Live birth from vitrified–warmed human oocytes fertilized with frozen–thawed testicular spermatozoa

A couple with male infertility due to non-obstructive azoospermia were referred to the fertility centre for treatment. Testicular biopsy was performed on the male partner and testicular samples were frozen. The female partner underwent ovarian stimulation and 31 mature oocytes were recovered by ultrasound-guided vaginal aspiration. Twelve oocytes were cryopreserved by the Cryotop vitrification method and 19 oocytes were inseminated by intracytoplasmic sperm injection (ICSI) using frozen–thawed testicular spermatozoa. Nine out of 19 oocytes were fertilized and the resulting embryos were cryopreserved by slow freezing. Four months later, two out of six thawed embryos were transferred, but no pregnancy resulted. One year later, the couple decided to attempt pregnancy using vitrified oocytes and frozen testicular spermatozoa. Six vitrified–warmed oocytes were injected with frozen–thawed testicular spermatozoa and four were fertilized. On the day of transfer, two cleavage stage embryos (4-cell, 2-cell) were obtained. Serum β-HCG test 14 days after embryo transfer was positive. Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation. A healthy baby boy weighing 3.09 kg was delivered by elective Caesarean section at 38 weeks of gestation. This case report demonstrates that oocyte cryopreservation by the Cryotop vitrification method does not compromise oocyte developmental competence.     

Birth of Healthy Twins Resulting from Donated Oocytes and Posthumous Use of Frozen–Thawed Spermatozoa Obtained Prior to Chemotherapy

The improvement in life expectancy, following better cancer therapy combined with new options in treating male infertility have increased the use of sperm freezing. We describe a rare case of twin pregnancy of a childless widow, using donated oocytes after ICSI with her deceased husband's banked frozen spermatozoa. Sperm was frozen before chemotherapy treatment and a written consent for future thawing and injection to donor oocytes was given. Use of thawed spermatozoa after death was not mentioned in the consent form. During the husband's illness years, ICSI of donated oocytes fertilized with thawed spermatozoa yielded three embryos, but no pregnancy was achieved after ET. While waiting for a subsequent oocyte donation, the male partner died of his malignancy. The woman expressed her wish to be treated with the frozen spermatozoa of her deceased husband. Since the original informed consent did not cover postmortem use of the husband's sperm, a special application for its use was made and subsequently approved by the Israeli Ministry of Health legal advisor. Another six donated oocytes were fertilized with the deceased's thawed spermatozoa, and the transfer of four embryos to the uterine cavity resulted in a twin pregnancy and later delivery of two healthy babies. This case emphasizes the importance of written informed consent signed by patients with a life-threatening disease at the time of banking spermatozoa, and discussing the possibility of its posthumous use. Several legal and ethical concerns are discussed.     

Twin pregnancy obtained with frozen-thawed embryos after in vitro maturation in a patient with polycystic ovarian syndrome

Purpose : A twin pregnancy was obtained in a patient with polycystic ovary syndrome after the transfer of three in vitro maturation-derived day 3 embryos that has been frozen and thawed. Methods : The patient had received mild hMG stimulation followed by hCG injection. After culture for 24–48 h, mature oocytes were fertilized by ICSI. Embryos were cultured until day 3; supernumerary embryos were cryopreserved using a slow protocol. Results : Among 15 nonatretic oocytes, 9 matured, 8 were fertilized. Four embryos were transferred but they did not implant. The subsequent transfer of three frozen–thawed embryos resulted in the delivery of two healthy girls. Conclusions : These results indicate that a pregnancy could be obtained with in vitro maturation-derived day-3 frozen–thawed embryos.     

Frozen Embryos Generated from Surgically Retrieved Sperm from Azoospermic Men: Are They Clinically Viable?

