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.

The effect of CRH and its inhibitor, antalarmin, on in vitro growth of preantral mouse follicles, early embryo development, and steroidogenesis.

In vitro growth systems of preantral follicles allow studying the effect of various endocrine, paracrine, and autocrine factors on follicular growth and oocyte maturation. CRH is a 41-amino-acid neuropeptide responsible for endocrine, autonomic, immunological, and behavioral responses of mammals to stress and has two receptors, CRH receptor type 1 (CRH-R1) and CRH-R2. Antalarmin, a CRH-R1 antagonist, has been used to elucidate the role of CRH in stress, inflammation, and reproduction. The present study describes in vitro growth of mouse preantral follicles, early embryo development, and steroidogenesis in the presence of CRH and its antagonist antalarmin. We cultured 732 follicles in control media, 1306 in CRH 10(-7) mol/liter, and 1202 in CRH 10(-7) plus antalarmin 10(-6) mol/liter. The culture medium was assayed on alternate days for 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin. Total RNA was extracted from preantral follicles as well as early preimplantation embryos and was assessed by real-time RT-PCR for the expression of CRH-R1 and CRH-R2 mRNAs. Hormone analysis showed that the CRH group had lower levels of 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin as the culture progressed, in comparison with the other two groups. RT-PCR demonstrated the presence of CRH-R1 and CRH-R2 in all stages of preantral follicle culture. Morula/blastocyst-stage embryos expressed only CRH-R1. In conclusion, CRH has an inhibitory effect on in vitro fertilized oocytes, resulting from cultured preantral follicles at all stages of preimplantation embryo development. Furthermore, the presence of CRH in the culture medium inhibits steroidogenesis by preantral mouse follicles cultured in vitro.

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.

Corifollitropin alfa: a novel long-acting recombinant follicle-stimulating hormone agonist for controlled ovarian stimulation.

The advent of recombinant technology has made the production of modified proteins with desired properties possible. Corifollitropin alfa is a successful example of the first available long-acting, follicle stimulating hormone. Corifollitropin alfa has prolonged half-life and a slower absorption rate, but has the same receptor-binding and biological activity as recombinant FSH (rFSH). Its application is associated with the arrival of a novel simplified approach for controlled ovarian stimulation in IVF patients. Different studies have proven the efficiency of a single corifollitropin alfa dose to initiate and sustain multiple follicular development in a gonadotropin-releasing hormone antagonist protocol. Finally, corifollitropin alfa is well tolerated and is not correlated with serious adverse events, except for the slightly higher incidence of ovarian hyperstimulation syndrome compared with traditional management with recombinant FSH.

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.

Pharmacogenetics in ovarian stimulation--current concepts.

Ovarian response to follicle-stimulating hormone (FSH) action differs considerably among women; this has prompted researchers to determine which factors modify this response. The challenge is to identify the genes that affect the response to FSH stimulation by the application of pharmacogenetics to assisted reproduction techniques (ARTs). Recently, new insights have been gained in the investigation of the variability in the gene that encodes the FSH receptor (FSHR) gene or genes of the estrogen pathway. Several polymorphisms of the FSHR gene have been discovered, but Ser680Asn and Thr307Ala are the two most studied. The Ser680Asn polymorphism of the FSHR gene has been found to influence the ovarian response to FSH stimulation in women undergoing in vitro fertilization (IVF), and in women with the genotype Ser/Ser, the FSHR appears to be more resistant to FSH action. The clinical implications of this finding are highly important; the ultimate goal is to apply genetic markers as routine diagnostic tests before ovarian stimulation to predict ovarian response, determine the required FSH dose, and avoid the possible complications related to FSH stimulation.

Different ovarian stimulation protocols for women with diminished ovarian reserve.

PURPOSE: To review the available treatments for women with significantly diminished ovarian reserve and assess the efficacy of different ovarian stimulation protocols. METHODS: Literature research performed among studies that have been published in the Pubmed, in the Scopus Search Machine and in Cohrane database of systematic reviews. RESULTS: A lack of clear, uniform definition of the poor responders and a lack of large-scale randomized studies make data interpretation very difficult for precise conclusions. Optimistic data have been presented by the use of high doses of gonadotropins, flare up Gn RH-a protocol (standard or microdose), stop protocols, luteal onset of Gn RH-a and the short protocol. Natural cycle or a modified natural cycle seems to be an appropriate strategy. Low dose hCG in the first days of ovarian stimulation has promising results. Molecular biology tools (mutations, single nucleotide polymorphisms (SNPs)) have been also considered to assist the management of this group of patients. CONCLUSIONS: The ideal stimulation for these patients with diminished ovarian reserve remains a great challenge for the clinician, within the limits of our pharmaceutical quiver.

Alternate day and daily administration of GnRH antagonist may prevent premature luteinization to a similar extent during FSH treatment.

BACKGROUND: This randomized controlled trial was designed to evaluate whether a GnRH antagonist given every other day could prevent premature luteinization in women undergoing IVF/ICSI treatment. METHODS: A total of 73 women receiving ovulation stimulation IVF cycles with recombinant FSH were allocated randomly on cycle day 7 to GnRH antagonist ganirelix in multiple doses (0.25 mg each), either daily (n = 37 women, group 1) or every other day (n = 36 women, group 2) until the day of HCG administration. RESULTS: Serum FSH, LH, estradiol and progesterone values showed similar trends in the two groups. During FSH stimulation, 13 (35%) of the women in group 1 had premature LH rises (> or = 10 IU/l) of which eight (22%) were after the start of antagonist administration. In group 2 there were 14 (39%) LH rises during FSH stimulation of which 10 (28%) were after the start of antagonist administration. Luteinization (serum progesterone >2 ng/ml) occurred in only one woman in each group overall (3%). A significantly smaller total dose of the antagonist was used in group 2 than in group 1 (P < 0.001). The study did not have power to evaluate differences in total dose of FSH, number of oocytes recovered and clinical pregnancy rate, all of which appeared similar in the two groups. CONCLUSIONS: Whether alternate day is as effective as daily administration of ganirelix in preventing premature luteinization should be addressed in a non-inferiority trial powered to evaluate live birth rate.

