Premature luteinization of follicles during ovarian stimulation for in-vitro fertilization

The structure and growth of developing follicles was monitored using vaginal ultrasound scanning in an outpatient programme of in-vitro fertilization (IVF) and embryo transfer (ET). Patients received either human menopausal gonadotrophin (HMG) alone or clomiphene citrate (CC) + HMG for controlled ovarian stimulation. Ultrasound data were compared with pre-ovulatory oestradiol (E2), luteinizing hormone (LH) and progesterone (P) levels. Hormonal parameters and results were classified according to the main indications of IVF-ET treatment. Twenty-one of the 271 patients in the study showed ultrasonic evidence of premature luteinization (PL) of follicles, thickening of the follicular wall and the appearance of irregular echogenic structures in the follicle. PL was preceded in eight cases by an indisputable LH surge and subsequent P elevation. In the remaining 13 cases PL occurred either due to an abortive LH surge not exceeding by 3-fold the baseline values or as a result of HMG administration. Special attention was paid to the P pattern prior to and after human chorionic gonadotrophin (HCG) administration. PL cycles demonstrated significantly (P less than 0.05) higher P levels before HCG administration and at the time of oocyte retrieval as well. Because implantation was not achieved in these cases, the cancellation of PL cycles is recommended. Vaginal ultrasound scanning seems to be helpful in the evaluation of minor changes in the follicular structure, correlating frequently with hormonal findings.     

Increase in transforming growth factor beta1 in ovarian follicular fluid following ovarian stimulation and in-vitro fertilization correlates to pregnancy

We have analysed the content of the growth and differentiation regulating peptide, transforming growth factor beta1 (TGFbeta1), in follicular fluid from patients undergoing in-vitro fertilization (IVF), and correlated concentrations of TGFbeta1 with the outcome of the IVF treatment and the concentrations of 17beta oestradiol in serum at ovum retrieval. A total of 88 women with infertility of >3 years duration and age <38 years participated in the study. During IVF treatment, follicular fluid and matched serum samples were collected at ovum retrieval and analysed for TGFbeta1, oestradiol, progesterone, follicle- stimulating hormone (FSH) and luteinizing hormone (LH) using radioimmunoassay and enzyme-linked immunosorbent assay. We found that the TGFbeta1 content in the follicular fluid at the time of oocyte retrieval correlated positively with subsequent pregnancy. In 29 women who became pregnant following IVF, follicular fluid TGFbeta1 values were significantly higher (P=0.005) than in 59 women where IVF was unsuccessful. In the pregnant group, TGFbeta1 values correlated positively with oestradiol at ovum retrieval. TGFbeta1 also correlated positively with the number of fertilized oocytes. TGFbeta1 may thus be important for successful human pre-embryo development, contribute to successful embryo implantation and development and may be necessary for the establishment of pregnancy.      

An improved use of buserelin in ovarian stimulation for in-vitro fertilization

In earlier IVF programmes, subcutaneous buserelin (Suprefact, Hoechst) was initially administered three times per day (200 micrograms x 3); then twice daily (300 micrograms x 2). We now suggest that a single administration of 600 micrograms daily may be equally effective. In a preliminary study, 20 patients were selected on the basis of tubal or idiopathic infertility and received 0.6 ml buserelin subcutaneously once a day, beginning on day 1 or 2 of the cycle. A sufficient pituitary desensitization was obtained on day 10 in 75% of patients and on day 16 for 100% and the ongoing pregnancy rate was 35% per treatment cycle. A randomized study comparing the effect of 600 micrograms of buserelin administered in one (n = 50) or two injections (n = 46), has been carried out and indicates that the results in terms of the ovarian suppression and pregnancy rates, were similar. Therefore, this protocol represents a simplification of the treatment with buserelin.     

Does ovarian stimulation for in-vitro fertilization induce a hypercoagulable state?

