Steroid production in cultured thecal cells obtained from human ovarian follicles

During follicular maturation there is a co-ordinated hormonalregulation of the theca and granulosa cells. It is generallybelieved that granulosa cell proliferation and differentiationare promoted mainly by follicle stimulating hormone (FSH) andthat luteinizing hormone (LH) regulates the function of thetheca cells. The aim of the present study was to examine theeffect of LH/human chorionic gonadotrophin (HCG) on steroidproduction in human thecal cells. Isolated follicles (5–20mm) were obtained during the follicular phase of the menstrualcycle in 10 women undergoing gynaecological laparotomy for reasonsunrelated to ovarian pathology. The leading follicle(s) wasexcised and dispersed cells of the theca interna layer wereisolated through combined mechanical and enzymatic techniques.The thecal cells were cultured 4–6 days with and withoutLH/HCG. Medium levels of androstenedione, testosterone and progesteronewere measured by radioimmunoassay. Isolated thecal cells, culturedfor 6 days, showed a high sensitivity to stimulation by LH/HCG.Steroid secretion was highest during days 0–2 and thendeclined gradually. LH/HCG stimulated steroid production ina dosedependant way with the maximal stimulatory effect of LHat a concentration of 1–10 ng/ml, and of HCG at 0.01–0.1IU/ml. The important question, especially in clinical situations,of the optimal level of LH for normal follicular maturation,remains to be answered. The present study is compatible withthe view that thecal cell steroidogenesis in vivo is close tomaximally stimulated by normal basal LH levels.     

Effect of clomiphene isomers on progestin synthesis in cultured human granulosa cells

The effect of en-clomiphene and zu-clomiphene (10^–9–10^–5M) on progestin synthesis in cultured human granulosa cellswas studied under basal conditions and in the presence of LH(100 ng/ml). Granulosa cells were obtained from either pre-ovulatoryfollides of clomiphene-HMG-stimulated cycles or from large folliclesof mid-to-late follicular phase of spontaneous cycles. The basaland LH-stimulated progesterone accumulation was dose-dependentlyreduced by en- and zu-clomiphene (10^–6–10^–5M) in cells from both groups studied, an effect similar to thatof oestradiol. In contrast to oestradiol, both en- and zu-clomiphene(10^–6–10^–5 M) reduced the basal and LH-stimulatedpregnenolone accumulation in cells of stimulated cycles. Theeffect of clomiphene on progesterone, and pregnenolone accumulationwas more pronounced in LH-stimulated cells than under basalconditions. There were no qualitative differences between thetwo isomers and the results were principally the same in cellsfrom both groups of follicles studied. It is concluded thaten-clomiphene and zu-clomiphene have similar inhibitory effectson progestin synthesis in human granulosa cells in vitro.     

Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I).

There is evidence that insulin-like growth factor I (IGF-I) is a potent regulator of oestradiol synthesis by human granulosa and luteal cells; however, the question of whether IGF-I regulates progesterone synthesis by these cell types has yet to be answered. As a first step towards this goal, we have compared the effects of IGF-I, follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) on progesterone production by human granulosa cells obtained from individual dominant and cohort follicles, and granulosa luteal cells from preovulatory follicles of patients undergoing in-vitro fertilization (IVF). Granulosa cells from normal, unstimulated follicles cultured in serum-free medium as controls (no additions) produced some progesterone spontaneously. In all cases, FSH stimulated basal progesterone levels (10-fold average increase) and the effect was dose-dependent (ED50 of FSH = 9.1 +/- 3.9 ng/ml). Similar effects were observed when granulosa cells from large follicles were incubated with HCG (ED50 of HCG = 6.9 +/- 2.8 ng/ml). By comparison, the effects of IGF-I on progesterone production were not marked, being absent in 80% of the follicles tested. However, granulosa cells from healthy follicles co-incubated with IGF-I and FSH or HCG produced more progesterone compared with cells treated with the gonadotrophins alone; this effect of IGF-I was dose dependent (ED50 of IGF-I = 10 ng/ml). When the effect of each agonist was tested on IVF granulosa luteal cells, HCG but not FSH or IGF-I stimulated basal progesterone levels but the HCG effect required a two-day lag phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiology: Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine

The culture of individual intact follicles in vitro, from smallpre-antral to pre-ovulatory stages, will improve our abilityto perform controlled experiments studying follicle growth andfemale gamete development. This study was undertaken to characterizefurther the conditions required for physiological follicle growthin vitro. We cultured a total of 398 pre-antral follicles ofimmature mice (aged 26–28 days) with an initial diameterof 140–340 µm for 4 days in vitro, using individualmicro-cultures under paraffin oil. Summarizing the results ofall groups, 50 follicles were damaged (12.6%) and, of thoseremaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%)became antral and 26 (7.5%) ovulated. The most advanced folliclesgrew to 400–500 µm diameter. The presence of follicle-stimulatinghormone (FSH) in the medium significantly stimulated folliclegrowth in vitro (P < 0.03), in a manner proportional to theinitial diameter over the range of 140–250 µm initialdiameter, with larger follicles being refractory. FSH also significantlyincreased the proportion of follicles forming antra (P <0.001) and their likelihood of ovulating in vitro (P < 0.01),and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclicAMP mimicked FSH, significantly stimulating growth of largefollicles (P < 0.05) and antrum formation (P < 0.01).Hypoxanthine also stimulated antrum formation (P < 0.01)but did not significantly affect follicle growth. Porcine relaxinhad no significant effect on mouse follicle growth or antrumformation. The optimal conditions for mouse follicle growthin vitro have not yet been defined, but selection of folliclesof < 250 µm diameter and inclusion of FSH or dibutyryl-cyclicAMP in the culture medium are recommended.     

Infertility: Predicting and optimizing success in an intra-uterine insemination programme

We analysed 381 consecutive cycles of homologous intra-uterineinsemination (IUI) in 215 infertile couples, resulting in 48pregnancies (12.6%/cycle, 22.3/patient). Cycle fecundity rangedfrom 0.11 to 0.14 in women aged 25–39 years, falling to0.04 beyond age 40 years. Of the 48 pregnancies, 43 occurredin the first three treatment cycles, in which fecundity was0.14, 0.16 and 0.10 respectively. Beyond three cycles, fecunditywas 0.07 (P = 0.05 versus first two cycles). The occurrenceof pregnancy varied with diagnosis (P = 0.04). Fecundity wassignificantly greater for women with ovulatory dysfunction (0.30)than for endometriosis, male factor, tubal factor, idiopathicinfertility or multifactorial (0.08–0.14). Ovulation inductionusing menopausal gonadotrophins offered significant advantageover natural cycles or cycles using clomiphene citrate withoutgonadotrophins (0.15 versus 0.03, P = 0.01). Cycles in whichpre-ovulatory surges were either induced or supported with humanchorionic gonadotrophin (HCG) were superior to spontaneous luteinizinghormone surges (0.13 versus 0.03, P = 0.05). Recruitment ofat least two mature (>1.6 cm) follicles was critical. Onlyone pregnancy occurred in 64 cycles characterized by one maturefollicle, compared with a pregnancy rate of 0.15 in cycles characterizedby two or more mature follicles (P = 0.006). IUI is not beneficialto women >40 years old, and has the best chance of successwithin three cycles. Multiple follicle recruitment using gonadotrophin-basedstimulation protocols and mid-cycle HCG are necessary to achievean acceptable pregnancy rate.     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17 (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.     

Endocrinology: Daily measurements and in-vitro effects of human chorionic gonadotrophin in the early luteal phase

The purpose of the present study was to analyse daily measurementsof human chorionic gonadotrophin (HCG) in in-vitro fertilization(IVF) cycles and to reproduce the effects of HCG in vitro usinghuman granulosa—luteinized cells from the same patients.The study population consisted of nine women undergoing IVFbecause of tubal infertility in whom blood was drawn every 24h from the day of the ovulatory dose of HCG (10 000 IU) until6 days after ovum pick-up. Granulosa—luteal cells fromthe follicular aspirates were collected and cultured in vitroup to 6 days in the presence of increasing concentrations (0,0.01, 0.1, 1.0 and 100.0 IU/ml) of HCG. Serum progesterone andHCG in vivo as well as progesterone accumulation in vitro ondays 2, 4 and 6, were the main outcome measures. Maximum HCGconcentrations (0.25 IU/ml) were reached the day before ovumpick-up, and continuously decreased until day 6 after ovum retrieval.HCG did not stimulate progesterone production in vitro at anydose tested until day 6 after ovum pick-up. Then, 0.01 IU/mlresulted significantly (P < 0.05) stimulatory compared tocontrols, while 1.0 IU/ml was inhibitory (P < 0.05). It isconcluded that HCG supplementation in an IVF cycle is unnecessaryuntil day 6 after ovum pick-up. On day 6, progesterone productionis stimulated with very low concentrations of HCG.     

Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin

Plasma prolactin levels rise in stimulated cycles. To clarifythe effects of gonadotrophin on the lactotrophs, three studieswere performed. First, plasma concentrations of prolactin duringclomiphene citrate (CC)-human menopausal gonadotrophin (HMG)-humanchononic gonadotrophin (HCG) treatment of women enrolled forin-vitro fertilization (IYF) were compared with those duringHMG-HCG administration while under pituitary suppression witha gonadotrophin releasing hormone (GnRH) analogue (buserelin).Women suppressed with buserelin had higher basal levels of PRLin plasma (14.4 ± 4.3 nglml versus 6.9 ± 1.4 ng/ml,P<0.001). Only buserelin-suppressed women showed a significantrise in plasma prolactin before HCG administration, while bothpatient groups had marked prolactin peaks after HCG injection.This peak was higher in the buserelin group (71.9 ± 50.7ng/ml versus 52.6 ± 29.7 ng/ml). The second study showedthat plasma levels of prolactin of 6 post- menopausal womenwere significantly increased 48 h after an injection of 5000IU HCG, i.m. (24.9 ± 17.4 ng/ml versus 12.4 ±6.2 ng/ml P<0.05). Third, plasma prolactin was studied in5 women over 30 days after surgical castration. An upward trendwas observed similar to that of endogenous gonadotrophin, withthe change in prolactin values closely correlating with thechange in concentrations of follicule stimulating hormone (P<0.005).All these findings suggest that human gonadotrophins stimulatelactotrophs.     

An aggressive philosophy in controlled ovarian stimulation cycles increases pregnancy rates

To assess the effect of timing of human chorionic gonadotrophin(HCG) administration in ovarian stimulation cycles, the serumoestradiol concentration and follicle profile were comparedwith the clinical pregnancy rate in 582 ovarian stimulation— intra-uterine insemination (OS—IUI) cycles and3917 in-vitro fertilization—embryo transfer (IVF—ET)cycles. The pregnancy rates increased exponentially with increasingoestradiol in both OS—IUI and IVF—ET cycles (R²= 0.720, P < 0.001) but then decreased in OS-IUI cycles whenthe oestradiol concentration exceeded 5000 pmol/l (R² = 0.936,P < 0.004) at HCG administration. In OS—IUI cyclesthe percentage of cycles with three or more mature follicles( 18 mm diameter) increased up to an oestradiol concentrationof 5000 pmol/l then declined, mirroring the pregnancy rate (R²= 0.900, P = 0.01). The exponential increase in pregnancy ratewith increasing oestradiol concentration in IVF—ET cyclessuggests that high oestradiol concentration does not have adeleterious effect on endometrial receptivity. The decreasein pregnancy rate in OS-IUI cycles when oestradiol concentrationexceeded 5000 pmol/l reflected fewer mature follicles, resultingfrom premature administration of HCG to avoid severe ovarianhyperstimulation syndrome (OHSS). We recommend that HCG administrationbe delayed until multiple follicles have reached maturity, andreducing the risk of severe OHSS by converting high risk OS—IUIcycles to IVF—ET, or if funds or facilities are unavailable,transvaginally draining all but four or five mature follicles.     

A double-blind cross-over controlled study to evaluate the effect of human biosynthetic growth hormone on ovarian stimulation in previous poor responders to in-vitro fertilization

