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The aim of this article is to gather 9 thought leaders and their team members to present their ideas about the future of in vitro fertilization and the andrology laboratory. Although we have seen much progress and innovation in the laboratory over the years, there is still much to come, and this article looks at what these leaders think will be important in the future development of technology and processes in the laboratory., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The in vitro fertilization and andrology laboratories are at the center of assisted reproductive technologies and the place where technicians and embryologists manipulate gametes and preimplantation-stage embryos with the goal of achieving the best embryo for transfer. Through the years, these laboratories have seen developments in technique, technology, and testing. The goal of this Views and Interviews series is to bring together the thought leaders in the field and envision what the laboratories will look like in the next 10 years., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Platelets modulate clinically relevant yet incompletely understood tissue regeneration processes, and platelet rich plasma (PRP) has been previously used with some success in various non-reproductive medical contexts. Here, we extended PRP application to ovarian tissue with a view to document impact on ovarian reserve among women attending for infertility treatment. PRP was freshly isolated from patients (n= 4) with diminished ovarian reserve as determined by at least one prior IVF cycle canceled for poor follicular recruitment response or estimated by serum AMH and/or FSH, no menses for ≥1 year. Immediately following substrate isolation and activation with calcium gluconate, approximately 5 mL of autologous PRP was injected into each ovary under direct transvaginal sonogram guidance. For each study subject, AMH, FSH, and serum estradiol data were recorded at two-week intervals post-PRP and compared to baseline (pre-PRP) values. In this pilot group, mean (±SD) patient age was 42 ± 4 years with infertility duration reported as 60 ± 25 months. Following this protocol of intraovarian PRP administration, increases in serum AMH (p = .17), decreases in FSH (p < .01), or both, were observed in all cases, sufficient to permit retrieval of 5.3 ± 1.3 MII oocytes. IVF occurred 78 ± 22 (range =  59-110) days after activated PRP injection, and results appeared independent of patient age, infertility duration, baseline platelet concentration or pretreatment antral follicle count. Each patient had at least one blastocyst suitable for cryopreservation. While autologous PRP has been successfully applied therapeutically to various tissues to accelerate healing and wound repair, this is the first description of direct injection of activated PRP into the human ovary of poor prognosis IVF patients. Evidence of improved ovarian function was noted in all who received intraovarian PRP, possibly as early as two months after treatment. Additional research is needed to clarify (and enhance) which PRP components are responsible for altered ovarian function, and to identify predictive characteristics for patients most likely to benefit from this intervention.

This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Among infertile couples, 25% involve both male and female factors, while male factor alone accounts for another 25% due to oligo-, astheno-, teratozoospermia, a combination of the three, or even a complete absence of sperm cells in the ejaculate and can lead to a poor prognosis even with the help of assisted reproductive technology (ART). Intracytoplasmic sperm injection (ICSI) has been with us now for a quarter of a century and in spite of the controversy generated since its inception, it remains in the forefront of the techniques utilized in ART. The development of ICSI in 1992 has drastically decreased the impact of male factor, resulting in millions of pregnancies worldwide for couples who, without ICSI, would have had little chance of having their own biological child. This review focuses on the state of the art of ICSI regarding utility of bioassays that evaluate male factor infertility beyond the standard semen analysis and describes the current application and advances in regard to ICSI, particularly the genetic and epigenetic characteristics of spermatozoa and their impact on reproductive outcome., (© 2017 Society for Reproduction and Fertility.)

Purpose: The purpose of this study was to investigate the utility of a combined GnRH-agonist (GnRH-a) and human chorionic gonadotropin (hCG) trigger in improving ICSI cycle outcomes in patients with poor fertilization history after standard hCG trigger in prior ICSI cycles., Methods: Retrospective cohort study. Patients with a fertilization rate of <20% in at least two prior ICSI cycles who subsequently underwent another ICSI cycle with hCG trigger were compared to those who underwent another ICSI cycle with a combined GnRH-a and hCG trigger. Oocyte maturity, fertilization, clinical pregnancy, and live birth rates were compared. A multiple linear regression model was used to explore the association between combined GnRH-a and hCG trigger (vs hCG trigger alone) and fertilization rate., Results: A total of 427 patients with mean age of 37.3 ± 1.94 years and mean baseline fertilization rate of 17.9 ± 2.03% were included, of which 318 (74.5%) and 109 (25.5%) patients underwent a subsequent ICSI cycle with hCG and combined GnRH-a and hCG trigger, respectively. The baseline parameters of the male and female partner were similar. The mean fertilization rate in the combined trigger group was 16.4% (95% CI: 7.58-25.2%) higher than the hCG trigger group, even after adjustment for confounders. Patients in the combined trigger group had higher oocyte maturity (82.1 vs 69.8%), higher clinical pregnancy (27.5 vs 5.67%), and higher live birth rates (20.2 vs 3.46%) compared to the hCG trigger group., Conclusions: Combined GnRH-a and hCG trigger in ICSI cycles increase oocyte maturity, fertilization, clinical pregnancy, and live birth rates in patients with a history of poor fertilization after standard hCG trigger alone.