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.

Comparison between "short" and "long" protocols in an ICSI programme.

In the present retrospective study we compare the efficacy of gonadotropin-releasing hormone (GnRH) agonist in a long protocol and a GnRH agonist in a short protocol administration for controlled ovarian hyperstimulation (COH) in an ICSI program. A total of 424 consecutive patients with a history of male factor were included in the present study. Three hundred and three patients were included in the long protocol and 121 in the short protocol. Patients treated with the short protocol were stimulated in a shorter time and achieved lower estradiol levels. A significantly higher percentage of oocytes transferred were found in the long protocol. The clinical pregnancy rate per embryo transfer was 39.3% in the long protocol and 19.2% in the short protocol (p=0.001).

A double embryo transfer on days 2 and 4 or 5 improves pregnancy outcome in patients with good embryos but repeated failures in IVF or ICSI.

The purpose of this paper was to evaluate the outcome of a double embryo transfer during the same cycle for patients who had had three or more implantation failures in IVF-ET or ICSI-ET programs after the transfer of good quality embryos in all attempts. Forty-five women who had had previous unsuccessful attempts in IVF-ET or ICSI-ET programs after transfer of good quality embryos (Group A) were included in the study. Group A was divided into two subgroups, Group A1 consisted of 34 patients who underwent embryo transfer on day 2 and day 4 after pick-up and Group A2 consisted of ten patients who underwent embryo transfer on day 2 and day 5 after pick-up. Forty-two other women with a similar unsuccessful history in IVF-ET (Group B) were studied as controls. The patients in this group had a day 4 or 5 only transfer without having an additional day 2 transfer. The outcome of the procedure was compared in the two groups. Double embryo transfer had beneficial effects on patients with good embryos but with previous failure attempts. These patients had a 38.2% clinical pregnancy rate and a 50% total pregnancy rate if the additional embryo transfer was done on day 4 and a 60% clinical and 60% total pregnancy rate if the additional embryo transfer was done on day 5. Our data showed that excellent pregnancy rates can be obtained with a commercially available medium and double embryo transfers on days 2 and 4 or 5 after pick-up for patients with good quality embryos that have had previous failure attempts in an IVF-ET program. Due to the fact that endometrial maturation varies considerably in each patient, an adequate endometrial maturation and improved uterine receptivity seem to be the reason for improved pregnancy rates with double embryo transfers. It was also shown that morullae have high viability and high potential for implantation and pregnancy.

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.

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 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.

Selective feticide of an affected fetus in the second trimester has comparable risks to those in the first trimester.

The broad acceptance of prenatal diagnosis of various genetic diseases leads to an ever-increasing number of parturients with twin gestations undergoing selective feticide of an affected fetus. In most of the cases, delayed diagnosis leads to second trimester reduction. The aim of the present study was to investigate whether this procedure can be performed in the second trimester with results comparable to those obtained when it is performed in the first trimester. There was a 5.6% miscarriage rate in the group reduced in the first trimester (n=18, Group A) and an 8.3% miscarriage rate in the group reduced in the second trimester (n=48, Group B). The mean weight of neonates in the first group was 2780 g, and in the second group 2620 g. The mean gestational age at delivery was 36.7 weeks for Group A and 35.1 weeks for Group B. No significant differences were observed for any two-paired values considered. There was no perinatal mortality in either group. We therefore conclude that selective feticide of an affected fetus is as safe in the second trimester as it is in the first.

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.

Adhesions: laparoscopic surgery versus laparotomy.

This study was undertaken to assess the effectiveness in pregnancy rates of microsurgery and operative laparoscopy in adhesiolysis. Adhesions were found to be the sole infertility factor in 15% of our patients. One hundred and ninety infertile patients with periadnexal adhesions as the only cause of their infertility were treated by microsurgery (86) or operative laparoscopy (104) and were followed up for 24 months. Our results indicate that advanced laparoscopic surgery in general is as effective as microsurgery in healthy infertile patients with adhesions but offers some advantages in comparison to laparotomy. Factors that adversely affect the postoperative success rates are the age of the women, the duration of infertility, and the severity of the adhesions.

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.

The adhesion molecule CEACAM1 (CD66a, C-CAM, BGP) is specifically expressed by the extravillous intermediate trophoblast.

CEACAM1 (CD66a, C-CAM, BGP) is an adhesion molecule of the carcinoembryonic antigen family which has been shown to be normally expressed at the apical pole of epithelial cells, including the apical pole of endometrial surface and glandular epithelia. The purpose of the present study was to investigate its expression pattern at the maternal-fetal interface, and thus to determine whether CEACAM1 could be implicated in the human implantation process. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody (mAb) as well as flow cytometry and Western blot on isolated trophoblast populations. On the maternal side of the maternal-fetal interface, CEACAM1 was present in epithelial cells of pregnancy endometrium as well as in small endometrial vessels, whereas it was absent from decidual cells. On the fetal side, CEACAM1 was strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture, as shown by flow cytometry and Western blot. Expression was also observed in placental villous core vessels but was absent from both villous cyto- and syncytiotrophoblasts throughout the pregnancy. We conclude that, given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial and/or trophoblast/endothelial interactions during the trophoblastic invasion of the endometrium.