Discontinuation of rLH two days before hCG may increase the number of oocytes retrieved in IVF

Background

Administration of recombinant luteinizing hormone (rLH) in controlled ovarian hyperstimulation may benefit a subpopulation of patients. However, late follicular phase administration of high doses of rLH may also reduce the size of the follicular cohort and promote monofollicular development.

Methods

To determine if rLH in late follicular development had a negative impact on follicular growth and oocyte yield, IVF patients in our practice who received rFSH and rLH for the entire stimulation were retrospectively compared with those that had the rLH discontinued at least two days prior to hCG trigger.

Results

The two groups had similar baseline characteristics before stimulation with respect to age, FSH level and antral follicle count. However, the group which had the rLH discontinued at least two days prior to their hCG shot, had a significantly higher number of oocytes retrieved, including a higher number of MII oocytes and number of 2PN embryos.

Conclusions

When using rLH for controlled ovarian hyperstimulation, administering it from the start of stimulation and stopping it in the late follicular phase, at least two days prior to hCG trigger, may increase oocyte and embryo yield.     

Gonadotropin regulation of rat ovarian lysosomes: Existence of a hormone specific dual control mechanism

Gonadotropic hormones PMSG (15 IU/rat), FSH (3 g/rat), LH (9 g/rat) and hCG (3 g/rat) were shown to decrease the free cytosolic lysosomal enzymes during the acute phase of hormone action in rat ovaries. When isolated cells from such rats were analyzed for the cathepsin-D activity, the granulosa cells of the ovary showed a reduction in the free as well as in the total lysosomal enzyme activities in response to FSH/PMSG; the stromal and thecal compartment of the ovary showed a reduction only in the free activity in response to hCG/PMSG. The results suggest the presence of two distinct, target cell specific, mechanisms by which the lysosmal activity of the ovary is regulated by gonadotropins.Abbreviations PMSG pregnant mare serum gonadotropin - FSH follicle stimulating hormone - LH luteinizing hormone - hCG human chorionic gonadotropin - GC granulosa cells - S/T stromal and thecal cells     

The impact of dose of FSH (Folltropin) containing LH (Lutropin) on follicular development, estrus and ovulation responses in prepubertal gilts

FSH is favored over chorionic gonadotropins for induction of estrus in various species, yet little data are available for its effects on follicle development and fertility for use in pigs. For Experiment 1, prepubertal gilts (n = 36) received saline, 100 mg FSH, or FSH with 0.5 mg LH. Treatments were divided into six injections given every 8 h on Days 0 and 1. Proportions of gilts developing medium follicles were increased for FSH and FSH-LH (P < 0.05) compared to saline, but follicles were not sustained and fewer hormone-treated gilts developed large follicles (P < 0.05). No gilts expressed estrus and few ovulated. Experiment 2 tested FSH preparations with greater LH content. Prepubertal gilts (n = 56) received saline, FSH-hCG (100 mg FSH with 200 IU hCG), FSH-LH5 (FSH with 5 mg LH), FSH-LH10 (FSH with 10 mg LH), or FSH-LH20 (FSH with 20 mg LH). FSH-LH was administered as previously described, while 100 IU of hCG was given at 0 h and 24 h. Hormone treated gilts showed increased (P < 0.05) medium and large follicle development, estrus (>70%), ovulation (100%), and ovulation rate (>30 CL) compared to saline. There was an increase (P < 0.05) in the proportion of hormone-treated gilts with follicular cysts at Day 5, but these did not persist to Day 22. These gilts also showed an increase in poorly formed CL (P < 0.05). FSH alone or with small amounts of LH can induce medium follicle growth but greater amounts of LH at the same time is needed to sustain medium follicles, stimulate development of large follicles and induce estrus and ovulation in prepubertal gilts.     