Introduction: Although the majority of singleton births after in vitro fertilization (IVF) are uncomplicated, studies have suggested that IVF pregnancies may be independently associated with low birth weight (LBW), preterm birth (PTB), and perinatal mortality. These outcomes complicate multiple gestations as expected, but have also been reported in singletons. A multiple embryo implantation model allows for assessment of the early in utero environment, and therefore, assessment of any maternal constraints on developing fetuses. We question whether adverse perinatal outcomes associated with assisted reproductive techniques (ART) occur as a result of maternal physiologic adaptations., Patients and Methods: This is a retrospective, single center study of ART cycles, specifically intracytoplasmic sperm injection (ICSI) cycles during a 16-year period. For each positive pregnancy test 9-11 days after embryo transfer, an ultrasonogram was performed at 7 weeks of gestation to record the number of implanted fetal poles with cardiac activity. Controlled ovarian stimulation (COS), hCG trigger, oocyte retrieval and sperm injection were performed as per our standard protocols. First trimester implantation sites that resulted in live births were defined as "true" to distinguish them from those that spontaneously reduced called "virtual." Birth outcomes analyzed included birth weight and gestational age at delivery., Results: A total of 17,415 cycles were analyzed. The average maternal age was 36.9 (±5.0) years. An overall fertilization rate of 73.4% generated approximately 48,708 good quality cleavage-stage embryos. In most patients (92.8%), an average of 3 embryos were transferred. The clinical pregnancy rate was 39.2% (n = 6,281). The overall occurrence of multiple gestations was 38.2% (n = 2,608) consisting of 2,038 twin, 511 triplet, and 59 quadruplet pregnancies. Of these multiple gestations, 18.6% of twin, 54.2% of triplet and 76.3% of quadruplet gestations spontaneously reduced. Failure of the implanted embryo to progress was not related to maternal age. Singleton newborns resulting from multiple implantation sites had lower birth weights (P<0.01) and shorter gestational ages (P<0.01) than those from a single implanted embryo. The number of embryos transferred did not affect the gestational length of singleton newborns. Although the birth weights of singletons from multiple implantation sites (virtual singletons) were lower than true singletons, the birth weight of virtual singletons were comparable to the birth weights of true twin, triplet, and quadruplet live births. Multiple logistic regression revealed that virtual singletons were an independent risk factor for PTB (odds ratio: 4.55, 95% CI 2.23-9.29) and LBW (odds ratio: 3.61, 95% CI 1.78-7.32), even after controlling for the number of oocytes, stimulation protocol type, sperm source, total gonadotropins administered, age, embryo quality, and day of embryo transfer., Conclusions: Our study highlights that embryonic implantation sites during early gestation set the growth profile of each embryo, dictating later growth patterns. Specifically, spontaneous reduction of an embryo after multiple embryo implantations can confer greater perinatal risk in the form of LBW and PTB to the surviving fetus. Our findings suggest that maternal constraints or physiologic adaptations maybe one of the mechanisms mediating adverse perinatal outcomes when multiple embryo implantation occurs., Competing Interests: The authors have declared that no competing interests exist.

Early follow-up studies of IVF children showed that the frequency of birth anomalies resembled those arising with natural conception. More detailed analyses confirmed these findings, reinforcing the concept of the preimplantation period as teratologically "safe." The use of intracytoplasmic sperm injection (ICSI) to achieve fertilization introduced another variable.ICSI's safety has often been criticized because the fertilizing spermatozoon neither binds to the zona pellucida nor fuses with oolemma. Bypassing these physiologic steps together with the arbitrary selection of the spermatozoon has been reason for concern. Thus far, ICSI offspring undergoing adolescence and beyond has provided sufficient information to reassure these qualms. In fact, the health of the offspring generated through ICSI, once taken into consideration the gestational order, the age and the genetic makeup of the couples are generally reassuring.

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

For fertility patients undergoing in vitro fertilization (IVF), blastocyst culture brings a number of potential advantages over laboratory techniques leading to traditional cleavage-stage embryo transfer. Because day 2-3 embryos normally should transit the oviduct only, their direct exposure to an intrauterine microenvironment is physiologically inappropriate. This mismatch is obviated by blastocyst transfer. Moreover, the nutritional milieu inside the fallopian tube is not the same as within the endometrial compartment, a feature possibly antagonistic to implantation when a day 2-3 embryo is placed directly within the uterus. Delaying transfer to day 5-6 may also improve reproductive outcome by reducing risk of embryo expulsion, given increased myometrial pulsatility measured at day 2-3. However, rigid reliance on a blastocyst culture approach will more often result in treatment cancellation due to embryo loss (no transfer), or having fewer embryos for cryopreservation. The development of sequential media to support embryos in extended in vitro culture was a significant laboratory refinement, since it enabled direct observation of embryos to improve transfer selection bias. This approach, in tandem with blastocyst cryopreservation, leads to fewer embryos being transferred and reducing multiple gestation rate. This review discusses key features of human blastocyst culture and its application in clinical reproductive medicine practice.

