Addition of low-dose hCG to rFSH during ovarian stimulation for IVF/ICSI: is it beneficial?

PURPOSE: The aim of the study was to assess the eftect ot the addition or iow-cose numan cnononic gonauoiropm (hCG) to ovarian stimulation with recombinant follicle stimulating hormone (rFSH) on in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcome. MATERIALS AND METHODS: This retrospective clinical study was conducted on 141 women undergoing ICSI through a short GnRH-agonist protocol with rFSH and the addition of low-dose (100 IU/day) hCG. The control group consisted of 124 women undergoing ovarian stimulation with a similar protocol devoid of hCG. Statistical analysis in the study population along with a subgroup analysis for age 35 years and 36 years was performed. RESULTS: Women in hCG group were statistically significant older and with higher basal FSH compared to control group. This can be attributed to the Centre's latent tendency to add hCG in the stimulation protocol in poor prognosis patients. Despite this fact and the fact that several ovarian stimulation parameters, such as peak estradiol levels, number of oocytes retrieved, number of mature oocytes, and fertilization rates were in favor of the control group, the quality of transferred embryos and pregnancy rates were in favor of hCG group. Similar results were obtained in the subgroup analyses apart from peak estradiol levels, which did not differ among the study groups. CONCLUSIONS: The addition of hCG to rFSH may be associated with better quality embryos and higher pregnancy rates, even in women of advanced reproductive age with higher basal FSH levels, which are often considered to have poorer ovarian reserve.

Extrauterine pregnancy resulting from late spontaneous rupture of an unscarred gravid uterus: case report.

PURPOSE: Rupture of the unscarred grand uterus is a rare obstetric event associated with major perinatal mortality and a high incidence of maternal mortality and morbidity, particularly peripartum hysterectomy. METHODS & RESULTS: We present the case of a primigravida woman who was admitted at 38 weeks of gestation complaining of intermittent abdominal pain and vaginal bleeding. Although initial evaluation suggested that both mother and fetus were doing well, continuous assessment resulted in cesarean section due to variable decelerations and increasing abdominal pain. An unexpected abdominal pregnancy was discovered resulting from a complete uterine rupture. A healthy infant was delivered and hysterectomy was performed. CONCLUSION: Although extrauterine advanced abdominal pregnancy resulting from late uterine rupture is associated with high maternal and perinatal mortality, a high index of suspicion, close surveillance and ultrasonography can achieve good outcome for both mother and infant. We strongly believe, that this case report contributes to the insight and further knowledge of this rare pregnancy complication.

Small doses of LH activity are needed early in ovarian stimulation for better quality oocytes in IVF-ET.

OBJECTIVE: To examine whether exogenous LH administration has a beneficial effect on the quality of oocytes, fertilization potential, as well as pregnancy rate in IVF-ET cycles. A randomized trial comparing r-FSH versus r-FSH and LH was employed. STUDY DESIGN: Forty-six infertile couples entering IVF-ET for the first time (either tubal or male factor) were divided after prospective randomization into two groups. In both groups the long protocol with GnRH-analogs was used. In group A, ovarian stimulation started with r-FSH (200 IU/day) for the first four days. In group B, the stimulation protocol started with one amp hMG (75 IU FSH + 75 IU LH activity) daily for four days, with simultaneous administration of r-FSH (150 IU/day). The outcome was compared. RESULTS: Statistical difference was observed in the number of mature oocytes, the number of fertilized oocytes as well as the number of transferable embryos. In all cases, results were statistically significantly better (p < 0.05) in group B. Clinical pregnancy rate, finally, was similar in the two groups. CONCLUSION: The relatively small sample size does not allow a definitive conclusion for the important role of LH during early oocyte maturation. Our results, however, indicate a beneficial effect when small doses of LH are used for ovarian stimulation in IVF-ET cycles. This effect may be more important in cases in which few embryos are available for transfer.

Biological factors in culture media affecting in vitro fertilization, preimplantation embryo development, and implantation.