Purpose: To assess the viability of frozen-thawed embryos derived from intracytoplasmic sperm injection (ICSI) in azoospermic men.Methods: Retrospective analysis of 154 consecutive ICSI cycles using surgically retrieved sperm from azoospermic men and case-control comparison of subsequent frozen transfer cycles with those using embryos generated from ejaculated sperm.Results: Patient and fresh cycle characteristics were similar in both groups. There were no differences between the two groups in the proportion of pronucleate (54% and 62%), and cleavage-stage embryos thawed (46% and 38%), post-thaw survival rates (retrievals: 69%; ejaculated: 73%) or quality of frozen embryos subsequently transferred. Implantation was significantly lower in frozen cycles where embryos were generated from surgically retrieved sperm (0% versus 11.5%; p=0.03). Both clinical pregnancy rate (5% versus 21%) and livebirth rate (0% versus 21%) were lower in this group, but only the difference in LBR reached borderline statistical difference (p=0.10).Conclusion: This small series demonstrates a significant impairment in implantation in FET cycles using embryos generated from surgically retrieved sperm and a trend towards a poorer pregnancy outcome.     

Successful birth after injection of frozen human oocytes with frozen epididymal spermatozoa

A couple (female 31, male 42 years old) with infertility due to obstructive azoospermy returned to the clinic in order to attempt pregnancy using their frozen oocytes and epididymal sperm cells, which had been cryopreserved at the time of a previous IVF attempt. Two days before the scheduled transfer, eight oocytes were thawed; 5/8 (63%) oocytes survived and 4/5 (80%) oocytes fertilized after intracytoplasmic sperm injection (ICSI) with the previously frozen epididymal spermatozoa. All four fertilized ova cleaved (100%). On day 2 after thawing, four embryos were transferred; three with two cells (grade II) and one with three cells (grade III). Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation, and the patient was delivered by Caesarean section at 40 weeks of gestation; the baby boy weighed 3025 g, and measured 51 cm, with Apgar of 10 in the 1st and 5th min. The cryopreservation and warming protocol used for this study yielded very favourable results, comparing well with reports in the literature. This case report demonstrates that it is possible to obtain high rates of oocyte survival following thawing and high rates of fertilization after ICSI, with viable development of the resulting embryos.     

Comparison of Pregnancy Outcome of Pronuclear- and Multicellular-Stage Frozen–Thawed Embryo Transfers

Purpose: Our purpose was to determine if supernumerary embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) should be frozen (using 1,2-propanediol) at the pronuclear or multicellular stage. Methods: The study was a retrospective analysis conducted at the Dubai Gynaecology & Fertility Centre of the Department of Health & Medical Services, Dubai, U.A.E. One hundred forty-one women undergoing frozen–thawed embryo replacement cycles with IVF generated embryos and 84 women undergoing the same with ICSI generated embryos. Results: Supernumerary, IVF-generated embryos frozen at the multicellular stage had a significantly higher rate of survival on thawing (73.9%) than embryos frozen at the pronuclear stage (64.4%). The morphological grades of the embryos in the two groups were similar, but a significantly higher pregnancy rate was obtained with embryos frozen at the multicellular stage (22.8%) than with pronuclear-stage embryos (14.8%). Similarly, with ICSI-generated embryos, significantly higher survival was seen with multicellular-stage frozen embryos (74.8%) than pronuclear-stage embryos (64.4%). The morphological grades of the embryos and pregnancy outcomes of the two groups were similar. Conclusions: Supernumerary embryos generated by IVF and ICSI should be frozen at the multicellular stage so as to allow selection of the best embryos for transfer and embryo freezing of only robust embryos.     

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa

Purpose: In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems.Methods: Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA.Results: Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients.Conclusions: This result demonstrates that the successful pregnancies and live births can be established from embryos produced from {in vitro} matured oocytes that fertilized with testicular sperm.     

