Biopsy-induced inflammatory conditions improve endometrial receptivity: the mechanism of action.

A decade ago, we first reported that endometrial biopsy significantly improves the success of pregnancy in IVF patients with recurrent implantation failure, an observation that was later confirmed by others. Recently, we have demonstrated that this treatment elevated the levels of endometrial pro-inflammatory cytokines and increased the abundance of macrophages (Mac) and dendritic cells (DCs). We therefore hypothesised that the biopsy-related successful pregnancy is secondary to an inflammatory response, and aimed at deciphering its mechanism of action. Supporting our hypothesis, we found that the pro-inflammatory TNFα stimulated primary endometrial stromal cells to express cytokines that attracted monocytes and induced their differentiation into DCs. These monocyte-derived DCs stimulated endometrial epithelial cells to express the adhesive molecule SPP1 (osteopontin (OPN)) and its receptors ITGB3 and CD44, whereas MUC16, which interferes with adhesion, was downregulated. Other implantation-associated genes, such as CHST2, CCL4 (MIP1B) and GROA, were upregulated by monocyte-derived Mac. These findings suggest that uterine receptivity is mediated by the expression of molecules associated with inflammation. Such an inflammatory milieu is not generated in some IVF patients with recurrent implantation failure in the absence of local injury provoked by the biopsy treatment.

Mitochondrial DNA STR analysis as a tool for studying the green sea turtle (Chelonia mydas) populations: the Mediterranean Sea case study.

The Mediterranean population of the green sea turtle Chelonia mydas is critically endangered. Genetic analysis of this population using the ordinary haplotyping system, based on sequence analysis of a segment of the mitochondrial DNA (mtDNA) D-loop (control region), revealed very little variation. The most common haplotype, CM-A13, was observed in all but three individuals in hundreds of samples in previous studies. In search for a more informative marker we sequenced the 3' of the mitochondrial control region which contains an AT-rich microsatellite. We found a unique pattern that consists of four AT short tandem repeats (STRs) with varying copy numbers. This allowed us to construct a new haplotyping system composed of four different STR sizes for each mtDNA sequence. Our new mitochondrial STR (mtSTR) haplotyping approach revealed 33 different haplotypes within the nesting and stranded sea turtles along the Mediterranean Israeli seashore. The Israeli coast nesting females had 10 different haplotypes that can be used for monitoring and conservation purposes. The mtSTR haplotyping system can clearly assist in fingerprinting of individual turtles. Moreover, it can be used for estimating phylogenetic distances within populations. This case study shows that the mtSTR haplotyping is applicable for the study of global green sea turtle populations and could also be considered as markers of genetic variability in other species.

Microfaunal remains at Middle Pleistocene Qesem Cave, Israel: Preliminary results on small vertebrates, environment and biostratigraphy.

Preliminary results of the investigation of the microfauna at the Acheulo-Yabrudian Middle Pleistocene site of Qesem Cave, Israel, are presented. Thus far the assemblage includes ca. 10,000 bone and tooth fragments, of which 50% could be identified to the generic and some hundreds to the species level. Based on the current material, the fauna includes the following squamate reptiles: Laudakia sp., Chamaeleo sp., Gekkonidae indet., Lacertidae indet., Scincidae indet., Pseudopus sp., Varanus sp., Colubroidea indet. (at least three species) and micromammals: Suncus etruscus, Crocidura cf. leucodon, Crocidurinae indet. (large form), Chiroptera indet., Sciurus cf. anomalus, Cricetulus cf. migratorius, Microtus guentheri, Nannospalax ehrenbergi, Dipodillus cf. dasyurus, Meriones cf. tristrami, Gerbillidae indet., Mus cf. musculus, Apodemus cf. flavicollis. These results suggest that the fauna includes only taxa that occur recently in the territory of Israel. The ecological preferences of the nearest living relatives of the recorded taxa allow us to infer a paleoenvironment with a mosaic of open and woodland habitats. However, comparing the lower with the upper levels of the microfauna-bearing profile, a slight shift towards more wooded conditions might be detectable. Biostratigraphical inferences from the recorded micromammal taxa cover a rather wide age range, whereas the radiometric (U-series and preliminary TL) dating enable a provisionally estimated date for the microfauna-bearing levels at 360-300 ka. Detailed morphometric comparisons with material from other sites in the region are necessary and may yet provide further insights.