Optimal culture conditions are of paramount importance for in vitro fertilization of gametes, preimplantation embryo development, and implantation for all species. Water is the basis of all culture media, and ultrapure water should be employed. The main energy sources of a medium are lactate, pyruvate, and glucose. The concentrations of the first two vary in different media, whereas the latter is necessary mainly for the later stages (morula to blastocyst) of development. A fixed nitrogen source is essential for implantation embryo development whether this is provided by amino acids, albumin, or serum. Suboptimal culture conditions can block development. Pronuclear zygotes of most species (but not human) arrest at some point between the two-cell and the 16-cell stage. Modifying culture conditions can lead the embryos to develop through this block. Hypoxanthine also causes a two-cell block to mouse pronuclear zygotes, and this again depends largely on culture conditions. Simple culture media are bicarbonate-buffered systems with pyruvate, lactate, and glucose. Complex media, such as Ham's F-10, contain in addition amino acids and other elements found in serum. Human tubal fluid simulates the fallopian tube microenvironment. EDTA, gonadotropins, growth factors, and other substances can be included in the media to stimulate development. Coculture of embryos with oviductal cells has shown promising results.

Birth of two infants who were seronegative for human immunodeficiency virus type 1 (HIV-1) after intracytoplasmic injection of sperm from HIV-1-seropositive men.

OBJECTIVE: To report two cases of live births after intracytoplasmic sperm injection (ICSI) in two women who were seronegative for human immunodeficiency virus type 1 (HIV-1) after the use of processed semen from their seropositive husbands. DESIGN: Case reports. SETTING: University hospital IVF center. PATIENT(S): Two HIV-1 seropositive men and their HIV-1 seronegative female partners; all gave their informed consent in writing before undergoing the ICSI procedures. INTERVENTION(S): The men provided semen samples that were processed with the use of Percoll and swim-up techniques. Ovarian stimulation in the women was performed with the long protocol using GnRH analogs and recombinant FSH. ICSI was performed. MAIN OUTCOME MEASURE(S): Oocytes were fertilized by ICSI, and the resulting embryos were transferred to the patients. The mothers and babies were tested for HIV-1 antibodies. RESULT(S): In the first case, seven mature oocytes were collected and fertilized with ICSI, and three embryos were transferred; the woman became pregnant and gave birth to a healthy boy. Six months after the birth, testing for HIV-1 antibodies in the woman and the baby gave negative results. In the second case, 10 mature oocytes were collected and fertilized with ICSI, and four embryos were transferred; the second woman became pregnant and also gave birth to a healthy boy. Testing for HIV-1 antibodies at the baby's delivery also gave negative results. CONCLUSION(S): In women who are infertile because of fallopian tube obstruction or in men who have poor quality semen for artificial insemination, ICSI can be performed using processed semen. This method, which involves the use of only one spermatozoon per oocyte, provides HIV-1 seropositive men with the opportunity to have children with a minimal risk-if any-of infecting their female partners.

Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the relation of oocyte morphology with embryo quality and pregnancy rates (PRs) after intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective study of patients undergoing ICSI. SETTING: University Hospital IVF Center. PATIENT(S): Sixty-eight patients who underwent ICSI and had transfer of good-quality embryos (grade 3), 60 patients with transfer of both good- and poor-quality embryos (grade 3 and grade 2), and 18 patients with transfer of poor-quality embryos (grade 2). INTERVENTION(S): Comparison of the outcome of ICSI in the three groups of patients and the relation of oocyte morphology to embryo quality. MAIN OUTCOME MEASURE(S): Oocyte morphology and embryo quality (grade). Fertilization, cleavage, and pregnancy rates. Serum E2 on the day of hCG administration. RESULT(S): Oocytes with poor morphology (dark cytoplasm; many vacuoles or fragments in cytoplasm) led to poor-quality embryos and consequently to lower PRs (5.5% versus 29.4%). Serum E2 on the day of hCG administration was significantly higher in the group with good-quality embryos compared with that with poor-quality embryos (2,047 +/- 135.7 versus 1,651 +/- 164.8 pg/mL, respectively). CONCLUSION(S): Serum E2 on the day of hCG administration is a marker of embryo quality. Oocyte morphology correlates well with embryo quality and PRs after ICSI.