Intracytoplasmic sperm injection improving embryo quality: Comparison of the sibling oocytes of Non-Male-Factor couples

Objective: Our objective was to investigate whether the quality of embryos developed after intracytoplasmic sperm injection (ICSI) is better than that of conventional IVF embryos. Methods: Nine couples who previously achieved a normal rate of fertilization following IVF and four couples whose normal rate of fertilization was expected were involved in this study. The oocytes from those couples were randomly divided into two groups, group A by conventional insemination and group B by ICSI. The fertilization rate and quality of embryos were compared. Results: Normal fertilization was achieved in 61% of the oocytes (83/136) after conventional insemination. In group B, 69% of the oocytes (99/144) achieved normal fertilization, although only 127 metaphase II oocytes were injected using the ICSI technique. More grade A embryos were obtained when the ICSI technique was used for fertilization than by conventional IVF (35.4 and 24.3%, respectively;P=0.028). Conclusions: A similar fertilization rate can be achieved by ICSI in comparison with conventional IVF, when male factor is not involved. Embryos after ICSI have an improved quality.     

Comparison of cryopreservation outcome following intracytoplasmic sperm injection and conventional in vitro fertilization

Purpose Our purpose was to compare the success rate of transferring frozen-thawed embryos generated from either intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Methods A retrospective review of all frozen—thawed embryo transfer (ET) cycles between January 1995 and April 1997 was performed. There were 83 and 204 transfer cycles of frozen — thawed multicellular embryos generated from conventional IVF (group A) and ICSI (group B), respectively. The survival rate of frozen — thawed embryos and the outcome following ET in both groups were assessed. Results The groups did not differ in age (31.7±4.6 and 30.6±6.0; mean±SD) or number of embryos transferred (3.5±1.1 and 3.8±1.3 for groups A and B, respectively). An acceptable pregnancy rate per ET was achieved in both groups, but the rate was significantly higher (P = 0.04) for group A than group B, 32.5 and 20%, respectively. Group A included frozen embryos of a higher quality than those of group B, but the proportion of embryos surviving after thawing was significantly higher for group B than group A (92.5 and 85.6%, respectively; P = 0.0004). The abortion rate did not differ between the two groups: 22 and 26.8% for groups A and B, respectively. Conclusions Although an overall high pregnancy rate was achieved following frozen-thawed ET, it was lower for cycles in which embryos had been generated from ICSI. This difference may be attributed to a lower prefreezing embryo quality in the ICSI group. Embryos originating from ICSI were not vulnerable to cryopreservation and, when implanted, resulted in a comparable abortion rate to thawed embryos of conventional IVF.     

Live birth of twins after IVF of oocytes that were cryopreserved almost 12 years before

A 45-year-old woman received embryos from IVF by intracytoplasmic sperm injection (ICSI) with her own oocytes that were cryopreserved (slow freezing in a low-sodium medium) 11 years and 7 and a half months before, when she was 33 years old. From seven metaphase-II oocytes thawed, five survived, four were fertilized after ICSI and two cleaving embryos were transferred on day 3. A diamniotic dichorionic term pregnancy was achieved, ending with the delivery of two healthy girls. As far as is known, this case represents, to date, the longest storage period of cryopreserved human oocytes resulting in a live birth.     

Use of a Medium Devoid of Any Human or Animal Compound (SMART2) for Embryo Culture in Intracytoplasmic Sperm Injection

Purpose: Our purpose wax to evaluate the efficiency of a medium, devoid of any human or animal compound and specially designed for early embryo development (from the zygote to the eight-cell stage), SMART2, in intracytoplasmic sperm injection (ICSI) and to compare it with a medium containing human serum albumin (EllioStep2). Methods: Oocytes from 50 ICSI attempts were randomly placed, after sperm injection, into either SMART2 or EllioStep2. After a 48-hr incubation, the embryos were examined for quality scoring before transfer or freezing. Results: The percentage of normally fertilized oocytes per intact oocytes was slightly higher using SMART2 (139/199 vs. 135/224, respectively, for SMART2 and EllioStep2: P < 0.05). The distribution of embryo scores and the percentage of embryos with a fair morphology (71/143 vs. 72/148, respectively, for SMART2 and EllioStep2; not significant) were identical in both media. Conclusions: These data show that SMART2 medium can be successfully used for early embryo growth and, because it is devoid of any human or animal compound, offers better safety for patients than conventional media.     

