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Cutaneous vibratory stimulation and rectal probe electroejaculation are highly successful methods of obtaining semen in the anejaculate patient. We report a case in which spermatozoa were retrieved in a brain dead man by artificially stimulated ejaculation. The specimen was cryopreserved to be used at a later date in combination with assisted reproductive techniques.
Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey (Macacafascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that ovarian stimulation by GnRH-rFSH did not compromise the developmental competence of the oocytes; effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.
The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.
Advances in assisted reproductive techniques, specifically, development of protocols for production of in vitro matured, fertilized and cultured domestic bovine embryos, offer opportunities to apply these techniques to nondomestic bovidae in species preservation. Domestic bovine oocytes were inseminated with nondomestic bovine spermatozoa. Effects of heparin concentration, sperm concentration and their interaction on total and normal in vitro fertilization rates and on subsequent embryo development were evaluated. In different replications, semen from 3 Bos bison, 2 Bos gaurus, 1 Bos grunniens, and 1 Bos javanicus bulls was used. Treatment of spermatozoa included 2 heparin levels (2 and 8 micrograms/mL) and 3 sperm concentrations (1, 3 and 5 x 10(6)/mL). The B. grunniens bull exhibited excessive polyspermy in all treatments; therefore, 1 replicate was completed using 2 levels of heparin (0 and 1 microgram/mL) and 2 sperm concentrations (1 and 2 x 10(6)/mL). After 18 to 22 h, cumulus cells were removed from presumptive zygotes, and a portion thereof was compressed between a slide and coverslip and fixed in acetic acid:ethanol solution. Light microscopy was used to visualize pronuclei and the second polar body as a determinant of fertilization. Remaining presumptive zygotes were placed into embryo culture medium, and blastocyst development was assessed on Days 7 and 8 (fertilization = Day 0). Percentages of total and normal fertilization and of blastocyst formation were analyzed by a logistic regression model, isolating effects due to bull, heparin and sperm concentration, and to their interaction. Work presented here suggests that, just as in Bos taurus, the nondomestic bulls in the Bos species seem to have individual heparin and sperm concentration requirements for successful IVF. We conclude that each bull, domestic or nondomestic, needs to be evaluated individually. Preliminary sperm characterization using domestic cattle oocytes would result in a greater potential for generating purebred embryos of the desired species should scarce female gametes become available.
Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.
Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.
Effective therapies are available for the treatment of infertility owing to specific causes. Most hormonal imbalances can be readily identified and successfully treated. The treatment of men with unexplained idiopathic infertility remains difficult. The availability of a multitude of agents ranging from hormones to nutritional supplements emphasizes the fact that none are consistently effective. There is no good way to predict which patients will respond to a specific treatment. Idiopathic infertility may result from multiple discrete defects in sperm generation and maturation that are as yet unidentified. A better understanding of these defects will yield more effective treatment options and appropriate triage of patients to specific therapeutic regimens. Assisted reproductive techniques remain an option for patients with idiopathic male infertility; however, they are expensive and treat the female partner for a male problem. Multiple gestations and other complications are not infrequent. Initial management should be directed at improving the quality of semen to facilitate natural conception. Specific abnormalities should be corrected. If empiric pharmacologic therapy is to be used, treatment should last at least 3 to 6 months to incorporate a full 74-day spermatogenic cycle. The infertile couple should be advised of the inconsistent response to therapy and the low conception rate that may follow when compared with the results of ART. When empiric therapy is decided upon, antiestrogens may be used and are effective in a subset of patients. The authors prefer to use clomiphene citrate, 25 mg per day. The dose may be increased to 50 or 75 mg to raise testosterone levels to the upper normal range. Lack of a significant improvement in semen parameters or of a pregnancy after a 3- to 6-month treatment period may be an indication to proceed with ART.
Normal spermatogenesis is a complex process that depends on many factors. Genetics plays a major role in many of these factors including providing a normal hormonal milieu, the development of the testis and ductal system, and control of the stepwise maturation of sperm in the testis. The Y chromosome plays a key role in testis determination and control of spermatogenesis. Understanding how these genes work together can elucidate of the exact cause of infertility in some patients once thought to have idiopathic infertility. It is not only important that patients understand the cause of their infertility. Using sperm from these men to attain pregnancies by assisted reproductive techniques will probably result in infertile male offspring. Additional consequences are currently unknown but are the topic of research investigations.
BACKGROUND: Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero. METHODS: We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero. FINDINGS: 16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7). INTERPRETATION: Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.
