To identify trends in embryo catheter loading and embryo culture techniques performed worldwide.

Objective: To determine whether oocyte retrieval and in vitro maturation (IVM) is effective in girls undergoing fertility preservation before cancer treatment.Design: Cohort study.Setting: Tertiary university medical center.Patient(s): Patients <= 20 years old before gonadotoxic chemotherapy undergoing ovarian cortex cryopreservation.Intervention(s): Before ovarian cortex cryopreservation, oocytes in all observed follicles were aspirated, matured in vitro, and cryopreserved.Main Outcome Measure(s): Maturation of oocytes.Result(s): One hundred seventy-nine oocytes were detected in 17/19 patients (89%) aged 5-20 years. We found 7, 8, and 17 oocytes in patients 5, 8, and 10 years old, respectively. The median number of oocytes per patient was 9 (0-37). Maturation rate was 45/133 oocytes (34%). In total, 81 oocytes were cryopreserved. We cryopreserved 4 of 12 detected, 4 of 9 detected, 1 of 8 detected, and 4 of 9 detected IVM oocytes for patients aged 5-10,11-14, 15-17, and 18-20 years old, respectively.Conclusion(s): Patients undergoing ovarian cryopreservation could benefit from supplementary oocyte aspiration from the cortex. Surprisingly, oocytes were detected even in young premenarcheal girls. The number of oocytes detected, matured, and cryopreserved was not age dependent. Retrieved oocytes can be matured in vitro and cryopreserved. Because no pregnancy has yet resulted from this procedure it should be considered to be experimental. We describe the youngest patients to undergo ovum collection, IVM, and oocyte cryopreservation. (Fertil Steril (R) 2009;92:458-63. (C) 2009 by American Society for Reproductive Medicine.)

Current therapy of childhood cancer makes long-term survival a realistic outcome for most patients. However, some treatment regimens entail a significant risk of infertility. No established method for preservation of female fertility is currently available. Ovarian cryopreservation is an experimental technology that is being offered with increasing frequency to women undergoing cancer therapy. It has not yet been reported in children and adolescent girls. The aim of this review is to stimulate discussion on the possibility of performing ovarian cryopreservation in pre-menarcheal girls in advance of therapies that may induce ovarian failure. We present a multi-disciplinary discussion of the risks and benefits associated with the procedure and propose guidelines for its implementation. We propose that all girls about to receive treatment that has a high risk for infertility be offered consultation about the possibility of ovarian cryopreservation.

PURPOSE:The present study investigated the effect of bladder distension on in vitro fertilization and embryo transfer (IVF-ET) results.METHODS:The study comprised 796 patients after successful transvaginal oocyte pickup and IVF, who, on the basis of bladder filling for ET, were divided into two groups. In group E, 385 patients underwent ET with an empty bladder, and in group F, 411 patients underwent ET with a full bladder.RESULTS:Sixty-four pregnancies were achieved in group E (16.6%), compared to 110 pregnancies in group F (26.8%, P = 0.006). A similar pregnancy loss rate was observed in both groups, 13 in group E (20.3%) and 29 in group F (26.4%; P = NS).CONCLUSIONS:A significantly higher pregnancy rate was achieved with routine bladder distension before ET, probably attributable to the smooth and easy insertion of the ET catheter.

In order to reduce total fertilization failure in unexplained infertility, sibling oocytes were submitted to both conventional IVF and intracytoplasmic sperm injection (ICSI). Two groups of ICSI embryos were compared in unexplained infertility patients: those derived from ICSI when IVF had failed to fertilize, and those derived from ICSI while their sibling oocytes were fertilized by IVF. The outcome of oocytes fertilized exclusively by ICSI (essential ICSI, n = 749) was compared with those fertilized both by IVF and ICSI (non-essential ICSI, n = 957) in all IVF patients treated for unexplained infertility at the Hadassah Hospital (1999-2002). The latter group was further subdivided into ICSI and IVF embryos. Total fertilization rate was 54%. Fertilization rates by ICSI were lower in the essential ICSI compared with the non-essential ICSI group, at 65 and 73% (P < 0.025). Pregnancy rates per embryo transfer in the essential ICSI group (49%), ICSI derived embryos group (55%) and IVF derived embryos (44%) from the non-essential ICSI group, were similar. Implantation rates were lower in the essential ICSI group as compared with the non-essential ICSI group (21 versus 32% respectively; P < 0.05) and 26% for IVF embryos. In conclusion, essential ICSI was associated with lower fertilization and implantation rates.

