B. Schöpper

Comparison of cryopreservation outcome with gonadotropin-releasing hormone agonists or antagonists in the collecting cycle

OBJECTIVE To compare the pregnancy rates of frozen-thawed 2-pronucleate (2PN) oocytes obtained either in a long protocol or in an antagonist protocol and ovarian stimulation with either human menopausal gonadotropin (hMG) or recombinant follicular stimulating hormone (recFSH). DESIGN Retrospective data analysis. SETTING Academic infertility center. PATIENT(S) Three hundred forty-two infertile couples who underwent a transfer of cryopreserved 2PN oocytes. INTERVENTION(S) hMG (n = 194) or recFSH (n = 92) in a long protocol or hMG (n = 16) or recFSH (n = 40) stimulation under pituitary suppression with the GnRH antagonist Cetrotide was used. The 2PN oocytes were transferred after endometrial preparation using E(2) valerate and vaginal progesterone (Crinone 8% vaginal gel). MAIN OUTCOME MEASURE(S) Implantation, pregnancy, and abortion rates. RESULT(S) Implantation rates in the freeze-thaw cycles were 5.6% (hMG) and 3.8% (recFSH) with 2PN oocytes from the long protocol and 7% from the antagonist cycles, irrespective of whether hMG or recFSH was used. Pregnancy rates were similar independent of whether they resulted from the long-protocol cycles with hMG (15.4%) and recFSH (13.1%) or from the antagonist protocol cycles with hMG (25.0%) and recFSH (17.5%). CONCLUSION(S) The potential to implant is independent of the gonadotropin-releasing hormone analogue and gonadotropin chosen for the collection cycle when previously cryopreserved 2PN oocytes were replaced after thawing in the cleavage stage.

Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients.

The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4-10% in 38% of the cases, and > or = 10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates <60% (P < 0.05). However, such correlations were not found in cases with fertilization rates >60%. There were no live deliveries in patients with high DFI levels (>10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates.

Prospective, randomized study to evaluate the success rates using hCG, vaginal progesterone or a combination of both for luteal phase support.

Background. A prospective study was done to compare the efficacy of luteal phase support (LPS) using either three times hCG (group I, n=77), hCG on the day of embryo transfer (ET) in combination with daily vaginal progesterone (group II, n=62) or vaginal progesterone only (group III, n=70).

In-vitro-Maturation

ZusammenfassungMit Hilfe der Fähigkeit, Oozyten in vitro zu reifen, ließen sich neue Erkenntnisse über Follikulogenese und Oogenese gewinnen. Darüber hinaus eröffnet diese Technik eine alternative Perspektive für die Eizellgewinnung bei infertilen Frauen. In-vitro-Wachstum (IVG) und In-vitro-Maturation (IVM) stellen im humanen System aufgrund der langen Dauer der Follikulogenese mit komplexen zellulären Veränderungen der Eizellen und ihren umgebenden Zellen eine Herausforderung dar. Im Mausmodell wurden bereits Lebendgeburten nach Reifung und Fertilisation in vitro publiziert. Besonders im humanen System befinden sich die Verfahren noch im Anfangsstadium ihrer Entwicklung. Wenige Lebendgeburten wurden nach IVM unreifer Oozyten, die aus kleinen antralen Follikeln aspiriert wurden, berichtet. Darüber hinaus kann das Wachstum primordialer Follikel in Ovarialbiopsaten initiiert, deren Kultur bis zu präantralen Stadien unterhalten und die Antrumformation isolierter präantraler Follikel unterstützt werden. Die vollständige Substitution der Reifungsprozesse in vitro vom präantralen Follikel zu präovulatorischen Stadien ist heute noch nicht möglich. Dennoch zeichnet sich ab, dass die IVM die assistierte Reproduktion revolutionieren kann.AbstractThe ability to rescue oocytes and mature them in vitro would provide invaluable information about folliculogenesis and oocyte maturation and could provide oocytes for infertile women. In vitro growth (IVG) of follicles and in vitro maturation (IVM) of oocytes are challenging especially in the human because folliculogenesis is a lengthy process with many complex cellular changes in the oocyte and its surrounding follicle cells. Reports have been published on live births in mice after maturation and fertilization. This technique is still in its infancy especially for use in humans. A few live births have resulted from IVM of immature human oocytes aspirated from small antral follicles. Furthermore, it is possible to grow primordial follicles to preantral stages in slices of ovarian tissue and support antrum formation in isolated preantral follicles. Today we are still a long way from growing and maturing preantral follicles to preovulatory stages in vitro, but these techniques may revolutionize assisted reproduction in the future.