The effect of exogenous human biosynthetic growth hormone (HGH;12 IU/day; Norditropin, Novo-Nordisk) on the response to ovarianstimulation using a buserelin/human menopausal gonadotrophin(HMG) regimen was assessed in women who had previously showna ‘poor response’ in spite of increasing doses ofHMG. Forty patients were recruited into a prospective double-blindplacebo-controlled study. The serum follicle stimulating hormone(FSH) on day 2–5 of a menstrual cycle (< 10 IU/I) wasused to exclude any peri-menopausal candidates. The urinary24 h GH secretion was normal in all patients. Thirty-three patientscompleted the study with 21 patients having human chorionicgonadotrophin (HCG) in both arms, thus providing a completeset of placebo control data. Of these 21 patients, the administrationof HGH compared to the placebo cycle resulted in increased serumconcentrations of fasting insulin on the 8th (median 3.9 versus5.8 mU/I; P < 0.0005) and13th (median 4.4 versus 5.8 mU/I;P < 0.05) day of HMG in those cycles receiving HGH. After8 days of co-treatment with HGH the number of cohort follicles(14–16.9 mm) was significantly increased, but this changewas not sustained on the day of HCG administration. No statisticaldifference in the serum oestradiol on the 8th day of HMG orday of HCG, length of the follicular phase, total dose of HMGused, or the number of oocytes collected was seen between theplacebo or HGH cycles. This study demonstrates that HGH doesnot improve the ovarian response to ovulation induction in previouspoor responders.     

Pregnancy: Endometrial ultrasonography as a predictor of pregnancy in an in-vitro fertilization programme

The endometrial pattern and thickness were analysed by ultrasonographyin 139 cycles stimulated for in-vitro fertilization (IVF) onthe day of administration of human chorionic gonadotrophin (HCG).A semi-programmed schedule based on the pill + clomiphene citrate+ human menopausal gonadotrophin (HMG) was used in all cycles.On the day of HCG administration, endometrial pattern and thicknesswere assessed with an Ultramark 4 (ATL) ultrasound equippedwith a 5 MHz vaginal probe. Endometrial pattern I (a ‘tripleline’multilayer) was observed in a total of 105 cycles (76%), andpattern II (fully homogeneous and hyperechogenic in relationto myometrial tissue) in 34 (24%). The incidence of clinicalpregnancy did not differ (P = 0.52) between the groups withendometrial patterns I (23.8%) and II (29.4%). Endometrial thicknesson the day of HCG administration in the group with pattern I(8.4 ± 1.9 mm) was similar (P = 0.96) to that observedin the group with pattern II (8.4 ± 2.0 mm). In addition,the endometrial thickness of the patients who became pregnant(8.0 ± 1.7 mm) did not differ (P = 0.15) from that ofwomen who did not achieve pregnancy (8.6 ± 2.0 mm). Theconclusion from the present data is that ultrasonographic analysisof endometrial thickness and refringency on the day of HCG administrationhad no predictive value for conception in IVF cycles.     

Aneuploidy in human granulosa lutein cells obtained from gonadotrophin-stimulated follicles and its relation to intrafollicular hormone concentrations

Proliferation of granulosa cells is inversely related to differentiationand hormone production. The purpose of this study was to evaluatethe intrafollicular and serum steroid concentrations and tocompare these results to granulosa cell proliferation as measuredby DNA flow cytometry. Human granulosa lutein cells in follicularfluid of in-vitro fertilization (IVF) patients were investigatedwith regard to ploidy, percentage of S-phase cells and proliferationindex (PI: percentage of cells in the S- and G₂/M-phase). Thestudy was originally designed to indicate an additional markerfor the outcome of IVF treatment by DNA flow cytometric measurementsof granulosa lutein cells. Follicular fluids of 160 follicles(45 patients) were evaluated; 45.6% (n = 73) of the folliclesshowed aneuploid granulosa lutein cells and 5.6% (n = 9) ofthe follicles contained multiploid granulosa cells, definedas at least two aneuploid populations of cells with differentDNA indices. A total of 48.8% (n = 78) of the follicles hadonly diploid cells. Thus > 50% of the investigated folliclesshowed aneuploidy. In all, 73% (33 of 45) of patients had atleast one follicle containing aneuploid granulosa lutein cells.The PI of the aneuploid cell populations significantly exceededthat of the diploid cell populations (median: aneuploid: 15.5;diploid: 7.4; P < 0.0001). The intrafollicular concentrationsof testosterone, progesterone and dehydroepiandrosterone sulphate(DHEA-S) were significantly lower in follicles with aneuploidgranulosa cell populations. Luteinizing hormone concentrationwas significantly higher in follicles with aneuploid granulosacells. Intrafollicular concentrations of oestradiol, folliclestimulating hormone and the serum concentrations of all steroidhormones did not show any significant correlation to ploidy.Although aneuploidy has been reported for oocytes (in 17% ofthe oocytes), no study, to our knowledge, has observed sucha high incidence of aneuploidy in granulosa lutein cells aftergonadotrophin stimulation. Except for aneuploidy found in tissueswith some characteristics of neoplastic growth (colon adenoma,borderline tumours, endometriosis with atypic cells, etc.),it is unique for non-malignant human cells. The correlationwith intrafollicular steroid concentrations points to a possiblepathophysiological or physiological relevance of these findings.However, it was impossible to correlate the outcome of IVF withDNA flow cytometry results.     