Approximately 15% of human prion diseases are inherited disorders associated with PRNP gene mutations, such as familial Creutzfeldt–Jakob disease (fCJD). fCJD, a dominant late-onset neurodegenerative disorder with near 100% penetrance, is prevalent among Jews of Libyan descent having a common PRNP E200K founder mutation.1 Mutation analysis can be used for diagnostic or predictive (presymptomatic) genetic testing in affected families. Offspring of affected individuals have a 50% chance of expressing the disorder; nevertheless, the age at onset varies between individuals. Many patients at risk for fCJD prefer not to know their genetic status but still do not want to pass on the mutation, if it exists, to their children. Prenatal diagnosis through direct mutation analysis forces them to learn their own carrier status. A partial resolution for such problem was introduced by exclusion prenatal testing in which the fetus is tested for the presence of either allele of the relevant gene from the affected grandparent.2 This procedure is designed to avoid the birth of at-risk offspring to an individual who chose not to perform a predictive test. A major …

BACKGROUND To determine whether mouse embryos generated from frozen-thawed oocytes can successfully survive a second cryopreservation. METHODS Immature C57BL6*BALB/c female mice underwent superovulation and the collected oocytes were divided into three groups. Group A oocytes (n = 107) underwent IVF. Group B oocytes (n = 167) underwent IVF and embryos generated were then cryopreserved. Group C oocytes (n = 94) were cryopreserved, thawed and underwent IVF. Two-four-cell stage embryos were re-cryopreserved and thawed. Embryos from all groups were then cultured to the blastocyst stage. RESULTS Cleavage rates to the 2-4-cell stage were 78, 71 and 46% for groups A, B and C respectively. Blastulation rates from 2-4 cell-stage embryos were 37/83 (45%), 27/118 (23%) and 8/35 (23%) for groups A, B and C respectively. Development to blastocysts was observed in 37/107 oocytes (35%), 27/167 oocytes (16%) and only 8/94 oocytes (9%) for groups A, B and C respectively. CONCLUSION Oocyte cryopreservation results in reduced fertilization rates. Embryo cryopreservation reduces blastulation rates by half regardless of whether the oocytes were fertilized fresh or frozen-thawed. Nevertheless, embryos generated from cryopreserved oocytes can survive cryopreservation and develop to the blastocyst stage at rates comparable with embryos obtained from fresh oocytes.

Polycystic ovary syndrome (PCOS) with prolonged anovulation had resulted in endometrial carcinoma in a 43-year-old woman. Since she and her husband did not share common biological children, they requested fertility preservation. Due to the woman's age, high dose progesterone and postponing surgery were both considered inappropriate. We therefore proposed oocyte retrieval from the ovaries removed by staging laparotomy followed by in vitro maturation and ICSI. Surrogacy could then enable a future pregnancy. Fourteen of 17 (82%) retrieved oocytes matured in vitro. Following ICSI, eight embryos (two at the pronuclear stage and six cleaved) were cryopreserved. To the best of our knowledge, this is the first report of oocyte aspiration, maturation and fertilization from an ovary removed by laparotomy.

It is uncertain how long IVF units can keep frozen embryos. Few data exist on success of embryo transfer for embryos that have been cryopreserved for many years. We report the delivery of healthy twins following the transfer of embryos cryopreserved for 12 years. To the best of our knowledge, this is the longest reported successful human embryo freezing.

Objective: To examine the effect on pregnancy and implantation rates when highly purified, fermentation-based hyaluronic acid was the only macromolecule supplement to the transfer medium in a human IVF program.Design: Prospective randomized study.Setting: In vitro fertilization center in an academic medical institution.Patient(s): Eighty patients were included in this prospective randomized double blind study. Inclusion criteria were age ! 35 years, the availability of at least three embryos eligible for transfer on day 3 after fertilization, and no more than three previous embryo transfer attempts.Intervention(s): All embryos were cultured in PI medium containing 10% synthetic serum substitute (SSS) until day 3. Patients were randomly allocated to two groups; in treatment group A (40 patients), embryos were transferred to P1 medium supplemented with 0.5 mg/mL hyaluronic acid for 5-10 min before their intrauterine transfer. In the control group B (40 patients), embryos were transferred, as routinely performed, in PI medium containing 10% SSS.Main Outcome Measure(s): Clinical pregnancy and implantation rates.Result(s): The mean age of the female partner was 28.7 +/- 3.3 years and 29.7 +/- 3.8 years for groups A and B, respectively. In group A, 103 embryos were transferred and in group B, 97 embryos were transferred for a similar mean number of 2.6 +/- 0.6 and 2.4 +/- 0.5 embryos/transfer, respectively. Twenty-five pregnancies were achieved in group A, and 21 pregnancies in group B. This led to a comparable clinical pregnancy and implantation rates of 62.5% and 34% as compared to 52% and 26.8% for groups A and B, respectively.Conclusion(s): Hyaluronic acid can successfully replace albumin as a sole macromolecule in a human embryo transfer medium and result in high pregnancy and implantation rates. The use of this supplement is an important step in the development of human embryo culture media free of blood-derived additives.