Construction of artificial gametes may be made possible by transferring somatic cells into enucleated oocytes and inducing chromosomal halving of their nuclei. This study examines the possibility of constructing viable human gametes, and their potential for participation in normal fertilization. Spare germinal vesicle-stage oocytes were donated by consenting patients undergoing intracytoplasmic sperm injection (ICSI). Approximately 62% of in-vitro matured oocytes survived enucleation and subsequent cumulus cell injection. Following micromanipulation and subsequent activation, about 40% of the reconstituted oocytes yielded two pronuclear-like entities. This was not accompanied by extrusion of a polar body, but resulted in the formation of two 'putative haploid' pronuclei. Therefore selective removal of a female pronucleus marker was required to restore a balanced ploidy. Male pronuclei were identified by association with sperm mitochondria. Additional pronuclei were then removed, allowing further cleavage. Zygotes derived were 'putatively haploid' in approximately 38% of cases with a limited number of chromosomes assessed. However, on karyotypic analysis, blastomeres isolated from cleaving embryos showed a chaotic distribution of chromosomes. Oocytes could induce 'putative haploidization' of transplanted somatic cell nuclei independently of donor cell gender. Fertilization of artificial oocytes was followed by embryonic cleavage despite blastocyst development and chromosomal content possibly being compromised.

Background: Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested., Results and Discussion: We contrast experimental culture of single blastomeres obtained by 1) non-destructive biopsy of embryos destined for transfer, and 2) isolation of karyotypically normal blastomeres from disaggregated ("dead") embryos considered unsuitable for transfer, and evaluate these approaches with regard to production of ESCs. Pluripotency was confirmed by morphological criteria and by quantification of divergent homeodomain proteins specific to undifferentiated cell development. Following ESC isolation and identification, assessment was conducted according to a novel ESC grading system, also proposed here., Conclusion: The role of reproductive endocrinology in ESC research remains paramount. In this report, we hypothesize new and expand on existing strategies having the potential to enhance human ESC isolation, identification and in vitro maintenance.

Objective: To determine whether the presence of a Y microdeletion confers any adverse effects on in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) outcome., Design: Retrospective case-control study., Setting: Academic infertility center., Patient(s): A total of 17 patients with Y microdeletions who attempted IVF/ICSI cycles at our center between March 1996 and March 2002 were studied. Study patients were analyzed in two groups: those who underwent testicular sperm extraction (TESE) and those for whom ejaculated sperm was used., Intervention(s): The two patient study groups were matched to controls treated at the same time who had either nonobstructive severe oligozoospermia or azoospermia with normal Y chromosomes. Controls were matched for age of the female partner, sperm concentration, and number of embryos transferred., Main Outcome Measure(s): Fertilization and clinical pregnancy rates., Result(s): Sperm was only obtained from patients with azoospermic factor (AZF)c microdeletions (and from one patient with a partial AZFb microdeletion). A trend toward lower fertilization rates in patients with Y microdeletions was noted, which did not reach statistical significance. Clinical pregnancy rates per cycle and per transfer were similar to those for controls., Conclusion(s): Patients with AZFc microdeletions seem to have IVF/ICSI outcomes comparable to those of controls with normal Y chromosomes.

We report the delivery of healthy monozygous (MZ) twins in a 31 year-old nulligravida following gonadotrophin ovulation induction, ICSI, assisted hatching and fresh embryo transfer. Although a sonogram on day 35 confirmed that two of four transferred embryos had implanted, a second transvaginal sonogram 1 week later showed each gestational sac had two conceptuses-all four were associated with distinct amniotic compartments. Cardiac activity was observed in all four embryos. At 12 weeks and 5 days gestation, chorionic villus sampling was performed on fetuses 1 and 2 which were euploid for chromosomes 13, 18, 21, X and Y via fluorescence in-situ hybridization analysis. Subsequent KCl injection into sacs 3 and 4 resulted in asystole for these fetuses, while cardiac activity in sacs 1 and 2 remained unchanged after reduction. A twin vaginal delivery occurred at 36 weeks gestation, resulting in the birth of two male infants and one placenta. This case represents the first known report of human quadruplet pregnancy consisting of two MZ twin sets conceived by assisted reproductive techniques. Our report reviews proposed mechanisms for explaining twinning, with special emphasis on zona pellucida micromanipulation and subsequent MZ twin induction.

