[Autofluorescence and endometriosis optical trap or new hope in dianosis of endometriosis?].

Laparoscopic autofluorescence imaging of endometriosis is a new method to properly detect the disease. Autofluorescence in contrast to conventional white light laparoscopy enables us to visualize the entire extent of disease and to recognize extremely small or occult lesions. Autofluorescence imaging in fact reveals a world behind the world, a substantial new representation of endometriosis, which will have much impact on our future therapeutic strategies.

Preimplantation genetic diagnosis in Germany - ethical responsibility and law.

Preimplantation diagnosis (PGD) can be judged as early or brought forward prenatal diagnosis and brings up the ethical question in Germany whether potential selection of embryos and intentional killing of a human conceptus, showing a defect not or almost not compatible with life, is reconcilable with human dignity. Whereas prenatal diagnosis and its consequences are socially accepted in Germany, PGD is not compatible with the Embryo Protection Law and is still under debate since >5 years. On the other hand abortion is not subject to prosecution because the legal status of the mother outweighs that of the embryo or fetus. Here the conflict of two legally protected rights - the embryo's worth of protection and the woman's family planning - are accepted and form the basis of the potential decision of the prospective mother to carry the fetus to term or not. An obvious and deep contradiction of assessment is present between the legal requirements for the regulation of abortions in its amendment of 1995 and the Embryo Protection Law which exists unchanged since 1991. The killing of an embryo or fetus in vivo is not subject to prosecution although it is illegal, whereas the discard of an embryo in vitro after PGD was performed, also illegal, is subject to prosecution.

Preoperative reduction of uterine fibroids in only 16 days by administration of a gonadotrophin-releasing hormone antagonist (Cetrotide).

Ten premenopausal women with symptomatic uterine fibroids confirmed by magnetic resonance imaging (MRI) were treated with four injections (s.c.) of 3 mg of the gonadotrophin-releasing hormone (GnRH) antagonist cetrorelix every 4 days, starting on the first day of cycle. On every fourth day, blood samples were drawn for the measurement of gonadotrophins and sex steroids. On the 17th day of treatment after a final MRI control, myomectomy was performed laparotomically, laparoscopically or hysteroscopically. All patients showed a deep and sustained suppression of gonadotrophins and sex steroids over the treatment time. In three patients, no change or even an increase in uterine fibroids volume was observed according to MRI, and in one patient MRI did not allow a reliable interpretation. However, six patients showed a mean reduction of 31% in fibroid size after only 16 days of hormonal treatment. In nine patients laparoscopic or hysteroscopic myomectomy could be performed, while laparotomy was necessary only in one non-responder. Preparation of the cleavage plane during surgery was easy and blood loss was minimal. Patient compliance was excellent. No side-effects occurred. The GnRH antagonist Cetrotide(R), acting as an intermediate depot preparation at a dose of 3 mg, opens up a new avenue for preoperative short term treatment in a subgroup of patients with uterine fibroids, minimizing treatment time and patient discomfort.

Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. DESIGN: Prospective clinical study. SETTING: A university hospital. PATIENT(S): One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. INTERVENTION(S): The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy rates. RESULT(S): In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell-only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2. 9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. CONCLUSION(S): The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved.

A new indication for an intracytoplasmic sperm injection procedure outside the cases of severe male factor infertility.

OBJECTIVE: To show the success of intracytoplasmic sperm injection (ICSI) in cases of a low number of oocytes retrieved. STUDY DESIGN: 715 microinjection cycles, which were performed at our center, were analyzed retrospectively. RESULTS: Within the analyzed cycles, there were 50 cycles with less than four oocytes retrieved. Twelve, 18, and 20 cases were treated with one, two, and three oocytes, respectively. The rate of metaphase II oocytes injected and subsequently fertilized, and intact oocytes was similar in the three groups. The transfer rate was not significantly different from those cases with more than three oocytes (84% vs. 94%). Therefore, the rate of fertilization failure was not higher in the low-number group. The number of embryos transferred was statistically significantly lower in the group with one and two oocytes compared to the group with three oocytes (1, and 1.31 vs. 2.27; P < 0.01) and the group of all patients (2.63; P < 0.01), as well as between the group of less than four oocytes and all patients (1.63 vs. 2.63; P < 0.01). There was no statistically significant difference between the pregnancy rates of the three groups (36.4% vs. 20% vs. 22.2%), and also no difference was found between the group with less than four oocytes and the data of all patients (22% vs. 26.9%). CONCLUSION: In our opinion, these data show that ICSI guarantees a successful treatment even if only as many oocytes are present as embryos are planned to be transferred, i.e. three embryos. This has been accepted to be a new indication in our center. Therefore it should be possible to stimulate patients mildly, with e.g. clomiphene citrate, to avoid unnecessary hyperstimulation syndromes, and to lower the cost of hormonal superstimulation.