Intensive chemotherapy and radiotherapy improve long-term survival in young women with cancer. However, these treatments lead to severe depletion of the follicular reserve. In addition, women who undergo bone marrow transplantation have a very high risk for ovarian failure, causing premature menopause and infertility.Ovarian cortex cryopreservation has been proposed as a method to preserve fertility in young patients with cancer (1Gosden R.G. Low temperature storage and grafting of human ovarian tissue.Mol Cell Endocrinol. 2000; 163: 125-129Crossref PubMed Scopus (91) Google Scholar). However, when cryopreserved ovarian slices were surgically implanted, only the small primordial and primary follicles survived and developed further (2Sztein J.M. Sweet H. Farley J. Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries new approach in gamete banking.Biol Reprod. 1998; 58: 1071-1074Crossref PubMed Scopus (141) Google Scholar). Antral follicles did not persist after implant transfer, probably because of failure to vascularize appropriately before the onset of follicular atresia. Moreover, because of the size and cellular complexity of antral follicles, follicular somatic cells or oocytes may be damaged by incomplete permeation of cryoprotectants.If oocytes of antral follicles could be rescued from ovarian tissue before cryopreservation, and matured and fertilized in vitro, they could provide additional opportunities for producing offspring and would not be wasted. We observed that human ovaries removed for tissue cryopreservation sometimes contain a few antral follicles.In this study, oocytes at the germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stage were aspirated and isolated from antral follicles of ovaries before tissue cryopreservation. Most of these oocytes were cryopreserved, while some matured in vitro and were fertilized.Young women with a significant risk for ovarian failure due to chemotherapy were offered ovarian cortex cryopreservation. Hospital review board approval and patient consent were obtained.Unilateral laparoscopic oophorectomy was performed under general anesthesia before commencement of chemotherapy. The ovary was removed from the abdomen through a 12-mm trocar port in a plastic bag.The ovary was immediately transferred on ice to the IVF laboratory in Leibovitz medium (GibcoBRL, Paisley, United Kingdom) and placed under a dissecting microscope. Antral follicles observed on the ovarian surface were aspirated by using a 1-mL syringe and a 21-gauge needle flushed with P1 medium containing 10% synthetic serum (Irvine Scientific, Santa Ana, CA). Cumulus-oocyte complexes and the naked oocytes that emerged from the follicles were collected with micropipettes and incubated in P1 medium.Subsequently, the ovarian cortex was sliced from the medulla, collected into Leibovitz medium, and dissected into 5-mm3 (5 × 1 × 1 mm) cortical strips. Leibovitz medium supplemented with 1.5 M of ethylene glycol, 10% synthetic serum, and 0.1 M of sucrose, was used as the cryoprotectant solution. This solution was decanted into 15-mL tubes, into which the ovarian slices were transferred. The tubes were gently rolled for 30 minutes at 4°C to promote equilibration of the cryoprotectant.Ovarian slices were transferred to cryotubes containing 1 mL of the cryoprotectant, cooled from 1°C at a rate of −2°C/min to −9°C. After manual seeding, cooling at a rate of −0.3°C/min was performed down to −40°C. Rapid cooling at a rate of −10°C/min was performed down to −140°C, and cryotubes were then lowered into liquid nitrogen.After dissection of the ovarian cortex, oocytes were sought in the remaining medium. Any cumulus–oocyte complexes were transferred to P1 medium enriched with 10% synthetic serum and incubated under oil at 37°C in an atmosphere of 90% N2, 5% CO2, and 5% O2. In most cases, oocytes were denuded from the cumulus cells by a short exposure to hyaluronidase solution (60 IU/mL) (Sigma, Rehovot, Israel), and their maturity was assessed. In some cases, cumulus–oocyte complexes were left intact.Freezing of oocytes was performed according to the method of Fabri et al. (3Fabbri R. Porcu E. Marsella T. Rocchetta G. Venturoli S. Flamigni C. Human oocyte cryopreservation new perspectives regarding oocyte survival.Hum Reprod. 2001; 16: 411-416Crossref PubMed Scopus (414) Google Scholar). In two patients, in vitro maturation was attempted by incubating the oocytes for 24 to 48 hours in the same medium to obtain complete maturation.Donor semen samples were prepared using a density gradient (Isolate, Irvine Scientific). Metaphase II oocytes were inseminated by intracytoplasmic sperm injection (ICSI), and the embryos obtained were cryopreserved.Oocytes were sought in nine patients admitted for ovarian cryopreservation. The mean age of the patients was 22.3 ± 6 years. Patients were treated for advanced osteosarcoma (n = 2), acute myelocytic leukemia (n = 2), bone marrow transplantation for thalassemia major (n = 2), Hodgkin's lymphoma (n = 1), non-Hodgkin's lymphoma (n = 1), and acute vasculitis (the Sussak