Complete and partial luteinized unruptured follicle syndrome after ovarian stimulation with clomiphene citrate/human menopausal gonadotrophin/human chorionic gonadotrophin

A total of 31 clomiphene citrate/human menopausal gonadotrophin(HMG)/human chorionic gonadotrophin (HCG)-stimulated cyclesin 28 patients were investigated to determine the fate of eachof the matured follicles. A standard stimulation regimen wasadhered to, and ultrasound as well as hormonal monitoring wasperformed. All follicles were measured by vaginal ultrasoundat –12, +35 and +45 h relative to HCG administration andat 7 days after HCG administration. Of the 220 follicles, 107(48.6%) ruptured. The number of ruptured follicles per cyclewas correlated with the mid-luteal progesterone concentration(r = 0.63, P = 0.0005). The probability of follicular rupturewas related to follicular diameter at 12 h before HCG administration;6% of follicles <12 mm in diameter ruptured compared with87% of follicles 18–19 mm. A complete luteinized unrupturedfollicle (LUF) syndrome was observed in six cycles (20%). Inthese cycles, follicular growth and oestradiol, progesterone,luteinizing hormone (LH) and follicle stimulating hormone (FSH)concentrations at 12 h before HCG administration were similarto those in cycles with follicular rupture. However, mid-lutealprogesterone concentrations were lower in complete LUF cycles(46.97 ± 8.95 nmol/1 versus 108.74 ± 12.27 nmol/1;P = 0.02). These data demonstrate that in stimulated cyclesmany follicles, usually the smaller ones, fail to rupture, evenafter HCG administration. Complete LUF syndrome, despite a strongexogenous ovulatory signal, and the absence of any differencein peri-ovulatory hormonal parameters, indicates that the defectcausing LUF resides in the follicle itself and/or hormonal changesduring the follicular phase.     

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis

The objective of this study was to investigate the influenceof thyroid hormone on gonadotrophin-induced oestradiol and progesteronesecretion by human granulosa cells maintained in vitro. Granulosacells were obtained by aspiration of pre-ovulatory folliclesfrom women undergoing assisted reproductive technology. Ovulationinduction was performed with gonadotrophin-releasing hormoneagonist, human menopausal gonadotrophin and human chorionicgonadotrophin. Granulosa cells were maintained in vitro in adefined medium with added insulin. Between 48 and 72 h afterthe initiation of cell culture, oestradiol and progesteronesecretion into the medium was determined for granulosa cellsgrowing in serum-free medium with follicle-stimulating hormone(FSH)/luteinizing hormone (LH) and in serum-free medium withFSH/LH and thyroxine added in a concentration range of 10^–10–10^–7M. All concentrations of thyroxine used produced a statisticallysignificant increase in oestradiol (range 1.18–1.37 timesthe amount with FSH/LH alone) and progesterone (range 1.29–1.51times the amount with FSH/LH alone) secretion.     

Arachidonic acid and its lipoxygenase metabolites stimulate prolactin release in superfused pituitary cells

The direct effect of leukotrienes and other lipoxygenase productson prolactin release has been assessed. Arachidonic acid andits lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoicacid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetraenoic acid(15-HETE) stimulated the release of prolactin in superlusedrat pituitary cells in a dose-dependent manner. Leukotrienes(LT) A₄, B₄, C₄ and E₄ provoked a very marked biphasic and dose-dependentsecretion of prolactin from superfused cells. Maximal effectswere achieved with leukotrienes at a concentration of 3 x 10^–11to 3 x 10^–10 M but LTD₄ did not affect peptide releaseunder these conditions. The metabolites were more potent thanarachidonic acid in affecting hormone secretion. Pukes of 4mioutes duration of these fatty acids may even elicit a morepronounced response thao thyrotrophin-releasing hormone (TRH).Nordihydroguaiaretic acid (NDGA 10^10–6 M), a lipoxygenaseinhibitor, prevented the effect of arachidonic acid on peptidesecretion. Repeated TRH (10^–7 M) administration to pituitarycells led to a reduction in cell response, which may also beobserved in cells pre-treated with pulsatile 5-HETE or 15-HETE.These data support previous findings that arachidonic acid andits lipoxygenase metabolites may play a role in the secretorymechanism of prolactin release in pituitary cells.     