Effects on blood coagulation and fibrinolytic activity during ovarian stimulation for in-vitro fertilization (IVF) were examined in 12 women. Blood samples were taken prior to hormonal stimulation (days 2-3 of the menstrual cycle, mean serum oestradiol concentration 0.16 nmol/l) and the day after ovulation induction with human chorionic gonadotrophin (HCG) (days 10-12, mean serum oestradiol concentration 5.35 nmol/l). We measured whole blood clotting time, whole blood clot lysis time, plasma fibrinogen, factor VII and antithrombin III. The whole blood clotting time was slightly, but not significantly shortened after ovarian stimulation. A significant rise in plasma fibrinogen (P less than 0.001) and reduction in antithrombin III (P less than 0.001) were observed, whereas no change in factor VII was found. The blood fibrinolytic activity was significantly reduced as evaluated by an increase in the clot lysis time (P less than 0.02). These results indicate that ovarian stimulation for IVF may create a state of hypercoagulability.     

Ovarian stimulation for in-vitro fertilization

In-vitro fertilization (IVF) and embryo transfer has becomean accepted clinical method for the treatment of sterility.Different types of ovarian stimulation have been used successfully.The therapeutic principles behind the stimulation treatmentin an IVF programme are the same as those applied in the treatmentof normal and hypogonadotrophic ovarian insufficiency. Theseinclude clomiphene therapy with sub sequent HCG administration,combined clomiphene/HMG administration and stimulation withHMG alone, followed by HCG. A new approach to the stimulationof follicular development and oocyte maturation is the use ofpure FSH and GnRH analogues. The principal indications, results,advantages and disadvantages of these different schemes of ovarianstimulation for oocyte retrieval are discussed.     

The impact of ovarian cystectomy on ovarian response to stimulation during in-vitro fertilization cycles

This study was carried out to investigate whether ovarian cystectomyinterferes with follicular recruitment and the number of oocytesretrieved in an in-vitro fertilization (IVF) cycle. Patientswho had previously undergone unilateral ovarian cystectomy (n= 90) and control patients (n = 90) with no history of ovariansurgery were included in our study. The parameters comparedwere the number of follicles recruited and the number of oocytesobtained from each ovary. In patients who had undergone surgery,the normal ovaries recruited a significantly higher number offollicles (P < 0.001) and yielded a significantly highernumber of oocytes (P < 0.001) compared with the contralat-eralovaries which had undergone cystectomy. In the control patients,no significant differences were identified between the leftand right ovaries. These results demonstrate that ovarian cystectomyreduces follicle and oocyte numbers in ovulation induction cycles.     

The relationship between ovarian vascularity and the duration of stimulation in in-vitro fertilization

The role of transvaginal pulsed colour Doppler ultrasound in the assessment of ovarian vascularity was studied in 196 in-vitro fertilization (IVF) cycles. The changes in ovarian blood flow after gonadotrophin-releasing hormone agonist (GnRHa) down-regulation and human menopausal gonadotrophin (HMG) stimulation were determined. The data obtained showed that the ovarian blood flow was significantly improved by oestradiol secretion (P = 0.05) and human chorionic gonadotrophin (HCG) administration (P = 0.003). Folliculogenesis was affected by blood flow supply. The resistance index (RI) value was significantly different (P = 0.05) according to the duration of ovarian stimulation. Patients with a mean RI value >0.56 had a longer stimulation with a significantly lower mean number of oocytes retrieved (P = 0.01) despite the administration of a standard dose of HMG. The RI value is a good indicator of modifications in ovarian vascularization during stimulation. Doppler blood flow measurement could be used to determine the optimal timing for the beginning of HMG administration in patients undergoing ovarian stimulation after down-regulation for IVF treatment.      

The relationship between endogenous oestradiol and vitamin D3 metabolites in serum and follicular fluid during ovarian stimulation for in-vitro fertilization and embryo transfer.