Association of Vitamin D with outcome after intra cytoplasmic sperm injection

Objective: To observe effects of vitamin D levels on pregnancy outcome after intra cytoplasmic sperm injection (ICSI).

Method: It was a cross-sectional study conducted in Australian Concept Infertility Medical Center from July 2011 to August 2014. Estimation of 25-hydroxy cholecalciferol (25-OHD) of consented females (252) was done before treatment protocol for ICSI. Results of β hCG performed 14 days after embryo transfer categorized groups; Pregnant with ß hCG more than 25?IU/mL and rest included in non-pregnant group. Both groups were compared by independent sample t-test and Pearson’s Chi Square test. Binary Logistic Regression Analysis was used to estimate odds ratio of pregnancy outcome with its predictors including Vitamin D.

Results: The mean value of 25-OHD, number of oocytes, fertilized oocytes and endometrial thickness was significantly higher in pregnant women. A significant positive association of 25-OHD with clinical pregnancy and thickness of endometrium was observed. After adjustment with female age and BMI, positive association of vitamin D with endometrial thickness was observed.

Conclusion: Deficiency of 25-OHD in females hinders the accomplishment of optimal endometrial thickness required for implantation of embryo after ICSI. The improvement in vitamin D status can thus improve success results in assisted reproductive clinics.     

The Outcome of Cryopreserved Human Embryos After Intracytoplasmic Sperm Injection and Traditional IVF

Objective: Our objective was to analyze the outcome of cryopreserved embryos obtained after intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in terms of survival rate, implantation rate (IR), total and clinical pregnancy rate (PR) in a retrospective, comparative study. Methods: Three hundred seventy-five IVF and 463 ICSI surnumerary cleaved embryos, frozen on Day 2 with 1,2-propanediol, were thawed. Results: Thirty-two percent of the thawed IVF embryos survived and 11 pregnancies (8 clinical) were obtained from 68 transfers (16.1%). Fourty-seven percent of the ICSI embryos survived, with 19 pregnancies (18 clinical) from 116 transfers (16.4%). The IR was 8.5% (8/94) in IVF cycles and 10.8% (20/185) in ICSI cycles. Conclusions: A significantly better survival rate of ICSI embryos was observed but with no difference in PR, preclinical, and clinical abortion rate, or IR.     

Birth of a healthy male infant after transfer of vitrified-warmed blastocysts derived from intracytoplasmic sperm injection with vitrified-warmed oocytes and frozen-thawed spermatozoa

Purpose

To report a successful delivery of a healthy baby after transfer of vitrified-warmed blastocysts derived from introcytoplasmic sperm injection (ICSI) with vitrified-warmed oocytes and frozen-thawed sperm.

Methods

A female patient and her husband with non-obstructive azoospermia received a transfer of vitrified-warmed blastocysts from vitrified-warmed oocytes and frozen-thawed sperm. The main outcome measures were fertilization, pregnancy and birth.

Results

Nine oocytes were matured and vitrified. When the vitrified oocytes were warmed, six survived with good quality morphology. Using ICSI, frozen-thawed sperm was injected into the six warmed oocytes that survived, and the fertilization rate was 100%. The zygotes were cultured, and five of six early embryos became blastocysts. One of them was transferred, but pregnancy was not achieved. The second time around, two vitrified-warmed blastocysts were transferred resulting in pregnancy, and a healthy boy was delivered.

Conclusions

This is a rare case of a successful birth using a vitrified-warmed blastocyst grown after ICSI with a vitrified-warmed oocyte and frozen-thawed sperm.     