ICSI outcome in patients of 40 years age and over: a retrospective analysis.

OBJECTIVE: To report and analyse our experience with ICSI treatment in infertile women >/=40 years of age, with the intention of contributing to current debates on the effect of aging on the reproductive potential. STUDY DESIGN: 107 infertile couples in which the female partner was aged >/=40 years and who received ICSI treatment between January 1996 and December 1998. RESULTS: A total of 107 women underwent 171 treatment cycles during this period. Of 171 cycles initiated 33 were cancelled (cancellation rate=19.3%). In this way, 17 women did not have embryo transfer at all, while 90 patients had 138 cycles with oocyte retrieval and successful embryo transfer, with a mean number of embryos per transfer 2.36. Sixteen pregnancies occurred and eight of them ended in spontaneous abortion. The implantation rate was 4.9%, the pregnancy rate per initiated cycle was 9.35% and per transfer cycle 11.59%. The miscarriage rate was 50%. Moreover, 12 patients had supernumerary embryos, that were cryopreserved and transferred in 17 thawing cycles and resulted in two pregnancies ending in abortion. All pregnancies occurred when three embryos were available, except in two cases with two available embryos. The great majority of the total pregnancies (16 of 18) resulting in women aged between 40 and 42 years. CONCLUSION: Our data show that women 40 and older with existing ovarian function may benefit from ICSI treatment, even when the indication for treatment is male factor infertility. Supernumerary embryos, that are cryopreserved and transferred in subsequent cycles can improve the overall pregnancy rates per oocyte retrieval, although these women should be aware of the very high risk of miscarriage.

Preserved pituitary response under ovarian stimulation with HMG and GnRH antagonists (Cetrorelix) in women with tubal infertility.

OBJECTIVE: To examine the pituitary response in patients undergoing short-term application of the GnRH antagonist Cetrorelix in the mid-cycle phase for hypophysial suppression of premature LH surges within an IVF-program. DESIGN: Twenty patients suffering from primary or secondary tubal infertility were stimulated with hMG from cycle day 2. From day 7 till ovulation induction Cetrorelix was administered in two different dose regimens (15 patients 3 mg s.c. daily; 5 patients 1 mg s.c. daily). Three hours before ovulation induction a GnRH test was performed using 25 micrograms of native GnRH and the pituitary response examined by measurement of the serum LH concentration after 30 min. RESULTS: Premature LH surges could be avoided in the 3-mg group and in the 1-mg group, respectively. Due to this, none of the cycles had to be cancelled. Oestradiol profiles and ultrasound demonstrated a satisfactory follicular maturation. All patients showed pronounced suppression of the serum LH levels before ovulation induction. The mean increase of serum LH due to the performed GnRH test was 10 mIU/ml for the 3-mg group, while the average maximum in the 1-mg group was about 32.5 mIU/ml. CONCLUSIONS: The pituitary response is preserved by the treatment with the GnRH antagonist Cetrorelix. The extent of suppression of the adenohypophysis, as expressed by the different reactions on GnRH test, can be modulated by the dosage administered. This should allow ovulation induction by GnRH or one of its agonists instead of hCG, which could be beneficial in patients at high risk of Ovarian Hyperstimulation Syndrome (OHSS) and those suffering from Polycystic Ovary Disease (PCOD).

Outcome of ICSI cycles using frozen-thawed surgically obtained spermatozoa in poor responders to ovarian stimulation: cancellation or proceeding to ICSI?