syndrome) (n = 1).From seven of these nine patients (78%), a total of 32 oocytes (range, 3 to 7 oocytes) were found in the follicular aspirate or in the collecting medium. Table 1 summarizes the outcome of the retrieved oocytes.TABLE 1Outcome of oocytes from patients undergoing cryopreservation of ovarian cortex.PatientAge (y)DiseaseNo. of oocytes foundNo. of oocytes cryopreservedOocyte morphology at retrievalIn vitro maturationNo. of oocytes treated by ICSINo. with normal fertilization (2 pronuclei)No. of embryos frozenGVGVBDAttemptedMatured to metaphase II114Osteosarcoma0216Thalassemia major333No319Osteosarcoma5512No—419Thalassemia major6642No—525Hodgkin's lymphoma3??32312626Acute myelocytic leukemia0731Non-Hodgkin's lymphoma73344aThis patient requested that half of oocytes be cryopreserved without in vitro maturation and fertilization.24——828Acute myelocytic leukemia5bOne oocyte was degenerative. Revel. Oocyte collection from ovarian cortex. Fertil Steril 2003.432No923Acute vasculitis31231111Mean22.34.64.22.42.53.31.62.711.5Total32211215105823Note: GV = germinal vesicle; GVB = germinal vesicle breakdown.a This patient requested that half of oocytes be cryopreserved without in vitro maturation and fertilization.b One oocyte was degenerative.Revel. Oocyte collection from ovarian cortex. Fertil Steril 2003. Open table in a new tab In patient 2, three oocytes at the GVBD stage were found and cryopreserved. In patient 3, five oocytes were found. Three oocytes were denuded of cumulus cells. Two oocytes were at the GVBD stage, and one was at the GV stage. These three oocytes and the two intact cumulus–oocyte complexes were cryopreserved.In patient 4, six oocytes were found. After denudation of all oocytes, four were found to be at the GV stage and two at the GVBD stage. All oocytes were cryopreserved.In patient 5, three oocytes were found. This patient requested donor insemination. All oocytes were denuded from the cumulus cells, and in vitro maturation was attempted. The following day, one oocyte was at the GVBD stage and two were at the metaphase II stage. ICSI was attempted in all three oocytes. Normal fertilization was detected in one oocyte. However, 24 hours later, two embryos were observed and were frozen on day 2 of culture.In patient 7, seven oocytes were found. After denudation, we observed three oocytes at the GV stage and four at the GVBD stage. At the patient's request, three GV oocytes were cryopreserved. In vitro maturation was attempted in the four GVBD oocytes. Two of the latter oocytes reached full maturity (metaphase II) within 24 hours. Although ICSI was performed on both GVBD and metaphase II oocytes, none of these oocytes was fertilized.In patient 8, five oocytes were found (two at the GVBD stage and three at the GV stage). One GV oocyte was degenerative; the other four oocytes were cryopreserved.In patient 9, three oocytes were found (two at the GVBD stage and one at the GV stage). One of these oocytes matured to metaphase II within 48 hours, and after ICSI, a normal embryo was obtained and cryopreserved.Of the seven patients in whom oocytes were obtained, four preferred to freeze the oocytes and not to attempt in vitro maturation followed by fertilization. In the other three patients, five of eight GVBD oocytes matured in vitro. Three of these five in vitro matured oocytes developed into embryos which were cryopreserved.The option for preserving fertility in patients with premature ovarian failure is cryopreservation of embryos, oocytes, or ovarian cortical tissue (1Gosden R.G. Low temperature storage and grafting of human ovarian tissue.Mol Cell Endocrinol. 2000; 163: 125-129Crossref PubMed Scopus (91) Google Scholar). We describe for the first time an approach combining all three cryopreservation possibilities in humans.Mouse oocytes from frozen–thawed ovaries and from unfrozen ovaries could undergo in vitro maturation, fertilization, develop to the blastocyst stage, and produce normal pregnancies after transfer to pseudopregnant mouse foster (4Sztein J.M. O'Brien M.J. Farley J.S. Mobraaten L.E. Eppig J.J. Rescue of oocytes from antral follicles of cryopreserved mouse ovaries competence to undergo maturation, embryogenesis, and development to term.Human Reproduction. 2000; 15: 567-571Crossref PubMed Scopus (33) Google Scholar). We do not know the success rate of fertilization of immature human oocytes, although immaturity in theory protects oocytes from chilling damage. Because only antral follicles, which would probably not survive cryopreservation, are aspirated, we do not believe that this procedure damages the future ovarian follicular content. Alternatively, antral follicles could be aspirated from the ovarian cortex after thawing. In the mouse, however, threefold more oocytes were aspirated from fresh than from thawed ovaries (4Sztein J.M. O'Brien M.J. Farley J.S. Mobraaten L.E. Eppig J.J. Rescue of oocytes from antral follicles of cryopreserved mouse ovaries competence to undergo maturation, embryogenesis, and development to term.Human Reproduction. 