The use of recombinant human LH (lutropin alfa) in the late stimulation phase of assisted reproduction cycles: a double-blind, randomized, prospective study.

BACKGROUND: The effect of recombinant human LH (r-hLH; lutropin alfa) in women undergoing controlled ovarian stimulation with recombinant human FSH (r-hFSH) prior to IVF was investigated. METHODS: After down-regulation with the GnRH agonist, buserelin, 114 normo-ovulatory women (aged 18-37 years) received r-hFSH alone until the lead follicle reached a diameter of 14 mm. Patients were then randomized in a double-blind fashion to receive r-hFSH in addition to r-hLH, 75 IU s.c., or placebo daily for a maximum of 10 days prior to oocyte retrieval and IVF. The primary end-point was the number of metaphase II oocytes. RESULTS: There were no significant differences between treatment groups for the primary end-point. Serum estradiol concentrations on the day of HCG administration were significantly higher in the group receiving r-hLH plus r-hFSH than in the group receiving r-hFSH alone (P = 0.0001), but there were no significant differences between the groups in dose and duration of r-hFSH treatment required, oocyte maturation, fertilization rate, pregnancy rate and live birth rate. CONCLUSION: In this patient population, the addition of r-hLH during the late follicular phase of a long GnRH agonist and r-hFSH stimulation cycle provides no further benefit in terms of oocyte maturation or other end-points.

Temporal analysis of connexin43 protein and gene expression throughout the menstrual cycle in human endometrium.

OBJECTIVE: To analyze the pattern of connexin43 gene and protein expression in human endometrium throughout the menstrual cycle. DESIGN: Controlled clinical study. SETTING: An academic research center. PATIENT(S): Women with 28-day menstrual cycles who had mechanical infertility and failed to conceive after IVF treatment. INTERVENTION(S): Endometrial and blood samples were collected on days 8, 12, 14, 21, and 25 of spontaneous menstrual cycles. MAIN OUTCOME MEASURE(S): Endometrial expression of connexin43 protein and messenger RNA, endometrial thickness, and serum concentrations of gonadotropins and steroids. RESULT(S): The expression of connexin43 gene and protein decreased on day 12 and day 14 of the menstrual cycle and then increased on day 21 and day 25, respectively. A serum LH surge accompanied by a peak in the FSH concentration was observed on days 12-14. The progesterone concentration increased on days 21-25, but there was no significant change in the E2 concentration. The thickness of the endometrium increased between days 8 and 12 and did not change further between days 21 and 25. CONCLUSION(S): The expression of connexin43 gene and protein in human endometrium changes during the menstrual cycle in a pattern that is associated with the secretion of LH, FSH, and progesterone. This pattern may serve as a marker for implantation competence.

Ovarian stimulation and in vitro fertilization in women with mature cystic teratomas.

OBJECTIVE: To report the results of ovulation induction and in vitro fertilization-embryo transfer (IVF-ET) in patients with ovarian cystic teratomas. METHODS: Six women with ultrasonographically diagnosed ovarian cystic teratomas (mean diameter 2.4 cm) who presented with infertility underwent IVF-ET (n = 4) or ovulation induction (n = 2). Serial ultrasound examinations were used to determine the size of the cystic teratomas during therapy and throughout pregnancy. RESULTS: Ovarian stimulation was successful, as evidenced by the serum estradiol concentration on the day of hCG administration (mean in IVF-ET patients, 3558+/-1319 pg/mL) and the number of oocytes retrieved (10+/-4.24). Three patients having IVF-ET and both patients having ovulation induction conceived, and six healthy infants were born. Cyst sizes remained unchanged throughout treatment and pregnancy. There were no cyst-related complications during ovulation induction or IVF-ET, or during the entire course of pregnancy, labor, and delivery. CONCLUSION: The presence of ovarian cystic teratoma should not be considered a contraindication for therapy in women undergoing ovulation induction and IVF-ET.