Comparison of cryopreservation outcome with human pronuclear stage oocytes obtained by the GnRH antagonist, Cetrorelix, and GnRH agonists

This retrospective study was performed to examine the implantation and pregnancy rates of frozen-thawed pronuclear stage oocytes obtained with the use of a GnRH antagonist, Cetrorelix (Cetrotide((R)) ASTA-Medica, Frankfurt/M, Germany) used in a multidose protocol with hMG, and to compare these results with those obtained after a conventional long GnRH analogue protocol (Decapeptyl-Depot, Ferring, Kiel, Germany). The study population consisted of 31 infertile couples with frozen-thawed pronuclear stage oocytes after ICSI treatment using the GnRH antagonist Cetrorelix (Cetrorelix((R))) and 31 infertile couples with frozen-thawed pronuclear stage oocytes after ICSI treatment using the long GnRH analogue protocol. Patients underwent ICSI after down regulation with a GnRH agonist (Decapeptyl) and stimulation with hMG, or a GnRH antagonist (Cetrorelix) and hMG. The supernumerary pronuclear stage oocytes were cryopreserved and transferred in a later mildly stimulated cycle. The implantation and pregnancy rates for frozen-thawed pronuclear stage oocytes derived from the GnRH antagonist compared with the GnRH agonist were 3.26% versus 3.73% (P=1.0000) and 8.33% versus 10.25% (P=1.0000), respectively. To our knowledge we report here the first pregnancies obtained by the transfer of cryopreserved pronuclear stage embryos generated from ICSI using a GnRH antagonist in the collecting cycle. The use of Cetrorelix in a multiple dose protocol in combination with hMG does not demonstrate a negative effect on viability, implantation potential or pregnancy outcome as compared to 2PN conceptuses obtained from a long GnRH agonist-hMG protocol.

Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia

There is a lack of data regarding variables affecting the treatment outcome for non-obstructive azoospermia when spermatozoa from cryopreserved testicular specimens are utilized for ICSI. The objective of the present retrospective analysis was to investigate the effect of various parameters on treatment outcome in such cases. One hundred and sixty-five couples with non-obstructive azoospermic males undergoing a total of 297 cycles were included. In all cases the testicular tissue retrieved by multiple open-biopsy testicular sperm extraction was stored in liquid nitrogen and, after thawing, only mature spermatozoa were used for ICSI. When no motile spermatozoa were recovered, immotile spermatozoa were used. In 159 cycles, motile spermatozoa were utilized for ICSI, while in 138 cycles immotile spermatozoa were utilized. Higher normal fertilization rate (60.4 +/- 3.1 versus 51.3 +/- 1.6%, P < 0.05), number of embryos transferred (2.8 +/- 0.06 versus 2.6 +/- 0.04, P < 0.05), modified cumulative embryo score (31.2 +/- 1.6 versus 23.9 +/- 0.8, P < 0.001), and proportion of motile spermatozoa injected (67.8 versus 49.8%, P < 0.05) were observed in cycles that resulted in clinical pregnancies. Binary logistic regression analysis showed that sperm motility (odds ratio 2.06, 95% CI 1.1-3.9, P < 0.05), but not womans age, number of treatment cycle, type of GnRH-analogue used for pituitary suppression, number of oocytes retrieved or number of embryos transferred was a significant determinant of the likelihood of clinical pregnancy. In conclusion, sperm motility after freeze/thawing of testicular tissue is the major determinant of the success of ICSI in non-obstructive azoospermia.

