A case of delayed postoperative bleeding after excision of endometrial polyp using resectoscope in an infertile woman with von Willebrand disease:a case report and literature review.

Von Willebrand disease (VWD) is a bleeding disorder caused by a congenital quantitative reduction, deficiency, or qualitative abnormality of the von Willebrand factor (VWF). Here, we report a case of delayed postoperative bleeding in an infertile woman with endometrial polyps complicated by VWD. The patient was a 39-year-old infertile woman with type 2A VWD. At 38 years of age, she was referred to our hospital for infertility and heavy menstrual bleeding. Hysteroscopy revealed a 15-mm polyp lesion in the uterus. The patient was scheduled for transcervical resection (TCR) of the endometrial polyp. Gonadotropin-releasing hormone agonists were preoperatively administered to prevent menstruation. The VWF-containing concentrate was administered for 3 days according to guidelines. The patient was discharged on postoperative day 3 after confirming the absence of uterine bleeding. Uterine bleeding began on postoperative day 6. The patient was readmitted on postoperative day 7 and treated with VWF-containing concentrate for 5 days, after which hemostasis was confirmed. TCR surgery for endometrial lesions is classified as a minor surgery, and guidelines recommend short-term VWF-containing concentrate replacement. However, it should be kept in mind that only short-term VWF-containing concentrate replacement may cause rebleeding postoperatively.

Successful pregnancy and delivery after a vitrified-warmed embryo transfer in a woman with Kallmann syndrome: A case report and literature review.

Kallmann syndrome, a congenital disorder of idiopathic hypogonadotropic hypogonadism associated with anosmia, results in infertility because of anovulation. Assisted reproductive technology (ART) is considered when optimal ovulation induction therapy is difficult or when several cycles of ovulation induction therapy do not result in pregnancy. However, evidence is lacking regarding the optimal ART treatment for Kallmann syndrome. We report the case of a 33-year-old woman who successfully achieved pregnancy and delivery after ART treatment. At 29 years old, she was diagnosed with Kallmann syndrome due to hypothalamic amenorrhea and anosmia. At 33 years old, she revisited the hospital, desiring a child after one year of infertility. Due to anovulation, she was treated with gonadotropin therapy, but controlling follicular development was difficult, and thus ART treatment was initiated. The controlled ovarian stimulation (COS) protocol for ART treatment employed gonadotropins, recombinant follicular stimulating hormone/human menopausal gonadotropin plus human chorionic gonadotropin, to promote follicular growth. Four oocytes were retrieved, and two cleaved embryos were vitrified and cryopreserved. After vitrified-warmed embryo transfer of a morula stage embryo in a hormone replacement cycle, pregnancy was achieved but resulted in a miscarriage. A second oocyte retrieval was performed under the same COS; four oocytes were retrieved, and two cleaved embryos were vitrified and cryopreserved. Further, a pregnancy was achieved through the vitrified warmed embryo transfer. At 40 weeks and 6 days of gestation, a baby boy weighing 3,344 g with an Apgar score of 7/8 was delivered vaginally. The mother's postpartum course and neonate were free from adverse events. For women with Kallmann syndrome, ART treatment and selective embryo cryopreservation may be a reasonable and safe option.

Severe Hemoperitoneum After Oocyte Retrieval Due to Ovarian Hemorrhage Possibly Caused by Violent Cough Under General Anesthesia With Propofol and Pentazocine: A Case Report and Literature Review.