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines.

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.

Is hydrosalpinx fluid cytotoxic?

Accumulation of oviductal fluid in the ampullar lumen as a result of occlusion of the infundibulum is referred to as hydrosalpinx. A low pregnancy rate (10%) after in-vitro fertilization (IVF) in hydrosalpinx patients and a relatively high incidence (50%) of abortions during the first trimester suggested that leakage of this fluid into the uterine cavity may exert a cytotoxic effect on the developing embryo. To examine this possibility, we analysed the composition of the hydrosalpinx fluid and tested its effect on human granulosa cells and embryos. Hydrosalpinx fluids and granulosa cells were collected from IVF patients at ovum pick-up. IVF eggs containing three pronuclei (3PN) were employed for this study. Analysis of hydrosalpinx fluids revealed electrolyte concentrations similar to those in serum with lower amounts of total protein and albumin. No blood cells were detected and bacterial cultures were negative. Granulosa cells incubated in hydrosalpinx fluid-containing medium (diluted 1:1) were not morphologically different and showed a steroidogenic capacity that was higher than that of cells incubated in its absence. Fertilized 3PN eggs incubated in IVF culture medium successfully developed into 6- to 8- and 8- to 16-cell embryos within 48 and 72 h, respectively. This rate of embryonal development was not impaired by hydrosalpinx fluid (at either 50 or 100% concentration). In the absence of a demonstrable detrimental effect we suggest that the low implantation rate in hydrosalpinx IVF patients may not be due to an embryotoxic effect. We further suggest that constant passage of fluid into the uterine cavity in these patients could possibly introduce some mechanical interference that may result in implantation failure.

Association of the Lewis blood-group phenotype with infertility in women.

OBJECTIVE: To evaluate the distribution of Lewis blood group phenotype and secretor status among women treated for infertility. SETTING: In vitro fertilization unit of a university hospital. PATIENTS: Forty-seven consecutive infertile women with mechanical (n = 31) or unexplained (n = 16) infertility scheduled for IVF-ET. The control group was formed of 47 fertile women from our database and additional new women matched for age. MAIN OUTCOME MEASURES: Determination of ABO and Lewis blood group phenotypes. RESULTS: Of the 47 subfertile women, 12 had blood type A (25.5%), 10 type B (21.3%), 4 type AB (8.5%), and 21 type O (44.7%); 38 had Le (a-b+) (80.9%), 4 had Le (a+b-) (8.5%), and 5 had Le (a-b-) (10.6%). Of the 47 controls, 17 had type A (36.2%), 12 type B (25.5%), 4 type AB (8.5%), 14 type O (29.8%); 26 had Le (a-b+) (55.3%), 11 had Le (a+b-) (23.4%), and 10 had Le (a-b-) (21.3%). The difference in the proportions of the A, B, AB, and O phenotypes was not statistically significant. The proportion of combined recessive and nonsecretor phenotypes Le (a+/-b-) was significantly lower in subfertile women (9/47) as compared with fertile controls (21/47) (P = 0.014). The difference in the proportions of the Lewis blood group phenotypes between the unexplained and the mechanical infertility groups was not statistically significant. CONCLUSIONS: Subfertile women have an increased frequency of the Le (a-b+) blood group phenotype. Our hypothesis is that the presence of exposed fucosylated determinants such as Le(b) on the surface of endometrial cells may interfere with implantation.

Acute changes in endometrial thickness after aspiration of functional ovarian cysts.