OBJECTIVE: To investigate the ICSI outcome of the patients who produced four follicles or less after ovarian stimulation using frozen-thawed surgically obtained spermatozoa. STUDY DESIGN: The patient cohort of this study was composed of a carefully selected group of 20 men suffering from obstructive and non-obstructive azoospermia and in whom spermatozoa had been seen in their harvested epididymal aspirates and testicular tissues and the cryopreserved specimens had been used for subsequent ICSI cycles. This group of men represent those in whom the female partners produced only four follicles or less after ovarian stimulation. RESULTS: For the cases of obstructive azoospermia with MESA (n=6) a total of nine cycles was carried out. Four couples went through one cycle, one couple underwent two cycles, one couple accomplished three cycles. Out of the nine cycles, seven went to embryo transfer. No pregnancy occurred in the MESA-ICSI group of patients. The mean+/-S.D. number of oocytes per cycle was 2.556+/-1.236, the mean+/-S.D. number of embryos per transfer was 1.444+/-1.014. Two couples did not have an embryo replacement because of absence of fertilisation. The mean+/-S. D. number of gonadotropin ampoules was 53.88+/-37.30 and the mean+/-S.D. duration of ovarian stimulation was 13.38+/-4.534 days. For the cases of non-obstructive azoospermia with TESE (n=14) a total of 16 cycles was carried out. Thirteen couples went through one cycle, one couple accomplished three cycles. Out of the 16 cycles, 11 cycles went to embryo transfer. One pregnancy occurred in the TESE-ICSI group of patients, which produced live offspring. The mean+/-S.D. number of oocytes per cycle was 3.00+/-1.211, the mean+/-S.D. number of embryos per transfer was 1.313+/-1.195. Five couples did not have an embryo replacement, four of them because of absence of fertilisation and one because of abnormal fertilisation (3 PN). The mean+/-S.D. number of gonadotropin ampoules was 81. 77+/-53.40 and the mean+/-S.D. duration of ovarian stimulation was 16.71+/-3.667 days. CONCLUSION: In our opinion, these data show that it is possible to achieve satisfactory fertilisation rates using frozen-thawed epididymal and testicular spermatozoa obtained from men with obstructive or non-obstructive azoospermia, but the limiting factor in reaching the stage of embryo transfer is the poor ovarian response to stimulation induction. It therefore seems preferable to cancel these cycles, in the hope that a better response might be obtained in a subsequent cycle, avoiding in this way financial and emotional implications.

Gonadotropin-releasing hormone antagonist protocol: a novel method of ovarian stimulation in poor responders.

OBJECTIVE: To estimate the efficacy of gonadotropin-releasing hormone (GnRH) antagonist 'Cetrorelix' in poor responders comparing with the standard long protocol. DESIGN: The study population consisted of 21 poor responders who underwent ICSI and treated with Cetrorelix according to the multiple-dose protocol and who were compared with 21 poor responders treated according to the long protocol and who also underwent ICSI. Patients in both groups were matched for chronological age, the number of follicles found by ultrasound at the retrieval day and cause of infertility. Fifteen patients of GnRH antagonist group were treated with the combination of GnRH antagonist with clomiphene citrate (CC) plus gonadotropins, while six patients were treated with the combination of GnRH antagonist plus gonadotropins, but without CC. RESULTS: The use of GnRH antagonist in a multiple dose protocol gave a pregnancy rate of 14.28% which was in the range expected for patient with poor response, but with shorter treatment duration and with fewer ampoules of gonadotropins as compared with the use of a GnRH agonist protocol in a depot formulation. Within Cetrorelix group patients who received CC had a significant shorter duration of stimulation and needed fewer ampoules as compared with patients in the same group who did not receive CC. CONCLUSIONS: A GnRH antagonist multiple dose protocol may be the protocol of choice for the treatment of poor responders. The use of GnRH antagonist Cetrorelix ended with significantly less ampoules of gonadotropins and a shorter duration of stimulation.

The use of cryopreserved mature and immature spermatozoa in assisted reproduction.

Treatment of severe male subfertility has become available since the intracytoplasmic injection of a single sperm into an oocyte was successfully applied for the first time in 1992. Moreover, with the use of fresh and cryopreserved epididymal and testicular spermatozoa for this procedure, fertilization and pregnancies could be accomplished. This review addresses the development and performance of these techniques and discusses achievements and problems as well as future aspects of the feasibility of early spermatid injection. Furthermore, limitations of these procedures and concerns with regard to genetic and epigenetic risks of using immature gametes are discussed.

Comparison of poor responders with good responders using intentionally frozen-thawed epididymal spermatozoa in subsequent ICSI cycles.

