect in the hypophysectomized rat on the testicular LH receptor; 3) PRL and GH appear to act at different sites and by different mechanisms; and 4) hormonal regulation of L H receptor concentration appears to be distinct from hormonal regulation of testicular responsiveness to LH.

Endocrine regulation of sex-dependent hydroxysteroid dehydrogenase activities in rat kidney: NADP-dependent microsomal 3alpha- and 20beta-hydroxysteroid dehydrogenase.

The NADP-dependent microsomal kidney enzymes, 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSDH), which exhibit considerable sex differences in their activities (male:female activity ratios, 16:1 and 30:1 respectively), were investigated after interference with the pituitary-gonad and pituitary-adrenal systems. Prepubertal gonadectomy as well as hypophysectomy of mature male rats led to a decline in HSDH activity to almost that found in the normal female rat, whereas activities in female rats were unaffected. Testosterone induced typical male 3alpha-HSDH activity in both gonadectomized and hypophysectomized rats of either sex. Administration of 5alpha-dihydrotestosterone (5alpha-DHT) or 5alpha-androstane-3alpha, 17beta-diol to hypophysectomized male rats was equally effective in restoring full 3alpha- and 20beta-HSDH activities whereas 5alpha-androstane-3beta, 17beta-diol was less effective and dehydroepiandrosterone was ineffective. Simultaneous administration of cyproterone acetate did not block the inductive action of 5alpha-DHT. Administration of chorionic gonadotrophin, pregnant mare serum gonadotrophin or a combination of luteinizing hormone and follicle-stimulating hormone to hypophysectomized male rats all led to parallel increases in the weight of the seminal vesicles and in both renal enzyme activities; administration of growth hormone, prolactin or thyroid-stimulating hormone was ineffective. Adrenalectomy of gonadectomized, but not of hypophysectomized male rats, caused a further drop in activity to the normal female level. Adrenalectomy of otherwise intact rats did not affect either enzyme activity. The hypophysis was involved in the regulation of the two NADP-dependent renal HSDH activities through its gonadotrophic function in male rats; adrenal secretions were of little physiological significance.

Role of insulin, thyroxin and cortisol in luteinization of porcine granulosa cells grown in chemically defined media.

In order to determine the exact hormonal requirements for luteinizationin vitro, porcine granulosa cells obtained from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles were grown in serum-free media in the presence of various combinations of luteinizing hormone (LH), follicle stimulating hormone (FSH), insulin, cortisol and thyroxin. Morphology as well as progestin secretion was monitored throughout an 8-10 day culture period. The cells were grown in either medium “199D,” which consisted of medium 199 plus 0.4 percent bovine serum albumin (BSA) and 0.2 percent lactalbumin hydrolysate, or the serumless medium of Neuman and Tytell (“SMD”), which already contains 0.2 percent lactalbumin hydrolysate, plus 0.4 percent BSA. Additional cells were grown in the presence of 15 percent pig serum in a balance of medium 199 (medium Y). Cell adherence to the glass and cell maintenance were both less in the serum free media compared to serum-containing medium V. In contrast, progestin secretion expressed per cell was consistently greater when the cells were grown in serum-free compared to serum-containing medium V. Cells harvested from large follicles were able to secrete more progestin compared to cells obtained from small or medium-sized follicles in both types of serum-free media. If cells were grown in serum-free media, addition of 1 mU/mI insulin had some stimulatory (50-200 percent) action upon cell maintenance as well as a small, slow developing stimulatory action upon progestin secretion. The insulin treatment led to an increase in cytoplasmic vacuoles and lipid droplets. Addition of either iO’� M triiodothyronine or thyroxine or 0.01 Mg/mI cortisol along with 1.0-10 mU/mI insulin led to a 3-5 fold stimulation of progestin secretion by cells harvested from small and large follicles. The thyroxin in combination with insulin brought about an increase in oil red 0 and hematoxylin and eosin stainable material present within the cytoplasmic. The cortisol brought about an increase in distinctness of nucleolar material. Addition of either cortisol or thyroxin in the absence of insulin consistently brought about cellular necrosis. In cultures of cells grown in serum-free medium obtained from small follicles, addition of a mixture of 0.1 jig/mI human LH plus 0.1 jig/mI human FSH, 1.0 mU/mI insulin plus either i07 M thyroxin or 0.01 Mg/mI cortisol brought about morphological luteinization and a 4-14 fold increase in progestin secretion above when LH and FSH were added alone. The LH and FSH failed to bring about full morphological luteinization in the absence of these hormones. Addition of a complete mixture of all these hormones brought about the best morphological luteinization and stimulation in progestin secretion. It can be concluded that spontaneous and LH- and FSH-induced granulosa cell luteinization in serum-free culture media is enhanced by the inclusion of a mixture of insulin, cortisol and thyroxin.

Failure of gonadotropins to induce in vitro maturation of mouse oocytes treated with dibutyryl cyclic AMP

In sufficient concentration, dibutyryl cAMP (DBC) prevents the spontaneous in vitro maturation of mouse oocytes. The effects of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on this inhibition were tested in an oil-free chamber-slide culture system. Mouse oocytes devoid of cumulus cells were incubated in the presence of DBC and/or gonadotropins. Oocytes cultured with follicle cells were similarly treated. Whether follicle cells were present or absent, DBC (100 or 500 mug/ml) prevented germinal vesicle breakdown in more than 95% of the oocytes cultured. Neither LH nor FSH in a wide range of concentrations acted directly on the oocytes or indirectly through the follicle cells to initiate maturation in oocytes incubated with 100 mug DBC/ml. The combination of LH (5 mug/ml) and FSH (10 mug/ml) was also ineffective in overcoming the block induced by either 100 or 50 mug DBC/ml. Maturation of oocytes in each of the DBC-free LH or FSH treatments was comparable to that occurring in control medium which did not contain exogenous gonadotropins or DBC. It was concluded that cultured oocytes treated with DBC are not a satisfactory model for studying the steps by which gonadotropins trigger the resumption of meiosis in mammalian oocytes.