培养液中添加不同促性腺激素对卵母细胞体外成熟的影响

目的:比较卵母细胞体外成熟培养液中添加不同促性腺激素对未成熟卵母细胞体外成熟结局的影响。方法:将行卵母细胞体外成熟(IVM)的35例患者共42个新鲜取卵周期,随机分成A组:22个取卵周期将重组人促卵泡激素(果纳芬,rFSH)和重组人绒毛膜促性腺激素(艾泽,hCG)按1∶1的比例混合添加,终浓度为75 mIU/ml;B组:20个取卵周期添加终浓度为75 mIU/ml的尿源性促性腺激素(hMG),进行未成熟卵母细胞体外成熟培养。35例患者中新鲜取卵周期未移植或移植后未孕者行解冻胚胎移植。比较组间患者的卵母细胞成熟率、受精率、卵裂率、优质胚胎率、累计临床妊娠率及胚胎着床率。结果:取卵均于月经周期第12日或最大卵泡发育至10￣12 mm时进行,故所获卵均为未成熟卵。A组获卵181枚,经培养后成熟84枚,行卵胞浆内单精子注射(ICSI)84枚,受精60枚,卵裂55枚,优质胚胎20枚;新鲜胚胎移植9例,获1例临床妊娠,解冻胚胎移植5例,获1例临床妊娠,累计临床妊娠率为14.29%,胚胎着床率为7.14%。B组获卵176枚,经培养后成熟120枚,行ICSI 120枚,受精97枚,卵裂90枚,优质胚胎41枚,新鲜胚胎移植6例,获4例临床妊娠,解冻胚胎移植9例,获3例临床妊娠,累计临床妊娠率为46.67%,胚胎着床率为33.33%。结论:卵母细胞体外成熟培养液中添加尿源性促性腺激素可获得较添加重组人促卵泡激素和重组人绒毛膜促性腺激素高的卵母细胞成熟率、临床妊娠率及胚胎着床率。     

Pregnancy after immature oocyte donation and intracytoplasmic sperm injection

Objective: To report a case of pregnancy from in vitro-matured primary oocytes fertilized by ICSI. The pregnancy occured in a woman who was in an oocyte donation program; the woman's husband had normal sperm parameters.

Design: Case report.

Setting: Private general hospital affiliated with a university hospital.

Patient(s): A recipient with premature ovarian failure, a recipient's husband with normal sperm, and a pregnant woman who donated her oocytes.

Intervention(s): Aspiration of immature oocytes during cesarean section, in vitro culture for maturation, ICSI of matured oocytes, coculture of fertilized oocytes.

Main Outcome Measure(s): Fertilization of oocytes by ICSI, and cleavage of embryos by Vero cell coculture.

Result(s): Two of seven immature oocytes became metaphase II oocytes, and both were fertilized by ICSI. The two zygotes were cocultured on Vero cells to become grade 1 two-cell embryos. Pregnancy was obtained after transfer.

Conclusion(s): More studies are necessary to clarify whether ICSI can increase the fertilization rate of in vitro-matured primary oocytes, and to clarify the role of coculture in fertilization.     

Fertilization, Embryo Quality, and Cryosurvival in In Vitro Fertilization and Intracytoplasmic Sperm Injection Cycles

Purpose: Our purpose was to investigate the influence of semen quality on fertilization, embryo morphology, cleavage, and cryosurvival in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)programs. Methods: A retrospective analysis of 513 couples undergoing IVF and 255 couples undergoing ICSI was done. Results: Semen quality influenced fertilization in IVF and abnormal fertilization in IVF and ICSI, but no effects on the development, morphology, implantation capacity, or cryosurvival of embryos were found. Fertilization, embryo quality, and cryosurvival rates were similar after IVF and ICSI. The fertilization rate of mature oocytes in IVF was lower when cytoplasmic immaturity in the oocyte population was frequent. The speed of development of embryos was 2 hr faster after ICSI than after IVF. Two-cell–stage embryos survived best after cryopreservation with propanediol and sucrose on day 2. Conclusions: After fertilization, semen parameters had no effect on the quality or cryosurvival of embryos in either IVF or ICSI.     