OBJECTIVE: To evaluate the influence of aspiration of functional ovarian cysts on endometrial thickness. DESIGN: Prospective study. SETTING: An IVF Unit of an academic medical center. PATIENT(S): Twenty-two patients from our IVF program, in whom administration of a gonadotropin-releasing hormone agonist preparation in the "long protocol" failed to induce pituitary desensitization, as evidenced by a serum E2 concentration of >55 pg/mL and the presence of an ovarian cyst of >20 mm in diameter. INTERVENTION(S): Transvaginal ultrasonographic-guided cyst aspiration was performed, and 2 days later, serum E2 concentration and endometrial thickness were reassessed. MAIN OUTCOME MEASURE(S): The values of serum E2 concentration and endometrial thickness before and after cyst aspiration were compared. RESULT(S): Two days after ovarian cyst aspiration, the serum E2 concentration dropped from a mean (+/-SD) of 203 +/- 93 to 37 +/- 34 pg/mL. The mean (+/-SD) endometrial thickness was 9.6 +/- 2.0 mm before cyst aspiration and decreased to 5.9 +/- 2.4 mm after the procedure. CONCLUSION(S): Within 48 hours after ovarian cyst aspiration, a significant reduction in endometrial thickness occurs concomitant with a sharp decline in serum E2 levels. The phenomenon of acute reduction in endometrial thickness in response to acute estrogen withdrawal has not been described previously. The exact mechanism and endometrial component involved in the "shrinking" process should be further investigated.

Prospective evaluation of endometrial thickness as a predictor of pituitary down-regulation after gonadotropin-releasing hormone analogue administration in an in vitro fertilization program.

OBJECTIVE: To determine whether pituitary down-regulation after gonadotropin-releasing hormone analogue (GnRH-a) administration can be accurately predicted by transvaginal ultrasonographic measurement of endometrial thickness. DESIGN: Prospective study. SETTING: An IVF unit of an academic medical center. PATIENT(S): One hundred eighty-one patients undergoing 265 IVF-ET treatment cycles using GnRH-a in the long protocol. MAIN OUTCOME MEASURE(S): Serum concentrations of E2 were determined, and endometrial thickness was measured by transvaginal sonography. The accuracy of endometrial thickness for predicting pituitary down-regulation was calculated. RESULT(S): Pituitary down-regulation, defined as a serum E2 concentration of < or = 55 pg/mL, was achieved in 77% (204 of 265) of the cycles. An endometrial thickness of < or = 6 mm was found in 92.2% (188 of 204) of cycles in which down-regulation was achieved. An estradiol level of < or = 55 pg/mL was present in 95.9% (188 of 196) of cycles with endometrial thickness of < or = 6 mm. CONCLUSION(S): A state of relative hypoestrogenism after GnRH-a administration, indicative of pituitary down-regulation, can be predicted with a high degree of accuracy by ultrasonographic measurement of endometrial thickness. Thus, routine testing for serum E2 concentration may be safely omitted. This may allow further simplification of IVF protocols and increase both cost-effectiveness and patients' convenience.

Ovarian hyperstimulation following the sole administration of agonistic analogues of gonadotrophin releasing hormone.

Ovarian hyperstimulation following the sole administration of gonadotrophin-releasing hormone agonists (GnRHa) is exceedingly rare. We hereby report on two infertile patients undergoing in-vitro fertilization-embryo transfer who developed ovarian hyperstimulation under such circumstances. In both patients, GnRHa were administered using the 'long protocol' regimen. The first patient developed ovarian hyperstimulation on two occasions, with mid-luteal depot administration of triptorelin and with early follicular triptorelin, administered as daily subcutaneous injections. In both cycles, within 2 weeks of triptorelin therapy, massive ovarian multifollicular enlargement occurred, concomitant with high serum oestradiol concentrations, which resolved spontaneously following expectant management. The second patient developed ovarian hyperstimulation following daily injections of leuprolide acetate starting at the mid-luteal phase. The final stage of ovulation was triggered by human chorionic gonadotrophin (HCG) and 11 oocytes were retrieved. In-vitro fertilization resulted in embryo formation, but failed to result in pregnancy. The same phenomenon recurred in a subsequent cycle despite preventive pretreatment with an oral contraceptive. A negative GnRH test, performed just before HCG administration, suggested than an ongoing 'flare-up effect' was unlikely to cause ovarian stimulation. Ovarian hyperstimulation can occur following the sole administration of GnRHa irrespective of the preparation used and the administration protocol. Although spontaneous resolution is the rule, once this condition has developed, HCG administration and oocyte retrieval are feasible. This rare entity probably represents an exaggerated form of ovarian cyst formation following GnRHa administration, the underlying pathophysiology of which remains unresolved.

Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells.

Freshly isolated granulosa cells obtained from women undergoing in-vitro fertilization (IVF) become refractory to further gonadotrophin stimulation in culture due to their previous hormonal treatment. However, when precultured for 7 days in gonadotrophin-free medium they regain their response to both human chorionic gonadotrophin (HCG) and follicle stimulating hormone (FSH) with a 10-fold and 5-fold increase in progesterone production respectively, within an additional 7 days of culture. Forskolin, a direct activator of adenylate cyclase, increased progesterone levels 12-fold compared with non-stimulated cultures. Oestradiol formation was also significantly elevated (P < 0.005) following 48 h stimulation with luteinizing hormone (LH), FSH or forskolin. Intracellular cAMP levels rose 1.5-fold, 10-fold and 15-fold after 1 h of FSH, HCG or forskolin treatment. Expression of both cytochrome P450 side chain cleavage enzyme (SCC) and the steroidogenic transcription factor SF1/Ad4BP could be demonstrated by Western blotting. However, elevation of P450 SCC alone was evident following FSH and HCG stimulation. In the presence of serum, the ultrastructure of these cultured cells displayed numerous lipid droplets and well-developed mitochondria, characteristic of highly steroidogenic cells. The proportion of apoptotic nuclei in these cultures was < 30%. Removal of the serum increased apoptotic incidence to 40%, whereas addition of FSH prevented cell death significantly (P < 0.01). HCG and forskolin increased apoptosis to approximately 50%, while treatment with 8Br-cAMP led to 80% cell death. Our data suggest that, after prolonged culture, human granulosa cells can regain cAMP and steroidogenic response to gonadotrophin stimulation. Moreover, our experiments indicate that apoptosis and steroidogenesis can coexist in the same cell population while the interrelationship between these processes can be determined by the intracellular levels of cAMP.

Comparison of sperm parameters, in vitro fertilization results, and subsequent pregnancy rates using sequential ejaculates, collected two hours apart, from oligoasthenozoospermic men.

OBJECTIVE: To evaluate the effect of second consecutive ejaculate collected 2 hours after the first one from infertile men on sperm quality and fertilization and pregnancy rates (PRs) in IVF. DESIGN: A prospective case-control study. SETTING: In vitro fertilization unit of a university hospital. PATIENTS: Thirty-nine consecutive infertile patients with oligoasthenozoospermia scheduled for IVF-ET. MAIN OUTCOME MEASURES: Two consecutive ejaculates were obtained 2 hours apart and were assessed for volume, sperm count, motility, morphology, and quality of swim-up fraction. The subsequent fertilization, cleavage, and PRs (as defined by the appearance of intrauterine gestational sac) were compared between the two ejaculates. RESULTS: In 28.2% of the individuals the semen analysis of the first ejaculate precluded proceeding with IVF. A statistically significant improvement was shown in sperm cell motility (31.9% +/- 20.7% versus 15.6% +/- 15.3%) and in motile count after swim-up (4.9 +/- 4.5 versus 2.6 +/- 3.1 x 10(6) sperm). No improvement could be demonstrated in sperm density or morphology. The volume of the second ejaculate was decreased significantly as compared with the first one. The fertilization rate, the cleavage rate, and PR were all increased when oocytes were exposed to sperm from the second ejaculate compared with oocytes exposed to sperm from the first ejaculate. The overall PR in our series was 25.6%. CONCLUSIONS: We suggest that in the group of infertile men with oligoasthenozoospermia, whose partners are scheduled for IVF-ET, if on the day of retrieved oocytes insemination, the ejaculate is of unacceptable quality, a second ejaculate collected 2 hours after collection of the initial ejaculate may produce a sample that exhibits improvements in both semen parameters and reproductive potential.