The promise of medical innovation has long evoked social commentary, particularly when personal reproductive autonomy may be involved. Development of the oral contraceptive, effective and safe surgical sterilization, and later IVF and ICSI are among the revolutionary developments where the initial reactions were dubious but were accorded mainstream status with sufficient clinical experience. In each instance, debate about the moral and social implications of these treatments accompanied their introduction into the medical marketplace. This pattern appears to be repeating itself in connection with the use of preimplantation genetic diagnosis (PGD) for elective sex selection of human embryos. As with prior challenges in reproductive medicine, the development of meaningful "guidelines" for this latest controversy has proven to be a contentious task. Indeed, the progression of ethics committee reports from the American Society for Reproductive Medicine seems to echo the ambivalence within society at large regarding this issue. In this report, we chronicle sex selection claims based on sperm sorting, and describe how flow cytometry and especially PGD have facilitated this selection at the gamete and embryo stage, respectively. In doing so, we also explore market forces and practitioner considerations associated with the application of PGD for this; related ethical issues with particular emphasis on the progeny derived from such treatment are also reviewed.

In-vitro fertilization (IVF) has made great strides in the past 25 years including the development of micromanipulation techniques. Intracytoplasmic sperm injection (ICSI) for treatment of severe male factor infertility paved the way for the use of the micromanipulator. Micromanipulation techniques are now an integral part of an IVF program and are now also employed to biopsy embryos from patients harboring genetic abnormalities. Micromanipulation has also been used in the development of nuclear transfer techniques--a potential solution for creating "younger" oocytes for women of advanced maternal age and spermatozoa for azoospermic men. Other recent developments include the sequential media enabling the embryo to survive to blastocyst stage and also successful oocyte vitrification to preserve future fertility. This paper describes the latest technologies in in-vitro fertilization and relates them to the total problem of infertility, age-related and otherwise.

Objective: To investigate the efficacy of using intentionally cryopreserved epididymal sperm in selected cases of obstructive azoospermia., Design: A retrospective, nonrandomized study., Setting: Academic research environment., Patients: One hundred forty-one couples undergoing first-time IVF/ICSI using either fresh or cryopreserved epididymal sperm., Interventions: The epididymides were microsurgically aspirated., Main Outcome Measures: Clinical pregnancy rates., Results: Motile sperm were obtained from all men. For the fresh group, the mean total sperm aspirated was 99 x 10(6) with 5.5 vials frozen per patient after ICSI and 82 x 10(6) with 4.7 vials frozen per patient in the cryopreserved group. No statistically significant difference in oocyte fertilization rate or number of embryos transferred was noted between groups. Of 108 patients using freshly aspirated sperm, 72 (66.7%) achieved clinical pregnancy. Of 33 patients in the group using cryopreserved sperm, 20 (60.6%) achieved clinical pregnancy (P=0.47)., Conclusions: In selected ideal cases of unreconstructable azoospermia, elective open microsurgical epididymal sperm aspiration with cryopreservation yields pregnancy rates similar to that employing fresh sperm. The advantages of this method are: (1) Use of cryopreserved sperm obviates the logistics problems associated with the use of fresh sperm. (2) Abundant high-quality sperm can be cryopreserved in a single procedure for all future attempts at IVF/ICSI. Rarely, viable sperm will not be present after thawing, and fresh retrieval will be necessary.

To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.

The human zygote relies on the paternal gamete to provide the centrosome component essential for the first mitotic division. It is not known whether normal centrosome function requires an intact spermatozoon, or whether donation of an isolated paternal centrosome component can result in normal zygotes and embryos. To explore this possibility, mature human oocytes were microinjected with either intact or dissected spermatozoa. Fertilization and cleavage rates were documented; nuclear and cytoskeletal changes were observed with fluorescent immunocytochemistry; and chromosomal normality was assessed with fluorescent in-situ hybridization. A pilot study was performed to identify cytoskeletal features suggestive of centrosome function. Unfertilized oocytes and tripronucleate (3PN) zygotes from in-vitro fertilization or intracytoplasmic sperm injection were assessed to confirm the sequence of the landmarks of human fertilization. Oocytes injected with mechanically-dissected spermatozoa appear to be capable of normal pronuclear formation and embryonic cleavage, but do not undergo normal mitotic division. Although decondensed, apposed nuclei are noted in combination with diffuse cytoskeleton assembly, no spindle was detected in any zygote resulting from the injection of a dissected spermatozoon. Analysis of selected embryos resulting from dissected sperm injection revealed chromosomal mosaicism in the majority of specimens. The lack of a bipolar spindle, in combination with chromosomal mosaicism, suggests abnormalities of the mitotic apparatus when sperm integrity is impaired following dissection.

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.