Oocyte retrieval is an invasive procedure for patients undergoing assisted reproductive technology (ART). Complications regarding oocyte retrieval are rare; however, once they occur, patients often require hospitalization and, could in some cases, lead to life-threatening situations. We report a case of severe hemoperitoneum due to ovarian hemorrhage after oocyte retrieval. A 39-year-old patient wanted to have a second child and received ART treatment because of severe male infertility. Oocyte aspiration was performed under intravenous anesthesia with propofol and pentazocine. She suddenly coughed and violently moved her body while the puncture needle was inside the ovary. After increasing the propofol dose, the coughing subsided and oocyte retrieval continued. Five hours following oocyte retrieval, she revisited the clinic with severe abdominal pain. Transvaginal ultrasonography revealed echogenic findings in the Douglas' pouch; 300 mL of blood was aspirated by puncture of the Douglas pouch. She was transferred to our hospital because of suspected severe hemoperitoneum. Contrast-enhanced computed tomography revealed hematoma formation in the right ovary with a small amount of extravasation. Laparoscopic surgery revealed extensive hemoperitoneum due to right ovarian hemorrhage. The right ovary was enlarged to the size of a fist and contained a clot; the bleeding was due to a tear in the cortex of the ovary. The partially torn ovarian tissue was excised; hemostasis was performed using a bipolar device. The body movement caused by violent coughing during oocyte retrieval may have led to increased laceration at the ovarian puncture site. Although cough during general anesthesia using propofol and pentazocine is very rare, physicians and staff should be aware of the high risk of intra-abdominal hemorrhage when such complications occur.

Sho-Based Kampo Medicine Combined With Assisted Reproductive Technology Is Effective for Refractory Infertility and Early Recurrent Miscarriage: A Case Report.

Assisted reproductive technology (ART) is an effective treatment developed for infertile couples in the world. As a result, women suffering from infertility benefit from ART treatment. However, even when ART treatment is successfully performed, there are cases where conception is not achieved or maintained. Kampo medicine was originally developed in Japan, and Sho is the central part of Kampo concept. Although it is thought that Kampo medicine is useful for various women-specific symptoms in modern Japan, evidence is still lacking regarding the effectiveness of combination of Sho-based Kampo and Western medicine such as ART. In this article, we report a case of a patient with refractory infertility and early recurrent miscarriage (ERM) of unknown cause who successfully became pregnant with combination therapy of Kampo based on Sho and ART. The patient was a 34 year-old Japanese woman and had been treated with ART in a nearby clinic. In a 3 year period, she had undergone oocyte retrieval twice, frozen embryo transfer (FET) seven times and conceived twice. Since both conceptions ended in miscarriages and pregnancy could not be established thereafter, her clinic referred her to our hospital for Kampo treatment. As result of the diagnosis of her Sho-pattern, we chose Kampo medicine. Finally, she succeeded in conception 1.5 years after beginning treatment and was able to carry the fetus to term successfully. The current case showed that although our patient had been unable to give a birth after undergoing various western medical treatments for infertility, pregnancy was established and kept to term after addition of Sho-based Kampo treatment. Kampo medicine chosen by the Sho-patterns is useful for refractory infertility and ERM. It is important to note that examinations for evaluting the Sho-patterns are essential for selecting appropriate Kampo medicine. Sho-based Kampo leads to an increase in the effectiveness of ART treatment, and accumulation of evidence that clarifies Sho-pattern is required.

Effects of localisation of uterine adenomyosis on outcome of in vitro fertilisation/intracytoplasmic sperm injection fresh and frozen-thawed embryo transfer cycles: a multicentre retrospective cohort study.

BACKGROUND: Uterine adenomyosis is a benign disease, common among women in their 40 and 50 s, characterised by ectopic endometrial tissue in the uterine myometrial layer. Adenomyosis causes infertility and has a negative effect on the outcomes of in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) embryo transfer (ET) cycles. It has also been reported to have different characteristics depending on the adenomyotic lesion localisation. The effect of its localisation on IVF/ICSI-ET outcomes is unclear. This study aimed to investigate whether adenomyotic lesion localisation, assessed using magnetic resonance imaging (MRI), was associated with outcomes of IVF/ICSI-ET cycles. METHODS: This multicentre, joint, retrospective cohort study analysed the medical records of 67 infertile patients with adenomyosis who underwent IVF/ICSI with fresh and frozen-thawed ET at five participating facilities from January 2012 to December 2016 and for whom MRI data were available. Fifteen patients were excluded; therefore, the MRI data of 52 patients were evaluated by two radiologists. We assessed the localisation of and classified adenomyotic lesions into advanced (invades the full thickness of the uterine myometrium), extrinsic (localised on the serosal side), and intrinsic (localised on the endometrial side) subtypes. RESULTS: There were 40 advanced, nine extrinsic, and three intrinsic cases, and the outcomes of 100, 27, and nine ET cycles, respectively, were analysed. Pregnancy loss/clinical pregnancy and live birth rates of the advanced, extrinsic, and intrinsic groups were 64 % (16/25) and 9 % (9/100), 33.3 % (3/9) and 22.2 % (6/27), and 50 % (1/2) and 11.1 % (1/9), respectively. A logistic regression analysis adjusted for age, prior miscarriage, and body mass index showed that the extrinsic group had fewer pregnancy losses (odds ratio 0.06; 95 % confidence interval [CI]: 0.00-0.54, p = 0.026) and more live births (odds ratio 6.05; 95 % CI: 1.41-29.65, p = 0.018) than the advanced group. CONCLUSIONS: Adenomyotic lesions exert different effects on IVF/ICSI-ET outcomes. Thus, MRI assessments of adenomyosis in infertile patients are beneficial. Establishment of treatment plans based on adenomyotic lesion localisation should be considered.