Hormonal treatments for increasing the oocyte and embryo production in an OPU-IVP system

The objective was to enhance the inherent developmental ability of bovine oocytes retrieved by ultrasound-guided transvaginal aspiration. Various hormonal regimes were utilized to produce partially matured oocytes in vivo, in order to improve embryo development following IVF. In the first experiment, a two-by-two factorial design was used with FSH (multiple versus single dose) and im administration of LH (yes versus no) 6h prior to OPU. In all protocols (which lasted for nine consecutive weeks), ovarian stimulation was performed in the presence of a CIDR. One FSH administration was adequate for ovarian stimulation (9.33+/-0.7 and 10.14+/-0.7 follicles per cow per OPU session); however, multiple injections increased (P<0.05) follicular response (12.97+/-0.7 and 13.97+/-0.7). In the second experiment, a two-by-two factorial design was used to compare the effects, during ovarian stimulation, of the presence or absence of CIDR, and iv treatment with LH 6h prior to OPU (yes versus no), on oocyte competence (judged by blastocyst development rates following IVF). Presence of CIDR during superstimulation had no effect on the follicular response. Administration of LH 6h prior to OPU increased (P<0.05) the oocytes of higher morphological grades, and in the absence of a CIDR, improved (P<0.05) blastocyst development rate. Treatment with LH, 6h prior to OPU without the use of CIDR during ovarian stimulation, resulted in 2.89+/-0.4 blastocysts per cow per OPU session as compared to 1.56+/-0.4, 1.56+/-0.4 and 1.33+/-0.4 for all other groups. In conclusion, compared to single administration, multiple FSH administration increased (P<0.05) available follicles for aspiration. Moreover, when ovarian stimulation in the absence of CIDR was followed by administration of LH 6h prior to OPU, it increased (P<0.05) the number of blastocysts per OPU session.     

Hormonal regulation of testicular prolactin receptors and testosterone synthesis in golden hamsters

A study was conducted with hypophysectomized hamsters to determine effects of administration of prolactin (PRL), luteinizing hormone (LH), and follicle-stimulating hormone (FSH)-alone or in combination-on testicular PRL receptors and in vitro testosterone production. Hormonal injections commenced the second day after hypophysectomy, and hamsters were killed on Day 5, approximately 13 h after the last hormonal injection. PRL receptor numbers were reduced by hypophysectomy, and PRL administration alone lessened the extent of this decrease. By themselves, neither LH nor FSH affected PRL receptors, but a combination of PRL + FSH + LH produced the greatest effect on these receptors. Receptor affinity was only modestly affected by any treatments. In vitro testosterone synthesis was measured after addition of 0, 2, 10, and 50 mIU of human chorionic gonadotropin (hCG) to incubations of testicular tissue. Neither PRL nor FSH by themselves in vivo affected basal or hCG-stimulated testosterone production. However, PRL + FSH increased (p less than 0.05) the magnitude of the in vitro testosterone response to hCG, as well as the sensitivity of that response (slope of the dose-response curve). LH alone increased both basal and hCG-stimulated testosterone production. PRL + LH provided no additional increase in the magnitude of the testosterone response, but increased (p less than 0.05) the sensitivity. PRL + FSH + LH in vivo provided for the greatest sensitivity of the testosterone response to hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of an LHRH antagonist on the time of occurrence and amplitude of the preovulatory LH surge, progesterone and estradiol secretion, and ovulation in superovulated Holstein heifers

Beginning on Day 10 or 11 of the estrous cycle, mature Holstein heifers were given a superovulatory regimen of twice-daily injections of porcine FSH, together with injections of PG with the fifth and sixth FSH injections. Every 12 h from 24 to 60 h after PG administration, the animals received im injections of different doses of the LH releasing hormone antagonist [N-Ac-D-Nal(2)(1), D-pCl-Phe(2), D-Trp(3), D-Arg(6), D-Ala(10)]-LHRH or vehicle. Follicular development was monitored by transrectal ultrasonography every 12 h from 24 to 120 h after PG administration. All animals were given hCG at 72 h after PG injection, and were artificially inseminated. At Day 7 of gestation, the corpora lutea were counted by ultrasonography, and embryos were collected by nonsurgical flushing of the uterus. Treatment with the antagonist resulted in a dose-dependent decrease in the amplitude of the LH surge and in delays in the time of occurrence of the LH surge, ovulation and the shift from estradiol to progesterone production. These results indicate that LHRH antagonists can be used to delay the LH surge and ovulation in superovulated heifers. This finding may be beneficial to studies in the superovulation of cattle.     