The impact of intracytoplasmic sperm injection (ICSI) on cryopreserved zygotes and embryos was evaluated by comparing embryo survival and implantation between embryos derived from ICSI and those derived from standard insemination procedures. The study included patients whose excess zygotes and embryos were cryopreserved between September 1993 and December 1994 and who subsequently underwent a frozen embryo transfer. Embryo survival, clinical pregnancy rates per transfer and pregnancy outcome were compared. Three hundred and thirty eight cryopreservation cycles, during which 1471 embryos were cryopreserved, were included in this study. Of those, 961 were derived from oocytes fertilized by insemination in vitro and 510 were derived from oocytes fertilized by ICSI. A total of 690 of the embryos (451 in the insemination group and 239 in the ICSI group) have since undergone a thaw cycle. The embryo survival rates were similar between the two groups (70.5 and 73.2%, insemination and ICSI respectively) and were not significantly affected by the stage at cryopreservation. There was no significant difference in pregnancy rates per transfer (31.8 and 32.3%), the preclinical pregnancy loss rate (16.7 and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%) between the insemination and the ICSI groups respectively. It is concluded that ICSI does not have an adverse impact on the survival and successful implantation of cryopreserved and thawed embryos.

With the introduction of intracytoplasmic sperm injection (ICSI), couples with severe male factor infertility have achieved fertilization and clinical pregnancy rates comparable to other in-vitro fertilization (IVF) patients. However, failure of fertilization still occurs in some patients despite the utilization of microsurgical sperm injection techniques. How such fertilization failure after ICSI might impact later ICSI treatment(s) is unknown. In this investigation, couples with complete fertilization failure after ICSI treated from August 1993 to August 1996 were identified (index cycle, n = 21). Additionally, fertilization data from any previous or subsequent infertility treatments were evaluated. Seven patients (33%) had at least one IVF treatment before the index cycle, although no deliveries occurred. Of patients with complete fertilization failure in the index cycle, 48% (n = 10) underwent at least one subsequent ICSI cycle which proceeded to oocyte retrieval. The remainder (n = 11) elected to discontinue treatment. Although six subsequent cycles were cancelled due to poor follicular response (< or = 2 mature oocytes), all patients electing to continue treatment eventually achieved a subsequent embryo transfer. The clinical pregnancy rate per transfer was 45.4% for this group; the delivery and ongoing pregnancy rate per transfer was 36.3%. Review of semen parameters, superovulation characteristics or other clinical parameters during the three study cycles (pre-index, index, and post-index) was not prognostic of fertilization success or reproductive outcomes in later treatments. Fertilization failure with ICSI therefore could not be predicted by prior cycle performance, although total immotility of spermatozoa at time of oocyte retrieval, total teratozoospermia, and low oocyte yield were common characteristics of couples experiencing complete fertilization failure with ICSI. These findings suggest that fertilization failure in one ICSI cycle does not preclude successful fertilization and delivery in a later ICSI treatment.

The purpose of this study was to investigate any influence of maternal and/or paternal age on gamete characteristics and pregnancy outcomes in intracytoplasmic sperm injection (ICSI) cycles. In all, 821 consecutive ICSI cases were analysed retrospectively. While a significant linear decline in semen volume was detected, no significant differences in the concentration, motility or morphology of the spermatozoa were found with paternal ageing. A significant decline in the number of oocytes retrieved and the number of mature oocytes obtained was found with advancing maternal age. An increase in the occurrence of digyny was noted with parental ageing, while no difference in single or bipronuclear fertilization was found. Older women had a decreased incidence of single pronucleus formation and an increase in digyny, but no significant difference in the percentage of oocytes that underwent two-pronuclear fertilization was detected with regard to maternal ageing. Pregnancy outcomes were not influenced by the age of the male partner, while a strong negative correlation was found with maternal ageing. To better analyse male partner ageing as a factor affecting pregnancy outcome, we analysed a subgroup of patients with a female partner aged <35 years who underwent ICSI. No paternal influence on ICSI pregnancy outcome was found in this subgroup of patients. We conclude that the influence on pregnancy outcome after ICSI is related mostly to maternal and not paternal age.

Objectives: To provide fertility for men with nonobstructive azoospermia., Methods: A retrospective review of treatment results at a university infertility center was undertaken. Sixteen couples entered an attempted in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycle for treatment of nonobstructive azoospermia. Each man was azoospermic, and the male factor diagnosis of nonobstructive azoospermia was made on testis biopsy for 14 men and on clinical grounds for 2 men. Sperm were retrieved by testicular biopsy on the day of oocyte retrieval. Results of testicular examinations, serum follicle-stimulating hormone levels, and testicular histology as well as evaluation of the success rates of sperm retrieval, fertilizations, and pregnancies were made., Results: Sperm were extracted from testis biopsies in 10 of 16 (62%) testicular sperm extraction (TESE) attempts. For cycles in which sperm were retrieved, normal fertilizations were achieved for 51 of 98 (52%) mature oocytes injected with testicular sperm in 10 couples. Biochemical pregnancies were achieved for 6 of 16 (38%) couples, with clinical pregnancies during 5 of 16 (31%) attempts at sperm retrieval, and ongoing pregnancy and subsequent live delivery for 4 of 16 (25%) attempts. CONCLUSIONS; Pretreatment clinical parameters are unable to predict which men with nonobstructive azoospermia will have spermatozoa retrieved by TESE. When sperm are found, clinical pregnancies can occur for half (5/10) of these couples using TESE with ICSI, with ongoing pregnancy and delivery for 4 of 10 (40%). Many men with nonobstructive azoospermia will have retrievable sperm with testis biopsy that are suitable for ICSI; however, 6 of 16 (38%) couples will not have sperm retrieved with TESE and may undergo an unnecessary IVF procedure.