First pregnancy and life after preimplantation genetic diagnosis by polar body analysis for mucopolysaccharidosis type I

Preimplantation genetic diagnosis (PGD) may help couples at risk to avoid pregnancies with known genetic diseases. In Germany, the only option to perform PGD is the analysis of polar bodies (PB). Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder. Q70X is one of the frequent diseases causing mutations of alpha-L-iduronidase (IDUA), leading to a severe phenotype with mental retardation and various somatic abnormalities, and making a request for PGD is understandable. Using five polymorphic DNA markers from the vicinity of IDUA, PGD on first PB was performed for a consanguineous couple, both heterozygotes of the Q70X mutation of IDUA. Sixteen first PB were obtained by laser assisted hatching of the zona pellucida. Genotyping led to the conclusion that 3/16 oocytes carried wild-type IDUA alleles. Only one of these oocytes showed pronucleus formation after intracytoplasmic sperm injection and was transferred on day 2 after oocyte retrieval. A singleton pregnancy was established. Prenatal diagnosis showed a fetus heterozygous for Q70X. For MPS I, PB analysis is a feasible way to perform PGD and it may be an acceptable alternative for couples with moral objections to embryo selection, or for countries in which genetic testing of the embryo is prohibited.

Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia

The aim of the present study was to describe a simplified and inexpensive method of testicular tissue freezing, to assess the cumulative clinical pregnancy rate (CPR) by this technique, and to provide useful information for counselling couples with non-obstructive azoospermia. One hundred and sixty-five couples with non-obstructive azoospermic males pursuing assisted conception, from December 1995 to December 2002, were included. In all cases, the testicular tissue retrieved by open multiple-biopsy (both sides, by testicular sperm extraction) was frozen using a simple liquid nitrogen vapour freezing technique and was stored in liquid nitrogen thereafter. Only mature spermatozoa were used for intracytoplasmic sperm injection (ICSI) after thawing. Expected CPR were calculated using the Kaplan-Meier survival analysis. A total of 281 cycles were performed resulting in 53 clinical pregnancies. Crude and expected CPR (95% confidence intervals) after three cycles were 32.1 (25.7-40.1) and 55.7% (37.0-74.4) respectively. In conclusion, this simplified method for freezing testicular tissue resulted in a satisfactory outcome after ICSI in cases of non-obstructive azoospermia.

Unilateral follicular aspiration and in-vitro maturation before contralateral oocyte retrieval: a method to prevent ovarian hyperstimulation syndrome

BACKGROUND Five women undergoing intracytoplasmatic sperm injection (ICSI) considered to be at high risk of developing an ovarian hyperstimulation syndrome (OHSS) from March to July 2002 underwent unilateral follicular aspiration. METHODS When >/=15 follicles of 12-15 mm diameter in each ovary and a serum estrodial level >/=2500 pg/ml was present, follicular aspiration was performed unilaterally without hCG administration. Ovarian stimulation was continued for 1-3 days in four women before human chorionic gonadotrophin (hCG) was given. In one woman hCG injection was administered at the evening after unilateral follicular aspiration. The oocyte retrieval from the contralateral ovary was performed 36 h after hCG injection. By unilateral follicular aspiration two to six germinal vesicle (GV) oocytes could be retrieved. After in-vitro maturation of those oocytes ICSI was performed. RESULTS In four women one to two oocytes were fertilized and cryopreserved. In one case only one triploid pronucleus (3PN) was observed. At the contralateral ovum-pick up after hCG injection a median of 10 could be retrieved. After transfer of a median of 3 embryos, no pregnancy was achieved. Four of five patients developed a severe OHSS and were hospitalized for a median of 3 days. CONCLUSION Unilateral follicular aspiration and continuation of stimulation therefore can not be recommended for the prevention of OHSS.

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.