Overriding follicle selection in controlled ovarian stimulation protocols: Quality vs quantity

Selection of the species-specific number of follicles that will develop and ovulate during the ovarian cycle can be overridden by increasing the levels of pituitary gonadotropin hormones, FSH and LH. During controlled ovarian stimulation (COS) in nonhuman primates for assisted reproductive technology (ART) protocols, the method of choice (but not the only method) has been the administration of exogenous gonadotropins, either of nonprimate or primate origin. Due to species-specificity of the primate LH (but not FSH) receptor, COS with nonprimate (e.g., PMSG) hormones can be attributed to their FSH activity. Elevated levels of FSH alone will produce large antral follicles containing oocytes capable of fertilization in vitro (IVF). However, there is evidence that LH, probably in lesser amounts, increases the rate of follicular development, reduces heterogeneity of the antral follicle pool, and improves the viability and rate of pre-implantation development of IVF-produced embryos. Since an endogenous LH surge typically does not occur during COS cycles (especially when a GnRH antagonist is added), a large dose of an LH-like hormone (i.e., hCG) may be given to reinitiate meiosis and produce fertilizable oocytes. Alternate approaches using exogenous LH (or FSH), or GnRH agonist to induce an endogenous LH surge, have received lesser attention. Current protocols will routinely yield dozens of large follicles with fertilizable eggs. However, limitations include non/poor-responding animals, heterogeneity of follicles (and presumably oocytes) and subsequent short luteal phases (limiting embryo transfer in COS cycles). However, the most serious limitation to further improvements and expanded use of COS protocols for ART is the lack of availability of nonhuman primate gonadotropins. Human, and even more so, nonprimate gonadotropins are antigenic in monkeys, which limits the number of COS cycles to as few as 1 (PMSG) or 3 (recombinant hCG) protocols in macaques. Production and access to sufficient supplies of nonhuman primate FSH, LH and CG would overcome this major hurdle.     

Differential effects of follicle-stimulating and luteinizing hormones on testosterone production by mouse testes

In adult mice, direct intratesticular injection of ovine follicle-stimulating hormone (o-FSH-13; AFP 2846-C, from NIAMDD, less than 1% LH contamination) at 10, 100 or 1000 ng significantly elevated concentrations of testosterone (T) within the testis. These effects were rapid, with peak values attained by 15 min, and transient, with return to values comparable to that in the contralateral, saline-injected testis within 90 min. Intratesticular injection of FSH (1 microgram) significantly increased testicular T levels in 15- and 60-day old mice. This contrasted with the effects of intratesticular administration of human chorionic gonadotropin (hCG), which stimulated T production significantly at 30 days of age through adulthood. In adult mice, the equivalent LH to the possible contamination in the FSH preparation (1 ng) had no effect. Intratesticular injection of 10 ng LH produced comparable stimulation to that by 100 ng FSH (approximately 7-fold). Systemic pre-treatment with a charcoal-treated porcine follicular fluid (PFF) extract for 2 days reduced plasma FSH levels [86 +/- 17 (5) vs 700 +/- 8 (6); P less than 0.05], but had no effect on plasma LH. Twenty-four hours after the last treatment, the response to intratesticular injection of hCG (2.5 mIU), FSH (100 ng) or LH (10 ng) was also significantly attenuated in these mice. Intratesticular injection of PFF had no direct effect on testicular T levels. In vitro T production in the presence of hCG, LH or FSH were differentially affected by the concentrations of calcium (Ca2+) or magnesium (Mg2+) in the incubation media. The stimulatory effects of FSH were apparent at significantly lower levels of Ca2+ or Mg2+, than were those of LH or hCG. The results of these studies indicate that FSH is capable of stimulating testicular T production. Furthermore, the responsiveness to FSH is qualitatively different than that to LH/hCG in terms of the age pattern, as well as the dependence on Ca2+ or Mg2+. In addition, plasma FSH levels appear to influence testicular responsiveness to direct exogenous administration of gonadotropins. These studies indicate that FSH stimulation of T production can be differentiated from those of LH, and that these effects of FSH can be observed under physiological conditions.     