Ovary and ovulation: A morphological and functional study of the effect of slow freezing followed by complete in-vitro maturation of primary mouse ovarian follicles

Mechanically isolated intact early preantral follicles (100–130 µm diameter) from 14 day old mice were cryopreservedby a slow freezing protocol with dimethyl sulphoxide and thenmatured in vitro for 12 days after rapid thawing. Minor freezedamage observed after 1 day of in-vitro culture included ablationof the theca cell layer and granu-losa cell dehydration, resultingin disruption of intercellular contacts with the oocyte andbetween granulosa cells. Of the follicles, 24% were irreversiblydamaged and had a collapsed oocyte. The remaining majority ofthe follicles had an intact oocyte as evaluated by ultrastructuralanalysis. Follicles with an intact oocyte were cultured in vitroand, after an initial retarded development, the final numberof fully grown oocytes ovulated in vitro was not different fromthat of unfrozen controls. Cryopreserved early preantral folliclesmatured in vitro responded to stimulus with human chorionicgonadotrophin in a similar way to controls, with mucificationof the oocyte-cumulus complex, germinal vesicle breakdown andextrusion of the first polar body of the oocyte. These cryopreserved,in-vitro matured oocytes had the potential to fertilize anddevelop to hatched blastocysts.     

Endocrinology: Luteal function following ovulation induction in polycystic ovary syndrome patients using exogenous gonadotrophins in combination with a gonadotrophin-releasing hormone agonist

The luteal phase was studied in 12 polycystic ovary syndrome(PCOS) patients following ovulation induction using exogenousgonadotrophins combined with a gonadotrophin-releasing hormoneagonist (GnRH-a). Human menopausal gonadotrophin (HMG) was precededby 3 weeks of treatment with GnRH-a (buserelin; 1200 µg/dayintra-nasally) and administered in a step-down dose regimenstarting with 225 IU/day i.m. GnRH-a was withheld the day beforeadministration of human chorionic gonadotrophin (HCG; 10 000IU i.m.). Blood sampling and ultrasound monitoring was performedevery 2–3 days until menses. The luteal phase was significantlyshorter in PCOS patients as compared to eight regularly cyclingcontrols: 8.8 (3.3–11.4) days [median(range)] versus 12.8(8.9–15.9) days (P = 0.01). Median peak values for progesteronedid not show significant differences comparing both groups:52.3 (17.1–510.3) nmol/l versus 43.0 (31.2–71.1)nmol/l, respectively (P = 0.8). The interval between the dayof the progesterone peak and return to baseline was significantlyshorter in the PCOS patients than in controls: 2.5 (0.3–4.9)days versus 4.2 (3.9–10.5) days (P < 0.005). Luteinizinghormone (LH) concentrations during the luteal phase as reflectedby area under the curve were significantly lower in PCOS ascompared to controls: 4.4 (1.6–21.0) IU/l x days and 49.0(27.8–79.6) IU/l x days, respectively (P < 0.001).In conclusion, patients with PCOS may suffer from insufficientluteal phases after ovulation induction using HMG/HCG in combinationwith a GnRH-a. The corpus luteum apparently lacks the supportof endogenous LH and may be stimulated only by the pre-ovulatoryinjection of HCG. Potential involvement of adjuvant GnRH-a medicationor HCG itself in luteal suppression of endogenous gonadotrophinsecretion, and the importance of luteal function for pregnancyrates following treatment, warrant further studies.     