Objective: To analyze the in vivo development of embryos conceived after intracytoplasmic sperm injection (ICSI), as well as obstetric outcome, occurrence of chromosomal abnormalities, and rate of congenital malformations in neonates born as a result of this treatment., Design: Retrospective study., Setting: University-based in vitro fertilization (IVF) clinic., Patients: A total of 751 couples in whom the male partners were presumed to be the cause of repeated failed IVF attempts or whose semen parameters were unacceptable for conventional IVF treatment., Interventions: Analysis of pregnancies resulting from 987 ICSI cycles; pregnancy outcome data were obtained from the records of obstetrician-gynecologists and/or pediatricians., Main Outcome Measures: Pregnancy rates, obstetric outcome, and frequency of chromosomal abnormalities and congenital malformations., Results: The overall clinical pregnancy (fetal heartbeat) rate was 44.3%, with a resultant delivery rate per ICSI cycle of 38.7% (n=382). In 8 of 11 miscarriages for which cytogenetic data were available, an autosomal trisomy was found, and 7 additional pregnancies were terminated because of a chromosomal abnormality after prenatal diagnosis. There was an equal distribution of vaginal vs cesarean deliveries (n=192 and n=190, respectively). Of the 578 neonates resulting from treatment by ICSI, 15 (2.6%) presented with congenital abnormalities (9 major and 6 minor abnormalities). However, this frequency of malformations is lower than that observed in offspring born after standard IVF at our institution. Furthermore, when pregnancy outcome of ICSI vs IVF was analyzed in terms of semen origin, no differences were found in the frequency of miscarriages or in the rate of congenital malformations., Conclusions: The evolution of pregnancies and occurrence of congenital malformations following treatment by ICSI were within the range observed with standard in vitro fertilization.

Objective: To describe the novel micromanipulation technique known as intracytoplasmic sperm injection (ICSI), which has been applied successfully to treat male factor infertility, even in patients with severely impaired sperm characteristics. This paper reviews the historical aspects that led to the development of ICSI in the animal model as well as the current experience in the human., Design: Before using assisted fertilization techniques to enhance fertilization of human gametes, it is imperative that practitioners gain extensive experience in the animal model. In addition, criteria for accepting individuals for treatment with ICSI are discussed along with other applications of the procedure in infertile couples who do not benefit from standard IVF., Results: Because ICSI resulted in limited success in animal models, it seemed unlikely that it would be successful in humans. Yet, ICSI now appears to be the most successful and significant innovation developed for dealing with male factor infertility since the emergence of IVF itself. To date, a relatively large group of healthy children have been born from this technology and there appears to be no increased incidence of congenital malformations., Conclusions: The consistently high success rate resulting from the application of ICSI to treat couples with male factor infertility is comparable to the results obtained using standard IVF techniques performed in nonmale factor couples. This finding indicates that spermatozoa obtained from subfertile men selected for intracytoplasmic injection are usually genotypically normal.

This study was conducted to determine whether the mode of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) influences fertilization by immature spermatozoa. Of the 837 ICSI cycles evaluated, 81 were performed with epididymal or testicular spermatozoa; 35 cycles with epididymal spermatozoa immobilized in the standard fashion resulted in fertilization and pregnancy rates of 48.3 and 51.4% respectively. When a more aggressive sperm immobilization technique (i.e. permanently crimping the sperm flagellum between the midpiece and the rest of the tail) was applied in 17 cycles, the resultant fertilization and pregnancy rates were significantly (P < 0.05) higher: 82.0 and 82.4% respectively. Similar increases in fertilization and ensuing pregnancy rates were also observed in ICSI cycles with the aggressive immobilization of frozen-thawed epididymal spermatozoa (eight cycles) versus standard immobilization (16 cycles). However, the fertilization rates for ICSI using testicular spermatozoa (five cycles) were basically the same, regardless of the immobilization technique. Furthermore, for ejaculated spermatozoa (756 cycles), the fertilization rates following aggressive sperm immobilization were also positively affected (73.4%), although no statistical differences in the clinical pregnancy rates were found. Because aggressive immobilization appears to affect sperm membrane permeabilization, the enhanced fertilization patterns observed in immature spermatozoa following aggressive immobilization may suggest a different membrane constitution in these spermatozoa. These findings indicate that immature gametes may require additional manipulation to enhance the post-ICSI events essential for adequate nuclear decondensation.