In this issue we make a review of our experience in the management of the sterile men: protocols used for the study of men's sterility, causes and specific treatments, surgical recovery of spermatozoa and screening before IVF or IVF-ICSI. We concluded that with an adequate protocol, it's possible to establish the aetiology of men's infertility in a 78.8% of the cases and it's also possible to initiate a specific treatment in almost a 47.8% of men. Results of specific treatment, with a previous selection of patients, could be better than those of assisted reproduction techniques. Best results are obtained in cases of vaso-vasostomy (vasectomy reversal) and obstruction of ejaculatory duct. It's also needed a study of genetics causes and bad prognostic circumstances in those men whose partner is going on with an IVF or IVF-ICSI cycle.
OBJECTIVE: To verify the hypothesis that the incidence of chromosomal abnormalities significantly increases in the products of conception of males with sub-fertility, treated with assisted reproduction techniques (ART). STUDY DESIGN: All removed products of conception tissues were analyzed with standard cytogenetic techniques. A karyotype of the abortions was possible in 35 cases of IVF and 29 of ICSI. RESULTS: 15/35 (43%) IVF abortions and 14/29 (48%) ICSI abortions, respectively, have shown a chromosomal abnormality. The most frequent abnormality was monosomy X (45,X0). CONCLUSION: No significant difference in the incidence of embryonic anomalies was found between IVF and ICSI group. However, both IVF and ICSI allow pre-implantation diagnosis and embryo chromosomal evaluation should be considered, before transfer. Genetic counselling and consideration of prenatal diagnosis is suggested as integral part of planning of treatment strategies for 'at risk couples'.
Due to the implementation of assisted reproduction techniques, the incidence of multiple pregnancies associated with fetal and neonatal complications has significantly increased. A woman in the 24th week of a triplet pregnancy came to the hospital because of premature rupture of membranes of one amniotic sack and she had a miscarriage of one of the fetuses the same day. After confirmation of the viability of the two fetuses, she was kept under observation with antibiotic therapy only. The woman gave live birth to these remaining fetuses in her 32nd week of pregnancy. The outcome of this case demonstrates that watchful expectancy may be a feasible alternative to invasive intervention. The aim of this report is to add to the currently very limited literature of an expectant (conservative) policy with regard to pregnancy outcome after the early loss of a fetus from a multiple pregnancy.
A heterotopic pregnancy is in effect a multiple pregnancy with one or more intrauterine pregnancies coexisting with an ectopic pregnancy and is rarely spontaneous. With the increasing popularity of ovulation induction performed during assisted reproductive techniques, it will not be surprising to observe that this phenomenon has increased significantly. However, diagnosis is often delayed because of its rarity and difficulty. We report a case of a woman with a viable intrauterine pregnancy who had a complication of ovarian hyper-stimulation syndrome secondary to ovulation induction following in vitro fertilization and embryo transfer, but who, during hospitalization, presented with clinically progressive abdominal pain. An unruptured ectopic pregnancy of the right fallopian tube was diagnosed accidentally by laparoscopy and laparoscopic salpingectomy was immediately performed. Post-operative follow-up revealed that the intrauterine pregnancy continued normally. She delivered a normal female baby at 38 weeks of gestation. The promising neonatal outcome might suggest that laparoscopy might be safely performed to aid differential diagnosis in an uncertain condition during pregnancy: therefore, laparoscopic surgery might be an appropriate method to manage some carefully selected patients with HP. A brief review of the published literature on the role of laparoscopy in the diagnosis and management of heterotopic pregnancy is given.
Many genes that are required for fertility have been identified in model organisms (). Mutations in these genes cause infertility due to defects in development of the germ cell lineage, but the organism is otherwise healthy. Although human reproduction is undoubtedly as complex as that of other organisms, very few fertility loci have been mapped (). This is in spite of the prevalence of human infertility, the lack of effective treatments to remedy germ cell defects, and the cost to couples and society of assisted reproductive techniques. Fifteen percent of couples are infertile and half of all cases can be traced to the male partner. Aside from defects in sperm production, most infertile men are otherwise healthy. This review is divided into two distinct parts to discuss work that: (i) led to the identification of several genes on the Y chromosome that likely function in sperm production; and (ii) implicates DNA repair in male infertility via increased frequency of mutations in DNA from men with meiotic arrest.
Oocyte donation is an effective treatment modality for women lacking functioning ovaries, but also for women in whom repetitive in-vitro fertilization (IVF) cycles did not result in the development of adequate number of oocytes as well as for those at risk of transmitting genetic diseases. In women with ovarian failure, artificial menstrual cycles are required in order to produce endometrial growth and differentiation similar to that in women with normal ovarian function. Synchronization of donor's and recipient's cycles is mandatory, since the window of implantation is rather limited. The excellent results of oocyte donation treatment confirm that this assisted reproduction technique can provide a novel approach for the treatment of infertility in these groups of patients. Nevertheless, pregnancies in women of advanced reproductive age are associated with significantly more obstetrical complications and higher perinatal morbidity and mortality rates. Furthermore, aging parents have considerably higher chances to develop serious or life-threatening diseases. Thus, careful medical screening and extensive counselling is mandatory, taking into account the psychosocial ramifications of the procedure and, especially, the best interest of the child-to-be.