Human chorionic gonadotropin administration is associated with high pregnancy rates during ovarian stimulation and timed intercourse or intrauterine insemination

Background  

There are different factors that influence treatment outcome after ovarian stimulation and timed-intercourse or intrauterine insemination (IUI). After patient age, it has been suggested that timing of insemination in relation to ovulation is probably the most important variable affecting the success of treatment. The objective of this study is to study the value of human chorionic gonadotropin (hCG) administration and occurrence of luteinizing hormone (LH) surge in timing insemination on the treatment outcome after follicular monitoring with timed-intercourse or intrauterine insemination, with or without ovarian stimulation.     

Bimodal effects of luteinizing hormone and role of androgens in modifying superovulatory responses of rats to infusion with purified porcine follicle-stimulating hormone

Prepubertal (28-30 days old) female rats were infused s.c. over a 60-h period with a purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH specific activity 8.4 times that of NIH-FSH-S1 and luteinizing hormone (LH) specific activity less than 0.005 times that of NIH-LH-S1, based on radioreceptor assays. When the FSH infusion rate of this preparation was increased over the range of 0.5-2 units/day (mg NIH-FSH-S1 equivalent), an all-or-none response was observed, with the threshold dose for superovulation being between 1 and 2 units/day. Eleven of twelve rats receiving the 2 units/day dose ovulated a mean +/- SEM of 67 +/- 8 oocytes on the morning of the third day after the beginning of FSH infusion. Addition of human chorionic gonadotrophin (hCG), as a source of LH activity, to a subthreshold (1 U/day) FSH infusion rate resulted in 20% of rats ovulating at an hCG dosage of 50 mIU/day; increasing the hCG infusion to 200 mIU/day concomitant with the subthreshold FSH infusion rate increased ovulation rate to a mean of 69 +/- 8/rat, with 100% of rats ovulating. To determine the effect of varying both FSH infusion rates and LH:FSH ratios, FSH was infused at several rates, with hCG added to give varying hCG:FSH ratios for each FSH infusion rate. Administration of hCG alone was ineffective in causing ovulation except at the highest infusion rates. Adding hCG to FSH to reach a ratio of 0.2 IU hCG/U FSH significantly increased the superovulatory response to an intermediate, 1 U/day FSH dose, but not to the low, 0.5 U/day dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.     

Administration of follitropin alfa and lutropin alfa combined in a single injection: a feasibility assessment

Background  

Gonadotrophins are routinely administered in assisted reproductive technology (ART) treatment protocols. Recombinant human follicle-stimulating hormone (r-hFSH; follitropin alfa) and recombinant human luteinizing hormone (r-hLH; lutropin alfa) can be administered individually or in a fixed combination. The ability to vary the FSH to LH dose ratio in a single injection without compromising the bioactivity of either gonadotrophin or generating losses of active principle is important for physicians and patients alike.     

Direct evidence for de-novo synthesis of LH receptors in cultured pig granulosa cells in response to FSH

FSH plus insulin, cortisol, and thyroxine (IFT) stimulated incorporation of dense isotope-containing (2H, 13C, 15N) amino acids into soluble 125I-labelled hCG binding sites. Evidence of new synthesis of binding sites appeared as early as 3 h after the beginning of the pulse-labelling period. By 48 h the majority of detectable soluble 125I-labelled hCG binding sites appeared to be newly synthesized. Studies with FSH + IFT and puromycin indicated that FSH + IFT stimulated synthesis of new LH/hCG binding sites, and that internalization or degradation of LH/hCG binding sites may also require protein synthesis.     

Effect of dexamethsone on luteinizing hormone and follicle stimulating hormone responses to LHRH and to clomiphene in the follicular phase of women with normal menstrual cycles.

In an attempt to elucidate the mechanism of suppressive action of glucocorticoids on the hypothalamo-pituitary-ovarian axis, we studied the effects of short-term high dose dexamethasone administration of the LH and FSH responses to LHRH and to clomiphene in healthy women with normal menstrual cycles. Seven women, 21--35 years of age, received 100 micrograms of LHRH i.v. on day 6 of two consecutive menstrual cycles, once with and once without pre-treatment with dexamethasone 2 mg orally every 6 hrs. on days 2 through 5 of the menstrual cycle. Seven other women (ages 21--35 years) received clomiphene citrate 100 mg on days 2 through 5 of their menstrual cycle, once with and once without simultaneous administration of dexamethasone 2 mg orally every 6 h. The administration of dexamethasone suppressed baseline serum levels of LH and FSH and blunted LH and FSH response to both LHRH and clomiphene. The results indicate that short-term administration of pharmacological doses of glucocorticoids suppress the secretion of LH and FSH by a direct effect on the anterior pituitary and possibly by an effect at the hypothalamic level with inhibition of the release of LHRH.     

Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat

Pregnant rats were injected twice daily for 1-3 days (Days 13-16 of pregnancy) with various doses of ovine LH. Follicular maturation was determined by the ability of the follicles to ovulate in response to 10 i.u. hCG as well as by endogenous production of oestradiol-17 beta and inhibin. In control animals, no ovulation was induced by hCG given on Day 16 of pregnancy. An injection of hCG on Day 16 of pregnancy, however, induced ovulation in LH-treated animals (6.25-50.0 micrograms LH per injection, s.c. at 12-h intervals from Days 13 to 16). Concentrations of oestradiol-17 beta and inhibin activity in ovarian venous plasma increased after the administration of LH, indicating that development of ovulatory follicles had been induced. Abolishing the decline in plasma LH values therefore induced maturation of a new set of follicles or prevented the atresia of large antral follicles usually seen at this time of pregnancy. Plasma and pituitary concentrations of FSH decreased in LH-treated animals compared with those in control animals. Concentrations of progesterone, testosterone and oestradiol-17 beta in the peripheral plasma were not significantly different between the two groups. These results suggest that the increase in inhibin secretion from the ovary containing maturing follicles after LH treatment may suppress the secretion of FSH from the pituitary gland. These findings indicate that (1) the development of ovulatory follicles can be induced by the administration of exogenous LH during mid-pregnancy in the rat and (2) basal concentrations of FSH are enough to initiate follicular maturation even in the presence of active corpora lutea of pregnancy, when appropriate amounts of plasma LH are present.     

Developmental programming: exogenous gonadotropin treatment rescues ovulatory function but does not completely normalize ovarian function in sheep treated prenatally with testosterone

Prenatal testosterone treatment leads to LH excess as well as ovarian follicular and ovulatory defects in the adult. These disruptions may stem from LH excess, abnormal FSH input, compromised ovarian sensitivity to gonadotropins, or intrinsic ovarian defects. To determine if exogenous gonadotropins rescue ovarian and ovulatory function of testosterone-treated sheep, the release of endogenous LH and biopotent FSH in control and prenatal testosterone-treated sheep was blocked with a GnRH antagonist during the first two breeding seasons and with LH/FSH coadministered in a manner approximating natural follicular phase. An acidic mix of FSH was administered the first 36 h at 2-h intervals and a less acidic mix for the next 12 h at 1-h intervals (different FSH preparations were used each year), and ovulation was induced with hCG. Circulating FSH and estradiol responses to gonadotropins measured in 2-h samples differed between treatment groups in Year 1 but not in Year 2. Ovarian follicular distribution and number of corpora lutea (in ewes that ovulated) tracked by ultrasonography and luteal progesterone responses were similar between control and prenatal testosterone-treated females but differed between years. Furthermore, hCG administration induced large cystic and luteinized follicles in both groups of females in Year 2, although the growth rate differed between control and prenatal testosterone-treated females. Our findings provide evidence that 1) ovulatory response in prenatal testosterone-treated females can be rescued with exogenous gonadotropins, 2) resultant follicular response is dependent on the nature of gonadotropic input, and 3) an abnormal follicular milieu may underlie differences in developmental trajectory of cystic follicles in prenatal testosterone-treated females.     

Effect of mammalian gonadotropins on testosterone secretion in male alligators

Male farm-reared alligators were injected with mammalian FSH, LH, hCG, prolactin, or saline. A blood sample was taken immediately prior to injection of hormone and at 24 h postinjection. Testosterone concentrations in the plasma were then determined by radioimmunoassay. Only the alligators injected with FSH showed a significant increase in plasma testosterone. In a second series of experiments male alligators were injected with ovine LH, ovine FSH, or saline and bled at 0, 2, 4, 16, and 24 h postinjection. Again, only the alligators injected with FSH showed significant increases in plasma testosterone at 16 and 24 h postinjection. Mammalian LH does not appear to stimulate testosterone secretion in male alligators.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.