PURPOSE: To compare the outcome of intracytoplasmic sperm injection (ICSI) cycles performed with frozen-thawed epididymal spermatozoa between patients who respond poorly and patients who respond well to ovarian stimulation. METHODS: 17 patients suffering from obstructive azoospermia underwent microsurgical retrieval of epididymal spermatozoa (MESA) and the spermatozoa were frozen. The frozen-thawed spermatozoa were used in subsequent ICSI cycles. In six patients, the female partners responded poorly to ovarian stimulation. They accomplished nine ICSI cycles. In 11 patients, the female partners responded well to ovarian stimulation and they accomplished 16 cycles. RESULTS: Poor responders were older than those who reponded well. The mean number of metaphase II oocytes collected was lower in the poor responder group. In the poor responders, two couples failed to fertilise the oocytes in two ICSI cycles. In the good responders, one couple failed to fertilise the oocytes in an ICSI cycle. There were no significant differences in fertilization rates between the two groups. The estradiol concentrations on the day of hCG administration were significantly higher in the good responders. There was no pregnancy in the poor responder group, while three patients who responded well conceived. Eight good responders had 34 supernumerary 2PN oocytes which were cryopreserved. CONCLUSION: Frozen-thawed epididymal spermatozoa from men with obstructive azoospermia are potent to achieve satisfactory fertilization rates. Poor ovarian response to stimulation induction appears to be the main limiting factor in reaching the stage of embryo transfer. It is preferable in older women to cancel cycles with poor response in the hope that a better response might be obtained in a subsequent cycle. Thus, the frozen-thawed epididymal sperm can be preserved and the most stressful and expensive phase of IVF-ICSI treatment can be avoided.

[Limitations of reproduction medicine in male subfertility. Treatment of severe spermatogenesis disorders]. / Grenzen der Reproduktionsmedizin bei männlicher Subfertilität. Die Behandlung der schweren Spermatogenesestörung.

Treatment of severe male subfertility has become available since the intracytoplasmic injection of a single sperm into an oocyte was successfully applied for the first time in 1992. Moreover, also with the use of testicular spermatozoa for this procedure fertilization and pregnancies could be accomplished. This review addresses the development of these techniques and discusses achievements and problems as well as future aspects of the feasibility of early spermatid injection are stressed. Furthermore it includes the basic elements of spermatogenesis and the major concerns regarding the underlying genetic reasons for spermatogenic failure.

[GnRH antagonists in gynecology: initial results within the scope of controlled ovarian hyperstimulation]. / Die GnRH-Antagonisten in der Gynäkologie: Erste Ergebnisse im Rahmen der kontrollierten ovariellen Hyperstimulation (COH).

OBJECTIVE: Applicability of the GnRH-antagonist Cetrorelix within controlled ovarian hyperstimulation (COH) to avoid the premature LH-surge should be examined. METHODS: 35 patients suffering from tubal infertility were stimulated for In Vitro Fertilization (IVF) by human menopausal gonadotrophins (HMG) and concomitant administration of Cetrorelix in different dosages (3 mg, 1 mg, 0,5 mg). RESULTS: No premature LH-surge could be observed. CONCLUSIONS: Short term administration of the GnGR-antagonists avoids the occurrence of a premature LH-surge.

[Morphology in intracytoplasmic sperm injection]. / Morphologie bei der intrazytoplasmatischen Spermieninjektion.

OBJECTIVE: Was to determine if severe morphological defects of spermatozoa in Oligo-Astheno-Teratozoospermia (OAT) have any impact on the fertilization process in intracytoplasmic sperm injection (ICSI) and, if there are any specific morphological characteristics of unfertilized oocytes after the ICSI procedure. METHODS: Ejaculates of males with severe subfertility and unfertilized oocytes after the ICSI procedure were investigated by transmissionelectronmicroscopy. RESULTS: ICSI provided a fertilization rate of 66% in cases of severe defective spermatozoa. The ultrastructure revealed severe head and neck alterations in particular. The ultrastructure of the unfertilized oocyte displayed mostly no signs of activation and lack of decondensation of sperm chromatin. CONCLUSIONS: Our findings support the hypothesis that not severe sperm defects but the lack of intracytoplasmic sperm-oocyte interaction displays the most critical role in the fertilization process in intracytoplasmic sperm injection.

Pregnancy outcome after assisted fertilization--a short survey.

Since 1992, intracytoplasmic sperm injection has been established as the treatment of choice for severe male subfertility. Worldwide results with regard to fertilization, pregnancy and pregnancy outcome, collected by the EHSRE Task Force on ICSI, are reported in this short survey. Aspects of genetic inheritance that are of major concern are addressed briefly.

Ultrastructure of gametes and intracytoplasmic sperm injection: the significance of sperm morphology.