2000; 15: 567-571Crossref PubMed Scopus (33) Google Scholar).In conclusion, because a few antral follicles can be found in the ovary at any stage of the cycle, we recommend rescuing oocytes by aspirating all antral follicles from ovaries before ovarian cortex cryopreservation. Immature oocytes aspirated from unstimulated ovaries removed for fertility preservation could be cryopreserved or in vitro matured. Intracytoplasmic sperm injection of these oocytes yields embryos that could subsequently be cryopreserved. This approach not only rescues these oocytes but also expands the range of cryopreservation possibilities, thus increasing the potential for urgent fertility preservation in patients at risk of ovarian failure. Intensive chemotherapy and radiotherapy improve long-term survival in young women with cancer. However, these treatments lead to severe depletion of the follicular reserve. In addition, women who undergo bone marrow transplantation have a very high risk for ovarian failure, causing premature menopause and infertility. Ovarian cortex cryopreservation has been proposed as a method to preserve fertility in young patients with cancer (1Gosden R.G. Low temperature storage and grafting of human ovarian tissue.Mol Cell Endocrinol. 2000; 163: 125-129Crossref PubMed Scopus (91) Google Scholar). However, when cryopreserved ovarian slices were surgically implanted, only the small primordial and primary follicles survived and developed further (2Sztein J.M. Sweet H. Farley J. Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries new approach in gamete banking.Biol Reprod. 1998; 58: 1071-1074Crossref PubMed Scopus (141) Google Scholar). Antral follicles did not persist after implant transfer, probably because of failure to vascularize appropriately before the onset of follicular atresia. Moreover, because of the size and cellular complexity of antral follicles, follicular somatic cells or oocytes may be damaged by incomplete permeation of cryoprotectants. If oocytes of antral follicles could be rescued from ovarian tissue before cryopreservation, and matured and fertilized in vitro, they could provide additional opportunities for producing offspring and would not be wasted. We observed that human ovaries removed for tissue cryopreservation sometimes contain a few antral follicles. In this study, oocytes at the germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stage were aspirated and isolated from antral follicles of ovaries before tissue cryopreservation. Most of these oocytes were cryopreserved, while some matured in vitro and were fertilized. Young women with a significant risk for ovarian failure due to chemotherapy were offered ovarian cortex cryopreservation. Hospital review board approval and patient consent were obtained. Unilateral laparoscopic oophorectomy was performed under general anesthesia before commencement of chemotherapy. The ovary was removed from the abdomen through a 12-mm trocar port in a plastic bag. The ovary was immediately transferred on ice to the IVF laboratory in Leibovitz medium (GibcoBRL, Paisley, United Kingdom) and placed under a dissecting microscope. Antral follicles observed on the ovarian surface were aspirated by using a 1-mL syringe and a 21-gauge needle flushed with P1 medium containing 10% synthetic serum (Irvine Scientific, Santa Ana, CA). Cumulus-oocyte complexes and the naked oocytes that emerged from the follicles were collected with micropipettes and incubated in P1 medium. Subsequently, the ovarian cortex was sliced from the medulla, collected into Leibovitz medium, and dissected into 5-mm3 (5 × 1 × 1 mm) cortical strips. Leibovitz medium supplemented with 1.5 M of ethylene glycol, 10% synthetic serum, and 0.1 M of sucrose, was used as the cryoprotectant solution. This solution was decanted into 15-mL tubes, into which the ovarian slices were transferred. The tubes were gently rolled for 30 minutes at 4°C to promote equilibration of the cryoprotectant. Ovarian slices were transferred to cryotubes containing 1 mL of the cryoprotectant, cooled from 1°C at a rate of −2°C/min to −9°C. After manual seeding, cooling at a rate of −0.3°C/min was performed down to −40°C. Rapid cooling at a rate of −10°C/min was performed down to −140°C, and cryotubes were then lowered into liquid nitrogen. After dissection of the ovarian cortex, oocytes were sought in the remaining medium. Any cumulus–oocyte complexes were transferred to P1 medium enriched with 10% synthetic serum and incubated under oil at 37°C in an atmosphere of 90% N2, 5% CO2, and 5% O2. In most cases, oocytes were denuded from the cumulus cells by a short exposure to hyaluronidase solution (60 IU/mL) (Sigma, Rehovot, Israel), and their maturity was assessed. In some cases, cumulus–oocyte complexes were left intact. Freezing of oocytes was performed according to the method of Fabri et al. (3Fabbri R. Porcu E. Marsella T. Rocchetta G. Venturoli S. Flamigni C. Human oocyte cryopreservation new perspectives regarding oocyte survival.Hum Reprod. 