The aim of this study was to analyse the various reactions displayed by the oolemma to the penetrating pipette during intracytoplasmic sperm injection (ICSI) and correlate them with clinical factors, oocyte survival and fertilization patterns. Three types of oolemma responses were observed: normal breakage, when the injection needle created an invagination that ruptured at the approximate centre of the egg; sudden breakage, when the membrane broke without creating a funnel; and difficult breakage, when the membrane did not break or broke after several penetration attempts. A total of 2928 oocytes were analysed with the following observations: 73.9% (n = 2164) experienced normal breakage, 11.8% (n = 345) sudden breakage, and 14. 3% (n = 419) difficult breakage. The survival rate and number of normally fertilized oocytes were significantly lower and the incidence of digynic oocytes was significantly higher in the sudden breakage group; furthermore, in this group a significantly shorter length of stimulation was observed along with lower serum oestradiol concentrations when compared to oocytes experiencing normal and difficult breakage patterns. These recorded patterns were predictive of the survival and fertilization ability of the injected oocytes, as well as the incidence of digyny. The link between membrane behaviour and various clinical parameters appears to indicate a correlation between the modality of stimulation and oolemma characteristics.

The purpose of this study was to assess the genetic status of abnormal zygotes following assisted fertilization. Dispermic, monopronucleated and digynic zygotes were allowed to cleave intact or after enucleation, and on the biopsied blastomeres, multiplex polymerase chain reaction and fluorescent in-situ hybridization were performed. It was found that the distal pronucleus was usually male in origin in dispermic embryos, and that the sex ratio was restored when they were enucleated; however, they became mosaic at metaphase and their genetic heterogeneity was not restored after enucleation. Monopronucleated zygotes derived from standard in-vitro insemination can be transferred to the patient, since they usually showed normal diploid complement in their cells. On the contrary, single-pronucleated zygotes derived from intracytoplasmic sperm injection were usually activated parthenogenetically, but not fertilized. Digynic embryos, unlike dispermic ones, had a very low incidence of mosaicism, and when present, such mosaicism originated at a later embryo division. Most of the digynic embryos were triploid, indicating that the first division was normal and bipolar; moreover, when the female pronucleus was removed, they became diploid and their genetic status was considered normal. The recognition and understanding of fertilization abnormalities allow the identification of methods leading to their avoidance or correction.

Objectives: To evaluate the importance of in vitro micromanipulation techniques, specifically intracytoplasmic sperm injection (ICSI), for the fertility treatment of men with congenital absence of the vas deferens (CAV) or other unreconstructable male reproductive tract obstruction., Methods: Results using ICSI during in vitro fertilization (IVF) were compared to previously published results of IVF alone and IVF with other micromanipulation techniques at the same infertility center. Main outcome parameters evaluated were: fertilization rate per oocyte, clinical pregnancy rate, and ongoing pregnancies and deliveries., Results: IVF with ICSI yielded a fertilization rate per oocyte of 140 of 312 (45%) and a clinical pregnancy rate of 14 of 27 (52%) per cycle of sperm and egg retrieval. Ongoing pregnancies or deliveries have occurred for 13 of 27 (48%) cycles with ICSI. These results were better than our previously published results of IVF alone or in conjunction with the micromanipulation techniques of subzonal insertion (SuZI) or partial zona dissection (PZD) that yielded a 119 of 631 (19%; P < 0.0001) fertilization rate, clinical pregnancy rate of 14 of 51 (27%; P < 0.001) and ongoing pregnancy or delivery for 12 of 51 cycles (24%; P < 0.001)., Conclusions: Epididymal sperm retrieval should be performed only when micromanipulation is available in conjunction with IVF to maximize chances of fertilization and subsequent pregnancies. The use of ICSI for epididymal sperm appears to maximize chances of pregnancy for couples with surgically unreconstructable obstructive male infertility.

Objective: To assess the effects of cystic fibrosis transmembrane-conductance regulator (CFTR) gene mutations on sperm function and fertility in men with bilateral congenital absence of the vas deferens., Design: Prospective., Setting: Division of urologic microsurgery and associated hospital-based IVF unit., Main Outcome Measures: Fertilization and pregnancy rates., Patients: Men referred to our fertility unit for treatment of bilateral congenital absence of the vas deferens, using sperm surgically retrieved from the epididymis with IVF and micromanipulation., Results: Of 45 men with bilateral congenital absence of the vas, 54% (19/35) tested were found to be carriers of CFTR gene mutations, with one compound heterozygote. Epididymal sperm from men affected with CFTR mutations fertilized 19% (29/152) of oocytes, whereas men without mutations fertilized 22% (44/204) of oocytes. Pregnancy rates (PRs) were 36% (5/14) for cycles involving men with CFTR mutations and 33% (5/15) for other patients with congenital absence of the vas deferens but without detectable CFTR mutations., Conclusions: The presence of detectable CFTR mutations does not affect fertilization rates or PRs for men with bilateral congenital absence of the vas deferens when IVF and micromanipulation are applied.