The effect of the duration of GnRH-agonist down regulation before ovarian stimulation on the biological and clinical outcome after intracytoplasmic sperm injection.

The object of this study was to compare the biological outcome (oocyte maturity, fertilization, cleavage) and the clinical outcome after a 'long' (15-24 days) and a 'long-long' (25-40 days) protocol of GnRH-agonist administration for intracytoplasmic sperm injection. Group A consisted of 51 patients with a 15-24-day down regulation period and Group B consisted of 35 patients with a 25-40-day down regulation period, all of which entered ICSI due to severe male factor infertility. Duration and amount of gonadotropin stimulation, serum E2 on the day of hCG administration, number of oocytes retrieved, oocyte maturity, fertilization rate, cleavage rate and pregnancy outcome were comparable for the two groups of patients. Therefore, a flexible period of pituitary desensitization can be employed, allowing us to simplify planning for patients and for the medical staff without affecting the outcome of the trial.

The effect of compounds altering the cAMP level on reversing the 2-cell block induced by hypoxanthine in mouse embryos in vitro.

The possibility of reversing the hypoxanthine induced 2-cell block in mouse embryos when cultured in conditions supplemented with compounds that increase (FSH, hMG, IBMX, hCG) or inhibit (GnRH-analogue) cAMP was assessed. When embryos were cultured in Ham's F-10 without hypoxanthine supplemented with each of the above compounds, no inhibition of blastocyst development was observed. Embryos were then cultured in Ham's F-10 with hypoxanthine supplemented again with each compound. For the addition of GnRH-analogue or FSH, the rate of blastocyst formation was comparable with that of the control medium with hypoxanthine alone. Instead, the addition of IBMX or hMG reversed the induced block. There was no reversible effect for the addition of 2 micrograms/ml hCG while the latter was observed with higher doses. The results from GnRH-analogue and IBMX addition show that, contrary to what was found for oocytes, stimulation of cAMP reverses the hypoxanthine-induced block in mouse embryos. FSH and hCG also had effects opposite to those observed for oocytes. It is unknown why hMG (FSH + LH) reverses the block. A lower cAMP degradation rate resulting in a higher cAMP level is a possible explanation. Our results provide further evidence that cleavage arrest by hypoxanthine has a different mechanism than the hypoxanthine-induced arrest of meiosis.

Oocyte donation to women over 40 years of age: pregnancy complications.

Recently, oocyte donation to women of advanced age has led to a considerable number of conceptions, thus increasing the age limit for becoming pregnant. A main consideration encountered by physicians, though, is the potential medical and obstetric complications of a pregnancy at an advanced age. In this study, the obstetric complications, as well as the perinatal outcome, of pregnancies of aged recipients (above 40) are presented and compared to those of younger recipients. A significantly higher incidence of gestational diabetes (P < 0.001), an increased incidence of pre-eclampsia (at the 10% level of significance) and an increased risk for thrombophlebitis (again at the 10% level) was observed in the older patients, but a careful follow-up during their pregnancy led to a highly satisfactory obstetric and perinatal outcome. A rigorous precycle medical screening (especially for cardiovascular diseases and diabetes) and a careful follow-up during pregnancy is, therefore, imperative so that oocyte donation to older women is not withheld and continues to provide fertility possibilities to otherwise sterile patients.

Combined GnRH-agonist and HMG therapy in patients with stimulation failure.

This study deals with the combined therapy of GnRH-agonist (GnRH-a) and HMG for stimulation in 15 patients who failed two prior in vitro fertilization attempts. Fifty-three patients who received HMG without GnRH-agonist suppression served as controls. Comparing the HMG group with GnRH-a/HMG cycles, the cancellation rate dropped from 35.5% to 13.2%. Oocyte recovery was similar in both groups, as were the fertilization rates, 88.4% in GnRH-a and 82% in HMG cycles, respectively. The number of embryos available for transfer was virtually identical in both groups (3.7 vs. 3.6). Embryo cleavage speed was higher in GnRH-a than in HMG regimens. The E2 rise was smooth in the GnRH-a group compared to the sharp rise observed in the HMG group. The pregnancy rate per transfer was 30.5% in the GnRH-a group versus 20.5% in the HMG group. GnRH-a seems to offer a clear improvement to a number of stimulation failures.