2001; 16: 411-416Crossref PubMed Scopus (414) Google Scholar). In two patients, in vitro maturation was attempted by incubating the oocytes for 24 to 48 hours in the same medium to obtain complete maturation. Donor semen samples were prepared using a density gradient (Isolate, Irvine Scientific). Metaphase II oocytes were inseminated by intracytoplasmic sperm injection (ICSI), and the embryos obtained were cryopreserved. Oocytes were sought in nine patients admitted for ovarian cryopreservation. The mean age of the patients was 22.3 ± 6 years. Patients were treated for advanced osteosarcoma (n = 2), acute myelocytic leukemia (n = 2), bone marrow transplantation for thalassemia major (n = 2), Hodgkin's lymphoma (n = 1), non-Hodgkin's lymphoma (n = 1), and acute vasculitis (the Sussak syndrome) (n = 1). From seven of these nine patients (78%), a total of 32 oocytes (range, 3 to 7 oocytes) were found in the follicular aspirate or in the collecting medium. Table 1 summarizes the outcome of the retrieved oocytes. Note: GV = germinal vesicle; GVB = germinal vesicle breakdown. In patient 2, three oocytes at the GVBD stage were found and cryopreserved. In patient 3, five oocytes were found. Three oocytes were denuded of cumulus cells. Two oocytes were at the GVBD stage, and one was at the GV stage. These three oocytes and the two intact cumulus–oocyte complexes were cryopreserved. In patient 4, six oocytes were found. After denudation of all oocytes, four were found to be at the GV stage and two at the GVBD stage. All oocytes were cryopreserved. In patient 5, three oocytes were found. This patient requested donor insemination. All oocytes were denuded from the cumulus cells, and in vitro maturation was attempted. The following day, one oocyte was at the GVBD stage and two were at the metaphase II stage. ICSI was attempted in all three oocytes. Normal fertilization was detected in one oocyte. However, 24 hours later, two embryos were observed and were frozen on day 2 of culture. In patient 7, seven oocytes were found. After denudation, we observed three oocytes at the GV stage and four at the GVBD stage. At the patient's request, three GV oocytes were cryopreserved. In vitro maturation was attempted in the four GVBD oocytes. Two of the latter oocytes reached full maturity (metaphase II) within 24 hours. Although ICSI was performed on both GVBD and metaphase II oocytes, none of these oocytes was fertilized. In patient 8, five oocytes were found (two at the GVBD stage and three at the GV stage). One GV oocyte was degenerative; the other four oocytes were cryopreserved. In patient 9, three oocytes were found (two at the GVBD stage and one at the GV stage). One of these oocytes matured to metaphase II within 48 hours, and after ICSI, a normal embryo was obtained and cryopreserved. Of the seven patients in whom oocytes were obtained, four preferred to freeze the oocytes and not to attempt in vitro maturation followed by fertilization. In the other three patients, five of eight GVBD oocytes matured in vitro. Three of these five in vitro matured oocytes developed into embryos which were cryopreserved. The option for preserving fertility in patients with premature ovarian failure is cryopreservation of embryos, oocytes, or ovarian cortical tissue (1Gosden R.G. Low temperature storage and grafting of human ovarian tissue.Mol Cell Endocrinol. 2000; 163: 125-129Crossref PubMed Scopus (91) Google Scholar). We describe for the first time an approach combining all three cryopreservation possibilities in humans. Mouse oocytes from frozen–thawed ovaries and from unfrozen ovaries could undergo in vitro maturation, fertilization, develop to the blastocyst stage, and produce normal pregnancies after transfer to pseudopregnant mouse foster (4Sztein J.M. O'Brien M.J. Farley J.S. Mobraaten L.E. Eppig J.J. Rescue of oocytes from antral follicles of cryopreserved mouse ovaries competence to undergo maturation, embryogenesis, and development to term.Human Reproduction. 2000; 15: 567-571Crossref PubMed Scopus (33) Google Scholar). We do not know the success rate of fertilization of immature human oocytes, although immaturity in theory protects oocytes from chilling damage. Because only antral follicles, which would probably not survive cryopreservation, are aspirated, we do not believe that this procedure damages the future ovarian follicular content. Alternatively, antral follicles could be aspirated from the ovarian cortex after thawing. In the mouse, however, threefold more oocytes were aspirated from fresh than from thawed ovaries (4Sztein J.M. O'Brien M.J. Farley J.S. Mobraaten L.E. Eppig J.J. Rescue of oocytes from antral follicles of cryopreserved mouse ovaries competence to undergo maturation, embryogenesis, and development to term.Human Reproduction. 2000; 15: 567-571Crossref PubMed Scopus (33) Google Scholar). In conclusion, because a few antral follicles can be found in the ovary at any stage of the cycle, we recommend rescuing oocytes by aspirating all antral follicles from ovaries before ovarian cortex cryopreservation. Immature oocytes aspirated from unstimulated ovaries removed for fertility preservation could be cryopreserved or in vitro matured. Intracytoplasmic sperm injection of these oocytes yields embryos that could subsequently be cryopreserved. This approach not only rescues these oocytes but also expands the range of cryopreservation possibilities, thus increasing the potential for urgent fertility preservation in patients at risk of ovarian failure. The authors thank Mrs. June Sher for editorial assistance.