The present study was undertaken to examine the effect of circulating oestradiol on serum levels of 25-hydroxyvitamin D3 (25-OHD3), 24,25-dihydroxyvitamin D3[24,25-(OH)2D3], and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] during gonadotrophin-induced ovarian stimulation in 10 healthy women undergoing in-vitro fertilization and embryo transfer (IVF). The presence of these metabolites in the follicular fluid was also investigated. Plasma oestradiol increased from 25 +/- 3.2 (mean +/- SE) pg/ml before initiation of treatment to 2563 +/- 328 pg/ml on the day of injection of human chorionic gonadotrophin (HCG) and 1641 +/- 299 pg/ml on the day of ovum retrieval (P < 0.01). Serum levels of 1,25-(OH)2D3 increased from 32.0 +/- 1.9 (mean +/- SE) pg/ml to 46.6 +/- 8.1 and 48.5 +/- 7.7 pg/ml (P < 0.05) on the day of HCG and ovum retrieval, respectively. No changes in blood levels of 25-OHD3 and 24,25-(OH)2D3 were found. The presence of vitamin D metabolites in follicular fluid is documented herein for the first time. All three metabolites were present in the follicular fluid but were significantly lower than in the concurrent serum (P < 0.01). A highly significant correlation was found between serum and follicular fluid levels: r = 0.787, P < 0.001 for 1,25-(OH)2D3; r = 0.738, P < 0.01 for 25-OHD3; and r = 0.751, P < 0.01 for 24,25-(OH)2D3. Our results suggest that raised levels of circulating oestradiol during gonadotrophin-induced ovarian stimulation are associated with a significant increase of serum 1,25-(OH)2D3.     

Physiology: Enhanced sensitivity of the extrinsic coagulation system during ovarian stimulation for in-vitro fertilization

The tissue factor activity in blood monocytes was investigatedduring ovarian stimulation for in-vitro fertilization (IVF)in 13 women. Blood samples were taken prior to hormonal stimulation(days 2–3 of the menstrual cycle, median serum oestradiolconcentration 70 pmol/1) and the day after ovulation inductionwith human chorionic gonadotrophin (days 11–13, medianserum oestradiol concentration 6270 pmol/l). The tissue factoractivity in unstimulated monocytes and factor VII concentrationwere unchanged during the treatment. However, the tissue factoractivity in lipopolysaccharide-stimulated monocytes was on averagemore than twice as high after stimulation (P < 0.02). A positivecorrelation was found between the tissue factor activity andthe serum concentration of oestradiol (r = 0.514, P < 0.02).The tumour necrosis factor (TNF)- increased during ovarian stimulation(P = 0.05), and there was a positive correlation between thechange in TNF- and the change in tissue factor activity (r =0.663, P < 0.05). Our results indicate an enhanced sensitivityof the extrinsic coagulation system during IVF treatment sincemore tissue factor is available upon stimulation. It is suggestedthat this may be important in thrombotic situations. Furtherstudies are necessary to elucidate the mechanism behind thisresponse.     

Ultrasound measurement of endometrial thickness on different ovarian stimulation regimens during in-vitro fertilization

Endometrial thickness was measured ultrasonographically in threegroups of patients undergoing in-vitro fertilization (IVF) onthree different ovulation induction regimens. The endometrialthickness was comparable on all three regimens and similar tothat observed in a group of spontaneously ovulating, normal,fertile controls. These patterns of endometrial thickness wereobserved despite significantly higher levels of serum oestradiol-17In all of the hyperstimulated cycles, suggesting that in thenormal cyde a maximum response in terms of endometrial developmentmay be achieved. In the three conception cycles endometrialthickness continued to increase throughout the luteal phase,whilst In non-conception cycles plateauing of thickness increaseoccurred in the mid-luteal phase and reduction in late lutealphase. Whether ultrasonographic evaluation of endometrium duringIVF stimulation cydes has any prognostic value regarding predictionof conception has yet to be detennined.     

Infertility: Semi-programmed ovarian stimulation as the first choice in in-vitro fertilization programmes