Elevated tissue type plasminogen activator in human granulosa cells correlates with fertilizing capacity

An increased production of plasminogen activators, able to convertplasminogen into plasmin, has been found in experiments in vivoon rat ovarian granulom cells at the time of ovulation, indicatingan involvement in follicular rupture. The graoulosa cells of49 follicles from 20 patients undergoing in-vitro fertilizationwere obtained by laparclscopy and tested for the content ofurokinase-type plasminogen activator (u-PA), tissue-type plasminogenactivator (t-PA) and inhibitor of plasmhogen activator (PAI).In the respective follicular fluids the concentrations of oestradiol(E₂), progesterone (P) and testosterone (T) were determinedand the levels of these enzymes and of the follicular steroidcontent were related to the fertilizing behaviour of the respectiveoacytes. Follicles containing oocytes which could be fertilized,revealed significantly higher follicular fluid E₂ and P levelsand significantly lower T levels than follicles with unfertilizedoocytes. The respective granulosa cells of fertilized oocytesexhibited higher levels of t-PA compared to their unfertilizedcounterparts, whereas no significant difference occurred inthe levels of u-PA and PAI. These data suggest that successfulfertilization of human oocytes is associated with a high contentof t-PA in granulom cells and high E₂ and P levels in the follicularfluid.     

Immunohistochemical localization of the LH/HCG receptor in human ovary: HCG enhances cell surface expression of LH/HCG receptor on luteinizing granulosa cells in vitro

We examined the immunohistochemical localization of luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) receptor (LH-R) in the human ovary using the anti-human LH-R monoclonal antibody, 3B5. In the antral follicles, LH-R was detected on theca interna cells. In pre-ovulatory follicles, granulosa cells also expressed LH-R. During corpus luteum formation, granulosa cells seemed to increase the expression of LH-R, and in corpus luteum of mid-luteal phase, large luteal cells expressed LH-R more intensely than small luteal cells. In the regressing corpus luteum, LH-R was almost undetectable on both luteal cells, whereas in the corpus luteum of early pregnancy, LH-R continued to be expressed on large luteal cells. The granulosa cells obtained from the patients undergoing in-vitro fertilization therapy were cultured for 3 days in serum-free medium, without or with HCG (10 IU/ml) and tumour necrosis factor (TNF)alpha (10 ng/ml). Flow cytometry showed that the expression of LH-R on the cell surface of luteinizing granulosa cells was enhanced by HCG, but was unaffected by TNFalpha. These results suggest that the main target cells for LH/HCG change from theca interna cells/small luteal cells to granulosa cells/large luteal cells during ovulation, corpus luteum formation, and differentiation into the corpus luteum of pregnancy, probably under the influence of LH/HCG.      

Tumour necrosis factor-alpha inhibits follicle stimulating hormone-induced granulosa cell oestradiol secretion in the human: dependence on size of follicle

This study investigated the effects of tumour necrosis factor-(TNF) on human granulosa cells taken from ovaries of 11 premenopausalwomen undergoing oophorec-tomy during the luteal phase of thecycle for reasons unrelated to ovarian pathology. Granulosacells from follicles ranging from 5–10 mm diameter (small)and from >10–25 mm (large) were subjected to culturefor 48 and 96 h. Granulosa cells were cultured with human FSH(1 ng/ ml), testosterone (1 µM) and human TNF (10 ng/ml),each alone, and in various combinations. In granulosa cellsof small follicles, FSH alone increased progesterone and cAMPaccumulation and the conversion of testosterone to oestradiol.In granulosa cells of large follicles, FSH increased progesteroneand cAMP accumulation but not the conversion of testosteroneto oestradiol. Only in granulosa cells of small follicles didTNF significantly inhibit FSH-induced conversion of testosteroneto oestradiol but it was not apparent until the second 48 hof culture and concomitantly TNF did not alter the ability ofFSH to stimulate progesterone and cAMP accumulation. In granulosacells of large follicles, TNF did not alter FSH-stimulated oestradiol,progesterone or cAMP accumulation. Interestingly, progesteroneaccumulation in the presence of TNF and FSH was significantlygreater in granulosa cells of large follicles than in granulosacells of small follicles. The results indicate that TNF suppressesFSH-induced oestradiol secretion in granulosa cells from smallfollicles and this modulatory effect of TNF appears to be independentof decreases in progesterone and cAMP. The potential physiologicalsignificance of these findings is that TNF may be a relevantcytokine in suppressing FSH-induced oestradiol secretion andfollicular growth during the luteal phase of the cycle.