Micromanipulation techniques have been successfully applied to human gametes to assist fertilization when normal function is impaired. The past few years have witnessed the rapid evolution and clinical incorporation of this technology. The unprecedented efficiency of intracytoplasmic sperm injection far surpasses all previous methods. This method has thus become the best treatment option for many forms of male infertility.

Objective: To demonstrate the effectiveness of intracytoplasmic sperm injection to treat male factor infertility and to report on the achievement of fertilization and pregnancy compared with standard in vitro insemination., Design: Controlled clinical study., Setting: Couples suffering from male factor infertility treated in an academic research environment., Patients: Two hundred twenty-seven couples in whom the male partners were presumed to be the cause of repeated failed attempts at IVF or whose semen parameters were unacceptable for conventional IVF., Interventions: Oocytes for this study were harvested after superovulation with GnRH agonist and gonadotropins. After removing the cumulus cells, a single spermatozoon was injected directly into the cytoplasm of metaphase II oocytes, and the outcome was analyzed in terms of semen parameters and origin of semen sample., Main Outcome Measures: Fertilization and pregnancy rates (PRs) in relation to sperm parameters and origin of semen sample., Results: Two hundred twenty-seven couples were treated by intracytoplasmic sperm injection resulting in an ongoing pregnancy rate of 37.0% per retrieval (84/227). There were 47 singleton pregnancies (5 of which were vanishing twin pregnancies), 30 twin gestations, 6 triplet pregnancies, and 1 quadruplet pregnancy. The concentration of the total motile spermatozoa present in the ejaculate as well as the origin of the samples influenced the fertilization rate but not the pregnancy outcome., Conclusions: Intracytoplasmic sperm injection can be used successfully to treat couples who have failed IVF or who have too few spermatozoa for conventional methods of in vitro insemination. Sperm parameters do not clearly affect the outcome of this technique.

The purpose of this paper is to elucidate the experimental steps that led to the development of intracytoplasmic sperm injection (ICSI) and its application in the human. ICSI has become the most successful micromanipulation procedure for treating male infertility. A total of 355 in vitro fertilization (IVF) cycles utilizing ICSI are described; 180 couples were previously treated in 509 IVF cycles but achieved no fertilization and 175 couples could not be treated by IVF because of extremely poor semen parameters. Of the 3063 metaphase II (M II) oocytes retrieved, 2970 were injected with a survival rate of 93.6%, yielding 1917 bipronuclear zygotes (64.5%). In 148 patients, a foetal heart was evidenced by ultrasound; 11 of these patients miscarried between 7 and 13 weeks of gestation. The ongoing pregnancy rate was 38.6% (137/355) per retrieval and 40.5% (137/338) per embryo replacement. At the time of writing, there were 22 deliveries and one therapeutic abortion for a trisomy 21 chromosomal abnormality. In addition, 66 singleton, 37 twin, 10 triplet and 1 quadruplet pregnancies were ongoing. The concentration of motile spermatozoa in the ejaculate only slightly influenced the fertilization rate (P < 0.001) and the pregnancy outcome (P < 0.01). A preliminary injection procedure utilizing intracytoplasmic injection of isolated sperm heads was performed in 35 M II human oocytes with resultant fertilization and cleavage rates of 74% and 73% respectively. Skills in ICSI were acquired by injecting hamster and unfertilized human oocytes with human sperm. ICSI can be used to successfully treat couples who have failed IVF or who have too few spermatozoa for conventional in vitro insemination.(ABSTRACT TRUNCATED AT 250 WORDS)

Uni-pronuclear embryos (n = 42) were analysed by fluorescence in-situ hybridization (FISH) with two to four chromosome pair-specific probes. Half of these embryos resulted from conventional insemination and half from intracytoplasmic sperm injection (ICSI). The majority of uni-pronuclear embryos from conventional insemination were normally diploid (61.9%) whereas only 9.5% of uni-pronuclear ICSI embryos (P < 0.001) were diploid. In addition, a significantly higher number of uni-pronuclear embryos from conventional insemination had a Y chromosome (10/21, 47.6%) when compared with ICSI embryos (2/21, 9.5%) (P = 0.015). It is concluded that the majority of uni-pronuclear embryos following regular in-vitro fertilization are fertilized, whereas those from ICSI are parthenogenetically activated. The latter embryos should not be considered for embryo replacement.