The objective of this work was to evaluate the results obtainedwith a protocol of semi-programmed ovarian stimulation (low-dosecontraceptive pill + clomiphene citrate + human menopausal gonadotrophin+ dexamethasone) used as the first-choice method for in-vitrofertilization (IVF). A total of 207 punctures was performedfor oocyte collection from 168 patients (mean age 31.0 ±4.0 years); mean infertility duration was 5.81 ± 3.30years. The infertility factors indicating IVF for this populationwere as follows: tubo-peritoneal factor, 68%; pure or associatedmale factor, 9.2%; endometriosis, 11.1%; ovulatory factor, 4.3%;idiopathic factor, 11.6%; others, 2.4%. No oocyte was foundon aspiration in five procedures (2.4%), with the mean numberof oocytes collected per cycle being 5.87 ± 3.3 (range0–18). The cancellation rate per puncture was 5%. Themean embryo cleavage rate was 60.2 ± 36.8%, with transferof at least one embryo occurring in 82.6% of all punctures.The mean number of transferred embryos was 2.52 ± 1.60(range 1–5). The clinical pregnancy rates per startedcycle and per puncture were 22.4 (218 ovarian stimulation cycles)and 23.6% (a total of 49 clinical pregnancies, 36 single, ninetwins and four triplets) respectively. The clinical pregnancyrate per embryo transfer was 28.6%. The embryo implantationrate was 12.6%. The abortion rate was 16.3%. The index of deliveriesper puncture was 19.8%. There were no cases of moderate or severeovarian hyperstimulation syndrome. The favourable results obtained,in addition to the low operational costs, confirm the validityof the use of semi-programmed cycles as the first choice forpatients undergoing the IVF process.     

Dynamics of human follicular growth and in-vitro oocyte maturation

The physiological trigger for meiotic resumption in the human oocyte is the surge of luteinizing hormone, but it can also occur spontaneously if oocytes are released from antral follicles and cultured in vitro. The development of novel techniques for the culture of murine oocytes has raised the possibility of growing human oocytes to maturity in vitro. Such a system could open the door to a number of techniques with revolutionary consequences. It would clearly be of benefit in basic physiological studies of follicular development, as well as being used to test the effect of toxicological substances on oocyte maturation. More significantly, such a system could provide a source of human oocytes for in-vitro fertilization (IVF) where immature or germinal vesicle oocytes are cultured to maturity before being fertilized. If this can be achieved, it might facilitate oocyte cryopreservation, where surplus oocytes are stored, thus avoiding the need for repeated superovulation. A combination of immature oocyte cryopreservation for later maturation and IVF will provide the opportunity to establish oocyte banks and help overcome some of the practical and ethical dilemmas that are currently shadowing the field of reproductive medicine.     

Recurrent heterotopic pregnancy after repeated in-vitro fertilization treatment

We report a rare clinical case of recurrent heterotopic pregnancy in the same patient following in-vitro fertilization treatments. A 27 year old woman, who suffered from infertility for the last 4 years due to male factor, was being treated by intracytoplasmic sperm injection which resulted in two episodes of combined intrauterine and tubal pregnancy, in a 1 year period. The first ended in emergency salpingectomy by laparotomy and missed intrauterine abortion. The second was managed by laparoscopic salpingectomy and the synchronous pregnancy ended in the delivery of twins. The possibility of heterotopic pregnancy and recurrent heterotopic pregnancy, though rare, should be considered by every gynaecologist, especially those who use infertility treatment on patients.      

Subcutaneous low dose leuprolide acetate depot versus leuprolide acetate for women undergoing ovarian stimulation for in-vitro fertilization

