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.

ESR1, ESR2 and FSH receptor gene polymorphisms in combination: a useful genetic tool for the prediction of poor responders.

PURPOSE: Previous studies in humans concluded that a multigenic model including specific FSHR, ESR1 and ESR2 genotype patterns may partially explain the poor response to FSH. The aim of our study is to analyse three different loci -polymorphisms in ESR1 Pvu II, ESR2 Rsa I and Ser680Asn FSH receptor gene- in a Greek population and their involvement in stimulation outcome and pregnancy rates. METHODS: Each locus was studied alone, and in combination with the others. We performed both restriction fragment length polymorphism analysis and real-time polymerase chain reaction. A total of 109 normally ovulating female patients underwent IVF or ICSI. RESULTS: Studying each locus alone, no significant results were drawn for ESR1 and ESR2 genes. Concerning the FSHR polymorphism, the women carrying the AA variant presented higher total amount of gonadotrophins used (P=0,048) and tended to have higher number of stimulation days (P=0,057). Considering the ESR1 and FSHR gene polymorphisms in combination, the TC/SA combination presents the highest number of pregnancies in poor responders group (3/4 pregnancies carried this genotype), in good responders group (4/12 pregnancies carried this genotype) and in the total population (10/26 pregnancies carried this genotype). Except the CC/AA combination, all other genotype combinations presented incidence of pregnancy, with TC/SA having the highest incidence. The CC/AA genotype presents the worst profile of ovulation induction, confirming a poor responder profile: the total amount of gonadotrophins used was highest in CC/AA group (P < 0,05). The peak E2, the number of follicles and of retrieved oocytes and the pregnancy rate were significantly lower (P < 0,05). This genotype combination seems to be over-presented in the poor responders group in a statistically significant way (P=0,038). Women with CC/AA combination have 1,5-2,4 times more risk to be poor responders in comparison with women that do not carry that combination. CONCLUSION: This study supports the hypothesis that a multigenic model, including the well studied ESR1 and FSHR genes is involved in the controlled ovarian stimulation outcome indicating that the CC/AA genotype presents the worst ovulation induction profile, while the TC/SA genotype presents the higher number of pregnancies in our population.

EGF and IGF-I as predictors of ICSI outcome in human preimplantation embryo cultures.

PURPOSE OF INVESTIGATION: Detection of EGF and IGF-I in human embryo cultures and their effect on ICSI outcome. METHODS: Collection of culture medium from embryos of 50 women under ICSI program. EGF and IGF-I were measured via enzyme immunoassay. RESULTS: ICSI outcome was independent of age, infertility years, FSH, LH, prolactine and E2. EGF detection was higher in 48- (32%), than in 72-hour embryos (14%) (p < 0.001). EGF negative embryos are likely to be arrested at the morula stage (p < 0.001) and are associated with poor pregnancy rates (p < 0.05). IGF-I was undetected in 48-hour embryos. CONCLUSIONS: For the first time human embryos were surveyed from fertilization until embryo transfer, regarding EGF and IGF-I production. IGF-I is not a predictor of ICSI outcome. EGF is present in one-third of human embryo cultures at 48 hours, but this ratio wanes at the morula stage. EGF negative embryos are associated with lower pregnancy rates.

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.

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.

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.

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 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.

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.

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.

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.