A comparative study of the effect of ovarian stimulation protocols with different gonadotropin preparations on the biological and clinical parameters of the outcome of intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is widely employed today in cases of severe male factor infertility. This technique requires denuding the oocytes from the surrounding granulosa cells prior to sperm injection. One can thus assess oocyte maturity more accurately and can study the effects of various ovarian stimulation protocols on egg maturation and the rest of the parameters of the outcome of ICSI. The aim of the present study was to compare the outcome of ovarian stimulation using human menopausal gonadotropin (hMG) with that achieved by using highly purified follicle stimulating hormone (pFSH). The biological and clinical parameters of the outcome of ICSI in 99 subfertile couples were studied. Group A consisted of 46 patients to whom hMG was administered and Group B consisted of 53 patients to whom pFSH was employed for ovarian stimulation. The fertilization rate was significantly higher in the pFSH group but all other factors were similar, including the percentage of mature oocytes and pregnancy rate. The latter does not seem to be affected by the gonadotropin preparation employed for ovarian stimulation. This is very helpful for the physician to know since a gonadotropin with a lower cost can be employed and, in addition, shortage of some preparations of gonadotropins occurs frequently.

Preovulatory effects of the progesterone antagonist mifepristone (RU486) in mice.

The progesterone antagonist mifepristone (RU486), was given in mice once on different days of pregnant mare's serum gonadotrophin-human chorionic gonadotrophin (PMSG-HCG) treatment and its action upon the induction of ovulation studied. RU486 administered on the day after PMSG significantly reduced the ovulation rate. Ovulation was completely inhibited when the progesterone antagonist was given simultaneously with HCG, but RU486 administered 4 h after HCG treatment remained ineffective. The development of two-cell zygotes harvested on day 2 post-coitum from mice treated with RU486 on the day after the PMSG treatment was followed in vitro and showed a significant decrease in the number of embryos developing to blastocysts. These results favour the involvement of progesterone in the ovulation process, indicating a direct effect of this hormone at the ovarian level via a progesterone receptor-mediated action.

The in vitro development of mouse embryos beyond the blastocyst stage into the hatching and outgrowth stage using different energy sources.

PURPOSE: The purpose of this study was to investigate the effect of male and female serum supplementation on the in vitro development of mouse embryos beyond the blastocyst stage until the outgrowth stage since the latter may be related to the nidation of the embryo. We also studied the effect of EGF addition on embryo culture and blastocyst outgrowth. METHODS AND RESULTS: The blastocyst and hatching rates of two-cell mouse embryos cultured in Ham's F-10 + BSA, Ham's F-10 + male serum, or Ham's F-10 + female serum were found to be comparable (P > 0.05). The outgrowth rate of hatched blastocysts was significantly increased, though, when they were transferred to 50% male serum compared to either 50% BSA or 50% female serum (P < 0.01 and P < 0.05, respectively). In the last experiment, either 100 or 150 ng/ml EGF was added to the culture medium from the two-cell stage till blastocyst development and the latter were cultured till outgrowth in 50% BSA, male serum, or female serum. For both concentrations of EGF, the outgrowth rate was significantly higher in male serum compared to the other conditions (P < 0.01 and P < 0.05, respectively). The outgrowth rate was also higher when EGF was used compared to plain medium before transferring the blastocysts to either male or female serum (P < 0.01 for both). CONCLUSIONS: We conclude that the development of embryos to the outgrowth stage is significantly enhanced by male serum. The addition of EGF from the two-cell stage also significantly improves the outgrowth success rate for both male and female serum conditions.

Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos.