BACKGROUND The objective of this retrospective study, which included 51 men with non-obstructive azoospermia, was to evaluate the predictive value of the results of the first sperm recovery attempt on the probability for sperm recovery in a second attempt. METHODS AND RESULTS A positive testicular fine needle aspiration (TEFNA) was defined as the recovery of any number of mature sperm. At the first and second TEFNA attempts, mature sperm were recovered in 33 (64.7%) and 25 (49%) of 51 patients respectively. In 23 of the 33 (69.7%) patients with a positive first TEFNA, sperm were recovered at both attempts, whereas in only two of 18 (11.1%) with a negative first TEFNA, sperm were recovered at the second attempt. Our analysis revealed a high predictive value of the first TEFNA for sperm recovery at the subsequent attempt, with a mean positive predictive value of 69.7%, with the highest probability being 90.9% in hypospermatogenesis, 72.7% in Sertoli cell-only pattern, 75% in tubular hyalinization, and the lowest being 28.6% in maturation arrest. The mean negative predictive value was 88.9%, which was high in all categories (80% in Sertoli cell-only pattern and 100% in maturation arrest and tubular hyalinization). CONCLUSION A second TEFNA attempt should be offered to all non-obstructive azoospermic patients with a positive first TEFNA. Patients with a negative first TEFNA may undergo a repeated attempt, but a donor sperm back-up is strongly advised.

Objective: The effects of Nitrous oxide (N2O) on fertility in general and in-vitro fertilization (IVF) in particular are unclear as a result of conflicting evidence. One study has reported an increased risk of miscarriage among female dental assistants exposed to N2O for 3 or more hours per week, although another study failed to show such an effect on midwives. As for IVF, several authors have reported adverse effects on IVF outcome following anesthesia for oocyte pick-up (OPU) with N2O, although other authors failed to show such an effect, both in an animal model and in humans. The objectives of this study were to evaluate the effect of exposure to N2O during OPU on IVF results.Design: This prospective randomized study comprised 218 patients undergoing general anesthesia for OPU and IVF.Materials and Methods: Patients were randomized to receive general anesthesia with or without N2O. One hundred and one patients did not receive N2O (group 0), and 114 patients did receive N2O during the anesthesia (group 1).Results: Both groups were similar in age (33.9 vs. 33.7 years, in groups 0 and 1, respectively), mean number of oocytes (8.3 vs. 8.5), mature oocytes (5.5 vs. 7.3), degenerated oocytes (1.2 vs. 1.6), immature oocytes (1.7 vs. 2), number fertilized (3.5 vs. 4.1), embryo quality, number of embryos transferred (2.3 vs. 2.6), pregnancy rate (26% vs. 31%), and the number of implanted fetuses (1.3 vs. 1.4).Conclusion: Exposure to N2O during OPU was not shown to have any adverse effect on the outcome of IVF, as similar results were observed with and without N2O. Objective: The effects of Nitrous oxide (N2O) on fertility in general and in-vitro fertilization (IVF) in particular are unclear as a result of conflicting evidence. One study has reported an increased risk of miscarriage among female dental assistants exposed to N2O for 3 or more hours per week, although another study failed to show such an effect on midwives. As for IVF, several authors have reported adverse effects on IVF outcome following anesthesia for oocyte pick-up (OPU) with N2O, although other authors failed to show such an effect, both in an animal model and in humans. The objectives of this study were to evaluate the effect of exposure to N2O during OPU on IVF results. Design: This prospective randomized study comprised 218 patients undergoing general anesthesia for OPU and IVF. Materials and Methods: Patients were randomized to receive general anesthesia with or without N2O. One hundred and one patients did not receive N2O (group 0), and 114 patients did receive N2O during the anesthesia (group 1). Results: Both groups were similar in age (33.9 vs. 33.7 years, in groups 0 and 1, respectively), mean number of oocytes (8.3 vs. 8.5), mature oocytes (5.5 vs. 7.3), degenerated oocytes (1.2 vs. 1.6), immature oocytes (1.7 vs. 2), number fertilized (3.5 vs. 4.1), embryo quality, number of embryos transferred (2.3 vs. 2.6), pregnancy rate (26% vs. 31%), and the number of implanted fetuses (1.3 vs. 1.4). Conclusion: Exposure to N2O during OPU was not shown to have any adverse effect on the outcome of IVF, as similar results were observed with and without N2O.