Intravenous albumin for the prevention of severe ovarian hyperstimulation syndrome in an in vitro fertilization program: a prospective, randomized, placebo-controlled study.

OBJECTIVE: To evaluate the efficacy of i.v. administration of human albumin solution for the prevention of severe ovarian hyperstimulation syndrome (OHSS). DESIGN: A prospective, randomized, placebo-controlled study comparing the effects of i.v. administration of human albumin solution versus sodium chloride 0.9% solution at the time of oocyte retrieval with patients undergoing IVF-ET who are at high risk for the development of severe OHSS. SETTING: Specialized assisted reproduction unit. PATIENTS: Thirty-one patients undergoing IVF-ET who had serum E2 levels of 1,906 pg/mL (> 7,000 pmol/L) and multiple follicular development on the day of hCG administration. INTERVENTIONS: After hCG administration, patients were randomized to receive i.v., either 50 g of human albumin diluted in 500 mL of sodium chloride 0.9% or 500 mL of sodium chloride 0.9% at the time of oocyte retrieval. MAIN OUTCOME MEASURES: Ovarian size as measured by pelvic ultrasonography, development of ascites, serum E2 concentrations during the luteal phase, and results of the IVF-ET cycles. RESULTS: Although no patient who had received human albumin solution developed severe OHSS, there were four such cases in the control group. All four were hospitalized with marked ascites and ovarian enlargement. There were no significant differences between the two groups comparing serum E2 levels on the day of hCG administration and during the luteal phase, the number of oocytes retrieved, fertilization, and pregnancy rates. CONCLUSIONS: Our preliminary results suggest that the administration of human albumin solution may help to prevent the development of severe OHSS in high-risk patients. Further research is needed to assess the potential of this novel approach.

Overnight secretion pattern of growth hormone, sex hormone binding globulin, insulin-like growth factor-1 and its binding protein in obese and non-obese women with polycystic ovarian disease.

The pathophysiological mechanism underlying polycystic ovarian disease (PCOD) is different in obese and lean women. In obese patients the basic disorder is insulin resistance and hyperinsulinemia. In non-obese women the dominant derangement is a relative excess of luteinizing hormone (LH) and growth hormone (GH) production. The levels of GH, LH, sex hormone binding globulin (SHBG) and insulin-like growth factor binding protein-1 (IGFBP-1) were significantly lower and insulin levels considerably higher in obese PCOD women as compared to their non-obese counterparts. There was, however, no difference in the mean IGF-1 levels found in these two groups. The present study was designed to investigate whether, in addition to the mean levels, the overnight pattern of GH, IGF-1, IGFBP-1 and SHBG differed in obese women with polycystic ovaries as compared to that observed in the non-obese PCOD patients. Eight women with PCOD diagnosed by clinical, sonographic and hormonal means were studied. Four had basal body mass index exceeding 27. Blood samples were collected every 20 min over a period of 8 h, starting at 23:00 h. Twenty-four samples were collected from each patient and examined in one batch for GH, IGF-1, IGFBP-1, SHBG and insulin. The secretion patterns of the above substances during the late night (23:00-03:00 h) and early morning (03:00-07:00 h) hours were examined and compared in obese and non-obese PCOD women. Neither GH nor IGF-1 showed a distinct overnight secretion pattern. The overnight secretion patterns of IGFBP-1 and SHBG were similar in obese and non-obese women--the former showing a constant rising during the night and the latter exhibiting a converse trend. The integrated insulin levels were much higher during the late night as compared to early morning hours in all patients. It is proposed that the specific secretion pattern of IGFBP-1 is not directly dependent on body fat mass but is regulated by insulin in both obese and non-obese patients.