The gonadotrophins LH and FSH are known to regulate gonadal growth, and differentiation, endocrine function and gametogenesis. The LH receptor is expressed in ovarian theca, granulosa and luteal cells, and in testicular Leydig cells. The FSH receptor is expressed only in ovarian granulosa cells and in testicular Sertoli cells. The expression of the FSH and LH receptors was analysed by RT-PCR to study the role of these receptors in early mouse development. After reverse transcription, strategically designed nested primers were used for amplification from cDNA. Transcripts for the receptors were present in mouse oocytes and preimplantation embryos. The presence of mRNA for FSH and LH receptors in oocytes, zygotes and preimplantation embryos indicates a potential role for the gonadotrophins in the modulation of meiotic resumption and completion of oocyte maturation, as well as a beneficial effect on early embryonic development in mice.

A flexible protocol for the induction of recipient endometrial cycles in an oocyte donation programme.

Synchronization of the availability of good quality oocytes from donors and adequate endometrial maturation of recipients are very important for the success of an oocyte donation programme. A flexible protocol for the endometrial preparation of recipients is important in timing embryo transfer between days 17 and 19 of the cycle ('window of receptivity'). The purpose of this study was to evaluate the effect of the length of oestradiol administration to recipients on pregnancy outcome. Oestrogen administration was 8 mg/day, but its length varied prospectively from 6 to 27 days, followed by the addition of progesterone (100 mg daily i.m.) for 2-4 days according to the availability of good quality oocytes. Pregnancy outcome was evaluated regardless of age, indication for oocyte donation or number of embryos transferred per patient. The pregnancy rate per cycle was comparable when oestradiol was administered from 6 to 11 days before progesterone addition, while it dropped significantly thereafter. The variation in progesterone administration did not affect pregnancy outcome. These findings provide us with a greater flexibility by allowing us to vary oestradiol administration to recipients from 6 to 11 days prior to progesterone, reducing considerably, therefore, the need to cancel embryo transfer because of oocyte unavailability. Thus we can arrange to transfer embryos between days 17 and 19 of the recipient's cycle so as to obtain the best possible clinical outcome.

Results of controlled ovarian stimulation for ART in poor responders according to the short protocol using different gonadotrophins combinations.

INTRODUCTION: Improving pregnancy rates in intricate cases of ovarian stimulation remains a challenge during IVF and intracytoplasmic sperm injection (ICSI). Different protocols of ovulation induction have been proposed. METHODS: The short protocol of ovarian stimulation using recombinant follicle-stimulating hormone (rFSH) with or without the use of luteinizing hormone (LH) in IVF or ICSI outcome in patients with many failed attempts and maternity age > or = 37 years was investigated. The prognostic significance of high but normal values of day 3 serum FSH concentrations was also evaluated. RESULTS: Results show that FSH levels of >9 mIU/ml are associated with poor results even with the use of human menopausal gonadotrophin (HMG). Results were generally comparable when rFSH was used alone or in combination with HMG, except for the quality and the number of embryos transferred, the later being better in the rFSH + HMG group. CONCLUSION: In conclusion intricate cases have good chances for achieving a pregnancy using the short protocol and the outcome is further improved when LH is added from the beginning of ovarian stimulation. A slight elevation of day 3 FSH seems to be a strong prognostic factor for a poor outcome.

Outcome of ovarian response after suppression with a gonadotropin releasing hormone agonist in different chronological periods prior to gonadotropin stimulation for in vitro fertilization.

Gonadotropin-releasing hormone analogues (GnRH-a) are currently used in combination with gonadotropins in ovarian stimulation for in vitro fertilization programs (IVF). The present study aims at evaluating the treatment cycles for IVF for which human menopausal gonadotropin (HMG) was initiated only when pituitary desensitization was confirmed regardless of the time of GnRH-a administration. Two groups of patients were examined. Patients in group A (n = 46) were commenced with HMG treatment on day 15 when E2 level was less than 40 pg/ml. Group B patients (n = 27) comprised the cycle treatments in which E2 levels were greater than 40 pg/ml on day 15. In these cases HMG was first given when E2 levels declined to less than 40 pg/ml. The fertilization rate was similar in both A and B groups, 71.6 and 67.7% respectively. The pregnancy rate per transfer was 27.5 and 34.6% in group A and B respectively. Multiple pregnancies were found at 18.1% in group A versus 44.4% in group B. It is concluded that postponement of HMG administration in patients with high levels of E2 on day 15 after GnRH-a administration seems to offer an improvement in embryo cleavage speed, pregnancy rate and multiple pregnancies.