A total of 100 women undergoing ovarian stimulation with gonadotrophin-releasinghormone agonist (GnRHa) and a human menopausal gonadotrophin(HMG) for in-vitro fertilization (IVF) participated in thisrandomized comparative study. Leuprolide acetate at a dose of0.5 mg/day s.c. (n = 52, group I), or low-dose leuprolide acetatedepot at a dose of 1.88 nig s.c. (n = 48, group II), was startedon days 21–23 of the cycle. Stimulation with 225 IU/dayHMG was started after pituitary desensitization had been achieved.The luteal phase was supported by human chorionic gonadotrophin(HCG) i.m. injection. There were nostatistical differences inbaseline oestradiol (24.5 ± 4.8 versus 21.9 ±4.5 pg/ml) and follicle stimulating hormone (FSH) concentrations(3.9 ± 1.9 versus 3.2 $ 1.8 mlU/ml), and concentrationson the day of HCG administration of oestradiol (1657 ±245 versus 1512$165 pg/ml), luteinizing hormone (LH; 6.2 ±4.8 versus 5.6 ± 4.3 mlU/ml) and FSH (10.6 ± 2.8versus 10.8 ± 3.6 mIU/ml). There were also no statisticaldifferences in the HMG dosage (26.8 ± 1.8 versus 28.5± 1.5), the number of oocytes retrieved (7.6 ±3.0 versus 8.1 ± 4.3), the number of oocytes fertilized(5.3 ± 2.1 versus 5.6 ± 3.0) and the number ofembryos transferred (3.5 ± 1.3 versus 3.4 ± 1.6).There was no evidence of a premature LH surge in either group,but two patients appeared to have a poor response in the leuprolideacetate group (group I). There were 11 pregnancies (21.2%) afterthe use of leuprolide acetate and 12 pregnancies (25.0%) inthose given leuprolide acetate depot; no statistical differenceexisted between these two groups. Thus, an s.c. low-dose leuprolideacetate depot injection may offer a useful alternative for pituitarysuppression in ovarian stimulation for IVF.     

Vascular endothelial growth factor in serum and in the follicular fluid of patients undergoing hormonal stimulation for in-vitro fertilization

A cross-sectional study regarding endocrine and cytokine parameters in human follicular fluid (FF) as compared to serum values following hormonal stimulation for in-vitro fertilization was conducted. The patients (n = 32) were treated sequentially with the luteinizing hormone-releasing hormone (LHRH) agonist buserelin followed by a combination of buserelin plus highly purified follicle stimulating hormone and finally human chorionic gonadotrophin, in order to induce ovulation. The FF content of pro-inflammatory (IL-1, IL-6), and anti- inflammatory (IL-1ra, IL-10) cytokines, of the immune response-related soluble interleukin-2 receptor (sIL-2R), as well as the mitogens vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) were determined. Routine evaluation included peripheral blood cell counts, morphological data of the ovary and ova, ovarian steroids, prolactin concentrations and thyroid function parameters [free thyroxine (fT4), thyroglobulin]. The concentrations of IL-6, IL1- ra, sIL-2R, VEGF and bFGF in the FF compartment were higher than in serum in the majority of cases. Regression analysis showed a significant association between the serum and FF concentrations of fT4 (P = 0.04; y = 0.37 + 0.34x) and IL-6 (P = 0.002; y = 0.78 + 0.5x). Multiple regression analysis revealed that progesterone played a role in determining VEGF concentrations in the FF (P = 0.07; y = 0.37 + 0.86x). Thyroglobulin concentrations within the FF were extremely low, whereas fT4 concentrations in the FF were similar to those in serum. Patients with a previously diagnosed hypothyroidism tended to have lower serum oestradiol and higher serum progesterone when compared to euthyroids. We conclude that the human FF represents a functional compartment that integrates endocrine, immunological, and mitogenic signalling that is unique for each ovarian follicle. The close association between progesterone and VEGF within the FF suggests a close association of this mitogen to gonadotrophin stimulation, confirming the ovary as a production site of VEGF.      

High dose gonadotrophin-releasing hormone antagonist (ganirelix) may prevent ovarian hyperstimulation syndrome caused by ovarian stimulation for in-vitro fertilization

This case report describes the first attempt to treat imminent ovarian hyperstimulation syndrome (OHSS) by using a gonadotrophin-releasing hormone (GnRH) antagonist. A 33 year old, normo-ovulatory woman undergoing in-vitro fertilization received daily subcutaneous injections of 150 IU of recombinant follicle-stimulating hormone (recFSH) from cycle day 2, together with GnRH antagonist (ganirelix) 0.125 mg from cycle day 7 onwards. On cycle day 10 the patient was found to have a serum oestradiol concentration of 16 500 pmol/l and, on ultrasound examination, four preovulatory (>16 mm) and nine intermediate sized (10-16 mm) follicles. RecFSH injections were discontinued, human chorionic gonadotrophin (HCG) withheld, whereas the ganirelix dose was increased to 2 mg/d. This regimen led to a rapid decrease in serum oestradiol concentrations and the decrease in ovarian size on ultrasound. Since GnRH antagonists will become clinically available for in-vitro fertilization programmes in the near future this suggested regimen might have a role in preventing severe OHSS.      