The aim of this study was to determine characteristic malformations of sperm ultrastructure in patients with severe subfertility undergoing intracytoplasmic sperm injection (ICSI). Although light microscopy (LM) can reveal major abnormalities of the three parts of the spermatozoon (head, mid-piece and flagellum), the various cell organelles of the spermatozoon and their fine structure remain unevaluated by LM. Insight into the submicroscopic organization of the spermatozoon and its complex organellar system may contribute to a better understanding of the preconditions for success or failure of fertilization. An in-depth evaluation of semen quality by transmission electron microscopy (TEM) can improve the diagnosis of male subfertility and can give substantial information about the fertilizing competence of spermatozoa. Thus, in this study 56 ejaculated sperm samples from patients with severe male subfertility or previous failed attempts at in-vitro fertilization were assessed by LM and TEM prior to ICSI to evaluate the most important sperm defects causing extreme subfertility. LM analysis was performed according to World Health Organization criteria. It could be confirmed that severe head defects are mostly involved in long-term infertility and fertilizing failure in classical IVF treatments. The most frequent head defects are disorders of the nuclear membranes and the acrosomal cap and disorganization of the chromatin structure. These defects of sperm fine structure seem to be associated with dysfunctional sperm-oocyte recognition, binding and fusion with the oolemma. Chromatin alterations and signs of decondensation or karyolysis are frequently associated with a deterioration of the nuclear membranes and may be due to impaired spermiogenesis. However, our results and the success of ICSI proved that severe sperm defects have no predictive value and do not impair the fertilization process, and also that the maturity of spermatozoa does not play an important role. Fine structure analysis revealed the pleiomorphology and heterogeneity of human spermatozoa.

Assisted fertilization--treatment of severe male subfertility.

Since 1992 direct injection of a single spermatozoon into the cytoplasm of the oocyte (ICSI) has developed as the treatment of choice in cases of severe male subfertility. The ICSI procedure displays high fertilization and pregnancy rates in cases of extreme oligoasthenoteratozoospermia. In combination with sperm aspiration from the epididymis or sperm retrieval by testicular sperm extraction, fertilization is possible even in cases of azoospermia. To date, a couple of thousand children have been born after ICSI. Despite the criticism about the lack of natural selection while breaking the zona pellucida and the cytoplasmic membrane with the injection pipette, there has been no increased incidence of malformations and chromosomal abnormalities.

New approaches to male infertility: IVF and microinjection.

The male factor represents a major problem in the treatment of infertility. New methods of assisted fertilization such as partial zona dissection (PZD), subzonal sperm injection (SUZI) and intracytoplasmic sperm injection (ICSI) are described and discussed in terms of their potential benefits for infertility treatment in comparison to classical in-vitro fertilization (IVF) and intra-uterine insemination (IUI).

Paracrine changes in the peritoneal environment of women with endometriosis.

During the past decade, macrophage-derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In particular, growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis. In this study, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and intercellular adhesion molecule 1 (ICAM-1), substances recently detected in the peritoneal fluid of women with endometriosis, were assessed with regard to their concentrations in different stages of endometriosis and changes of the peritoneal paracrine activity after medical treatment with a gonadotrophin releasing hormone agonist (GnRHa). Peritoneal fluid was obtained from patients with endometriosis during laparoscopy before and after a 4-month treatment with a GnRHa. VEGF, TGF-beta and ICAM-1 could be detected in all women presenting with various stages of active endometriosis. After GnRHa therapy, all patients showed significant decreases in mean concentrations of VEGF (194+/-77 pg/ml), TGF-beta (902+/-273 pg/ml) and ICAM-1 (157+/-52 ng/ml). Patients with stage III and IV endometriosis (according to the rAFS score) had much higher concentrations of VEGF and TGF-beta before treatment compared with those patients with mild endometriosis (rAFS stages I and II). The most striking decrease in concentration was for TGF-beta, from 902 pg/ml before to 273 pg/ml after therapy. These results indicate an important role for paracrine activity in the establishment and maintenance of endometriosis. Indeed, treatment with a GnRHa may reduce paracrine activity in the peritoneal cavity via hypo-oestrogenism and provide proof of successful therapy.

Principles of mammalian fertilization.

The principles of mammalian fertilization are defined in relation to studies of the intracytoplasmic injection of spermatozoa into human oocytes (ICSI). Sections are included on the production of mature oocytes and mature spermatozoa, sperm-oocyte interaction, and formation of the pronucleate oocyte. Current concepts in gametogenesis are discussed, including spermiogenesis, oogenesis, the biology and penetration of the zona pellucida including capacitation and the acrosome reaction, gamete fusion, the block to polyspermy and growth of the pronucleus. A concluding section relates the normal events of fertilization to those occurring after ICSI and discusses various types of anomalies found in the oocyte after ICSI.