A preliminary study of the effect of growth hormone on mouse preimplantation embryo development in vitro.

The role of growth hormone (GH) in follicular development, ovulation and embryo development is currently under reconsideration. In this study, we have tried to investigate the effect of GH on preimplantation development of mouse embryos in vitro. Zygotes and two-cell mouse embryos were cultured without (control) or with GH. For zygotes, the addition of 0.2 micrograms/ml of GH resulted in 77 +/- 1% of blastocysts formed and 66 +/- 3% rate of hatching (control 64 +/- 4 and 31 +/- 3%, p < 0.05 and p < 0.01, respectively). For two-cell embryos, the addition of 0.2 micrograms/ml of GH resulted in 87 +/- 2% of blastocysts formed and 60 +/- 4% hatching rate (control 76 +/- 4 and 47 +/- 5%, p < 0.05 for both). This positive effect of GH addition implies that the latter can support mouse preimplantation development in vitro and it suggests, along with its local action on the ovary and its possible effects, via the insulin-like growth factor system, on the tubal and uterine epithelium, a continuous role of this hormone in reproductive physiology from follicular maturation to embryonic development and, possibly, implantation.

Does pre-treatment with micronized progesterone affect the ovarian response to a gonadotropin releasing hormone agonist flare-up protocol?

The purpose of this study was to investigate the ovarian response and the receptivity of the endometrium in women pre-treated with micronized progesterone. Eighty-two normogonodotropic women undergoing in vitro fertilization were studied. Thirty received micronized progesterone 1500 mg/day from day 21 of the cycle for a minimum of 2 weeks, and 52 did not receive micronized progesterone (control group). A gonadotropin releasing hormone agonist (GnRH-a) was administered to all the patients in the follicular phase (flare-up). Twenty-five cycles were cancelled for fertilization failure due to male factor, 12 (40%) in the progesterone group and 13 (25%) in the control group (p = 0.271). There was no difference in the number of oocytes retrieved (7.3 +/- 5 vs. 8.2 +/- 4), fertilization rate (50.8% vs. 65%), clinical pregnancy rate (16.6% vs. 25%) or implantation rate (8% vs. 14%). In the progesterone group cases without fertilization, we performed two biopsies to evaluate the receptivity of the endometrium. Pinopode expression was noted 7 days after oocyte retrieval. It seems that the administration of micronized progesterone in the previous cycle does not affect the ovarian response to the combination of follicular phase GnRH-a and gonadotropins, nor the receptivity of the endometrium.

A preliminary trial of human zygote culture in Ham's F-10 without hypoxanthine.

PURPOSE: This study deals with the suitability of Ham's F-10 without hypoxanthine for early cleavage-stage embryo culture. A high percentage of mouse two-cell embryos developed into morula and blastocysts in Ham's F-10 formulated without hypoxanthine (75.3 and 71.6%, respectively); in contrast, in agreement with previous reports, only 15.4% developed beyond the two-cell stage in Ham's F-10 with hypoxanthine. To begin to evaluate the effect of hypoxanthine on human embryos, a total of 318 human oocytes was fertilized and cultured in Ham's F-10 minus hypoxanthine. RESULTS: The fertilization, cleavage, and pregnancy percentages in two ovulation induction protocols [human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG) and gonadotropin releasing hormone/hMG/hCG] were 82, 88, and 20 and 83, 91, and 30%, respectively. CONCLUSION: These results suggest that, in agreement with mouse embryo development, hypoxanthine does not appear to be necessary for human embryo cleavage and its omission from Ham's F-10 may enhance the conditions for culture of early-cleaving human embryos. These observations may lead to a better understanding of critical cell processes during early human embryonic development.