Follicular and luteal phase characteristics following early cessation of gonadotrophin-releasing hormone agonist during ovarian stimulation for in-vitro fertilization

Gonadotrophin-releasing hormone agonists (GnRHa) are widely used in in-vitro fertilization (IVF) for the prevention of a premature rise in luteinizing hormone (LH) concentrations. However, the administration of GnRHa during the follicular phase may also impair subsequent luteal function due to retarded recovery of pituitary gonadotrophin secretion. Therefore, luteal supplementation is generally applied. The present study was designed to determine whether a premature LH surge would still be prevented after early cessation of GnRHa during ovarian stimulation and whether subsequent luteal phase LH production would be sufficient to support progesterone synthesis by the corpus luteum. Sixty patients were randomized for three groups: (i) A long GnRHa/human menopausal gonadotrophin (HMG) protocol with luteal support by repeated human chorionic gonadotrophin (HCG) (n = 20), (ii) early follicular phase cessation of GnRHa without luteal support (n = 20), and (iii) a long GnRHa protocol without luteal support (n = 20). Frequent ultrasound and blood sampling was performed during the entire IVF cycle. Forty normo-ovulatory women served as controls. No premature LH surges were found after early cessation of GnRHa. In this group, some pituitary recovery occurred during the late luteal phase, but this did not affect corpus luteum function. Progesterone concentrations were shown to be dependent on disappearance of the pre-ovulatory bolus of HCG. Pregnancies occurred in all three groups. In conclusion, early follicular phase cessation of GnRHa is still effective in the prevention of a premature rise in LH. Although some pituitary recovery was observed thereafter, corpus luteum function is still abnormal due to early luteolysis.     

Infertility: Recurrent gestational trophoblastic disease following in-vitro fertilization

Recurrence of gestational trophoblastic disease (GTD) followingtwo attempts at in-vitro fertilization (IVF)/embryo transferis reported in a childless couple after 17 years of unsuccessfultrials of ovulation induction. The diagnosis of bilateral tubalobstruction was finally established, indicating IVF treatment.After the first IVF/embryo transfer treatment, the woman developedGTD and was treated with methotrexate. After a second IVF attempt,GTD was again diagnosed. This time there was no response tomethotrexate, thus necessitating second-line chemotherapy. Etoposide,methotrexate, actinomycin D, cyclophosphamide, oncovine wasused, and after only four treatment cycles the -human chorionicgonadotrophin (HCG) dropped to <5 mlU/ml. After 26 monthsof follow-up, the -HCG continues to be undetectable. Preimplantationevaluation and ovum donation are described as measures to minimizethe risk for GTD recurrence in a future IVF/embryo transfer.     

Ovarian response to repeated controlled stimulation in in-vitro fertilization cycles in patients with ovarian endometriosis

In-vitro fertilization (IVF) is an effective infertility treatment for women with endometriosis, but most women need to undergo several cycles of treatment to become pregnant. This case-control study was designed to assess how consistently women with ovarian endometriosis respond to ovarian stimulation in consecutive treatment cycles compared to women with tubal infertility. We compared outcome measures in 40 women with a history of surgically confirmed ovarian endometriosis and 80 women with tubal infertility, all of whom had at least three IVF treatment cycles. The groups were matched for age and early follicular follicle stimulating hormone (FSH) concentration at their first IVF cycle. Outcome measures included number of follicles, number of oocytes, peak oestradiol concentration and number of FSH ampoules required per follicle. Cumulative pregnancy and live birth rates were calculated in both groups. The ovarian endometriosis group had a significantly poorer ovarian response and required significantly more ampoules of FSH per cycle, a difference that became greater with each subsequent cycle. However, cumulative pregnancy (63.3 versus 62.6% by fifth cycle) and live birth (46.8 versus 50.9% by fifth cycle) rates were similar in both groups. In conclusion, despite decreased ovarian response to FSH, ovarian endometriosis does not decrease the chances of successful IVF treatment.