OBJECTIVE:IVF-ET provides unique controlled conditions for the study of seasonal influences on the human reproductive process. This study was designed to evaluate the effects of seasonality on fertilization rate, embryo quality, and conception rates.DESIGN:A retrospective observational cohort study. A chart review of all individuals undergoing IVF-ET from 1988 to 1991 at our institution was performed.SETTING:An assisted reproduction unit at a university-based tertiary medical center.PATIENT(S):657 women, for a total of 1074 IVF-ET treatment cycles, were evaluated. From this population, we chose 305 women undergoing their first IVF-ET cycle for reasons of pure mechanical infertility.INTERVENTION(S):IVF-ET cycles.MAIN OUTCOME MEASURE(S):Seasonal variability in fertilization rates and quality-A embryo rates, and the correlation with the absolute number of light hours, as well as the influence of temperature, humidity, and other environmental parameters.RESULT(S):A significant seasonal variability in the fertilization rate and the quality-A embryo rate was demonstrated. The highest fertilization and quality-A embryo rates were observed during the spring and the lowest in the autumn. These changes correlated with the absolute number of light hours and its increment over time, but not with the temperature, humidity, or other environmental parameters.CONCLUSION(S):Seasonality seems to have a significant influence on the fertilization process and on the quality of the human embryos that are obtained in vitro, possibly because of the light/dark variations over time. If this finding is confirmed, these seasonal changes should be taken into account when evaluating infertility data and in everyday clinical practice.

Objectives: To summarize our 5 years experience with testicular fine needle aspiration (TEFNA) for sperm recovery in non-obstructive azoospermia. Design: A retrospective chart review of all men suffering from non-obstructive azoospermia operated for sperm recovery in our unit from January 1995 to December 1999. Materials and Methods: A total of 152 men underwent 236 TEFNA operations, consisting of a mean of 15 punctures and aspirations in each testis, using 23 gauge butterfly needles, connected to a 20 ml syringe with an aspiration handle. Following the initial TEFNA, a second intervention was performed in 51 men, a third in 20 men, a fourth in 6 men, a fifth in 4 men and one patient underwent 8 consecutive procedures, leading to a total of 236 TEFNA cycles. Classified by testicular histology, the study population comprised men diagnosed as suffering from Sertoli cells only (SC) in 56 (36.8%) cases, maturation arrest (MA) in 38 (25.0%) cases, hypospermatogenesis (HS) in 36 (23.7%) cases, tubular hyalinization (TH) due to non-mosaic Klinefelter's syndrome (47, XXY) in 20 (13.2%) cases, and post-irradiation fibrosis (IF) in two (1.3%) cases. Results: Mature testicular spermatozoa could be recovered in 127 (53.8%) cycles The recovery rate by testicular histology was 42 out of 87 (48.3%) in the patients with SC, 22 out of 57 (38.6%) in MA, 52 out of 63 (82.6%) in HS, 11 out of 27 (40.7%) in TH due to Klinefelter's syndrome, whereas no spermatozoa were found in the two cases with IF. ICSI was performed in all 127 cycles. Transfer of embryos deriving from 2PN fertilization was performed in 84 cycles, resulting in 26 ongoing pregnancies, a pregnancy rate of 31%. No complications or major side effects were recorded. Conclusion: TEFNA was found to be highly efficient, easy to learn, safe and well tolerated by patients. We considered TEFNA as the first choice approach whenever sperm recovery is attempted in non-obstructive azoospermia.