OBJECTIVES: To describe characteristics of multiples pregnancies in southern Reunion Island. MATERIAL AND METHODS: A three-year [corrected] cross-sectional observational study aimed at describing risk factors, events and [corrcected] complications and associated with twin pregnancies from 22 weeks gestation onward, within a tertiary care hospital centre, the Groupe Hospitalier Sud-Réunion. RESULTS: The study included 241 multiple pregnancies out of 15 837 pregnant women. The regional incidence of multiple pregnancies was 1.5%. Indicators of eligible twin pregnancies (n=234) were mother age up or equal to 35 years (63% of women, OR 8.4; CI95% 6.3-11.1), infertility treatments (15%): ovarian stimulations (OR: 50.2; CI95% 24.7-102) and in vitro-fertilisations (OR: 44.3; CI95% 22.6-86.3). A low maternal corpulence before pregnancy (BMI<20) and celibacy were negatively associated with twin pregnancies. Prenatal cares for twin pregnancies were globally adequate. The need for hospitalisation was 50% (30% of whom for preterm delivery threats). Twin pregnancies increased risks for pre eclampsia (OR: 3.0; CI95% 1.6-5.7) and gestational diabetes (OR: 1.9; CI95% 1.2-2.8). Caesareans and instrumental delivery rates were 50% and 12% (vs 16% and 7%), respectively. Twin infants were preterm for 62% and very preterm for 18%. Last, they were more likely to die than singletons (perinatal mortality: 78 per thousand for monochorionic twins, 57 per thousand for bichorionic twins vs 17 per thousand, p<0.0001, respectively). CONCLUSION: In Reunion Island, the incidence of multiple pregnancies is similar to that seen in Europe, but seems less due to assisted reproduction techniques. In return, they are associated with higher morbidity and mortality rates.
Insemination of recipients for oocyte transfer and gamete intrafallopian transfer (GIFT) in five experiments were reviewed, and factors that affected pregnancy rates were ascertained. Oocytes were transferred into recipients that were (1) cyclic and ovulated at the approximate time of oocyte transfer, (2) cyclic with aspiration of the preovulatory follicle, and (3) noncyclic and treated with hormones. Recipients were inseminated before, after, or before and after transfer. Intrauterine and intraoviductal inseminations were done. Pregnancy rates were not different between cyclic and noncyclic recipients (8/15, 53% and 37/93, 39%). The highest numerical pregnancy rates resulted when recipients were inseminated with fresh semen from fertile stallions before oocyte transfer or inseminated with cooled transported semen before and after oocyte transfer. Oxytocin was administered to recipients before oocyte transfer when fluid was imaged within the uterus. Administration of oxytocin to recipients at the time of oocyte transfer resulted in significantly higher pregnancy rates than when oxytocin was not administered (17/26, 65% and 28/86, 33%). Intraoviductal and intrauterine inseminations of recipients during oocyte transfer resulted in similar embryo development rates when fresh semen was used (12/22, 55% and 14/26, 55%). However, embryo development rates significantly reduced when frozen (1/21, 5%) versus fresh sperm were inseminated into the oviduct. Results suggest that insemination of a recipient before and after transfer could be beneficial when semen quality is not optimal; however, a single insemination before transfer was adequate when fresh semen from fertile stallions was used. Absence of a preovulatory follicle did not appear to affect pregnancy rates in the present experiments. The transfer of sperm and oocytes (GIFT) into the oviduct was successful and repeatable as an assisted reproductive technique in the equine.
There is a pressing need to develop and use assisted reproductive techniques in wildlife species living in small and captive groups. We evaluated the effect of freezing on membrane integrity and fertilizing capacity of European mouflon (Ovis gmelini musimon) spermatozoa collected during the breeding season. After thawing, the percentage of live spermatozoa, stained with fluorescein isothiocynate labeled Pisum Sativum agglutinin and propidium iodide, was 47% of which 19% showed intact acrosomal membrane. After culture in TCM 199 + 10% FCS, the number of live spermatozoa was significantly (P < 0.01) lower than in a medium with oviductal epithelial cells. The absence of oviductal cells decreased significantly the fertilization rates (P < 0.05), 24.0 vs. 63.1 with oviductal epithelial cells and 59.1 in vivo of in vitro matured ovine oocytes. Polyspermic fertilization rate of oocytes was lower (P < 0.05) with oviductal epithelial cells (1.6) than in absence of cells (12.8). However, the percentage of embryos that reached blastocyst stage was significantly higher in vivo than in vitro. These results provide interesting preliminary data for the development of genetic resource banks for European mouflon.