Paternal Centrosomal Dynamics in Early Human Development and Infertility

Purpose: Our purpose was to demonstrate the dynamics of the human sperm centrosome during fertilization and cleavage. Methods: Human gametes, fertilized oocytes, and preimplantation embryos were examined by transmission electron microscopy. Results: The functional sperm centrosome containing a typical centriole (proximal) is inherited at fertilization and forms a sperm monoaster. It then replicates and is perpetuated during cleavage. It organizes the mitotic apparatus at each stage of cleavage up to the hatching blastocyst stage. Bipolar spindles are formed in all monospermic and most dispermic embryos. Occasionally, two sperm asters and tripolar spindles are formed in dispermic embryos. Centrioles are associated with pronuclei and nuclei at interphases when they duplicate and occupy pivotal positions at spindle poles during mitoses. The maternal centrosome is not functional. Conclusions: The human embryo shows paternal centrosome inheritance and perpetuation like most other animals. Inheritance of defective centrosomes may lead to abnormal cleavage and contribute to infertility.     

Successful delivery following cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome-like disease: a case report

Purpose : To estimate frozen zygotes, which developed from in vitro matured oocytes retrieved from polycystic ovarian syndrome-like disease. Methods : Oocyte retrieval was performed on Day 15 following withdrawal bleeding. The oocytes were incubated for 24 h in TCM-199 maturation medium supplemented with follicle fluid, E2, FSH, and hCG. Results : A total of 12 immature oocytes were collected. Seven of the 12 oocytes (58.3%) developed to the metaphase-II stage, and subsequently, all seven fertilized oocytes were frozen at the pronuclear stage. The remaining five oocytes failed to develop to the metaphase-II stage after an additional 24 h of incubation. Three of seven cryopreserved oocytes were thawed and developed to 2–8-cell cleaved stage embryos. The first pregnancy failed. However, the second frozen–thawed embryo transfer resulted in the delivery of healthy twins. Conclusions : Successful delivery using frozen zygotes from an anovulatory woman with polycystic ovarian syndrome-like disease.     

Intracytoplasmic sperm injection as a treatment for unexplained total fertilization failure or low fertilization after conventional in vitro fertilization

OBJECTIVE: To determine whether IVF or intracytoplasmic sperm injection (ICSI) should be the choice of treatment in case of a previous IVF attempt with unexplained total fertilization failure or low fertilization (<25%). DESIGN: Prospective study. SETTING: Leiden University Medical Center. PATIENT(S): Thirty-eight couples undergoing IVF and ICSI on sibling oocytes after a first IVF attempt with total fertilization failure or with low fertilization (<25%). INTERVENTION(S): Performing IVF and ICSI on sibling oocytes. MAIN OUTCOME MEASURE(S): Fertilization and (ongoing) pregnancy rate. RESULT(S): A total of 271 oocytes were collected in 24 oocyte retrievals in the total fertilization failure group. Hundred nine oocytes were randomly allocated to IVF and 12 were fertilized (11%); 162 sibling oocytes were allocated to ICSI and 78 were fertilized (48%). In 8 of the 24 patients fertilization occurred after IVF. The pregnancy rate after transfer of 1 IVF and 1 ICSI embryo (n = 3) was 67% and after the transfer of 2 ICSI embryos (n = 21) this was 52%. In the low fertilization group 169 oocytes were collected in 14 oocyte retrievals. Seventy-two oocytes were randomly allocated to IVF and 16 were fertilized (22%). Ninety-seven sibling oocytes were allocated to ICSI and 58 were fertilized (60%). In 7 of 14 patients fertilization occurred after IVF. The pregnancy rate after the transfer of 1 IVF and 1 ICSI embryo (n = 5) was 80% and after the transfer of 2 ICSI embryos (n = 9) this was 33%. CONCLUSION(S): Performing ICSI on some oocytes of a cohort may avoid total fertilization failures both in patients with a history of total fertilization failure and in patients with a history of low fertilization, as the percentage of fertilization is higher after ICSI compared to IVF and the recurrence of total fertilization failure and low fertilization is high after IVF treatment.