Successful Planned Pregnancy through Vitrified-Warmed Embryo Transfer in a Woman with Chronic Myeloid Leukemia: Case Report and Literature Review.

A 35-year-old female patient with chronic myeloid leukemia (CML) wanted to have a child. She had been treated with imatinib and had achieved major molecular remission, after which imatinib was intentionally discontinued, and interferon-α treatment was initiated. After three failed cycles of artificial insemination with her husband's semen, the patient underwent treatment with assisted reproductive technology. After two cycles of in vitro fertilization, two embryos (8-cell stage and blastocyst) were cryopreserved. The patient again had elevated major BCR-ABL mRNA levels; thus, infertility treatment was discontinued. After 18 months of dasatinib treatment, major molecular remission was again observed, and the patient underwent vitrified-warmed embryo transfer with a single blastocyst. After that, she became pregnant. Discontinuation of tyrosine kinase inhibitors combined with the timely initiation of infertility treatments, including assisted reproductive technology, might thus be useful for treating women with CML who wish to become pregnant.

Mild phenotypes associated with an unbalanced X-autosome translocation, 46,X,der(X)t(X;8)(q28;q13).

Unbalanced X-autosome translocation can result in various phenotypic manifestations. We present the first case of 46,X,der(X)t(X;8)(q28;q13) in a 34-year-old female with relatively mild manifestations, including congenital heart disease, epicanthal fold, mild intellectual disability, and menstrual irregularity. Our findings expand the known spectrum of unbalanced X-autosome translocations, for improved clinical management.

Influence of radiofrequency-electromagnetic waves from 3rd-generation cellular phones on fertilization and embryo development in mice.

The purpose of this study was to evaluate the effects of 3rd-generation (3G) cellular phone radiofrequency-electromagnetic wave (RF-EMW) exposure on fertilization and embryogenesis in mice. Oocytes and spermatozoa were exposed to 3G cellular phone RF-EMWs, 1.95 GHz wideband code division multiple access, at a specific absorption rate of 2 mW/g for 60 min, or to sham exposure. After RF-EMW exposure, in vitro fertilization and intracytoplasmic sperm injection were performed. Rates of fertilization, embryogenesis (8-cell embryo, blastocyst), and chromosome aberration were compared between the combined spermatozoa and oocyte groups: both exposed, both non-exposed, one exposed, and the other non-exposed. Rates of fertilization, embryogenesis, and blastocyst formation did not change significantly across the four groups. Considering that the degree of exposure in the present study was ≥100 times greater than daily exposure of human spermatozoa and even greater than daily exposure of oocytes, the present results indicate safety of RF-EMW exposure in humans. Bioelectromagnetics. 38:466-473, 2017. © 2017 Wiley Periodicals, Inc.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

STUDY QUESTION: Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? SUMMARY ANSWER: The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. WHAT IS ALREADY KNOWN: To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. STUDY DESIGN, SIZE, DURATION: This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. LIMITATIONS, REASONS FOR CAUTION: Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. WIDER IMPLICATIONS OF THE FINDINGS: Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Effects of 1950 MHz W-CDMA-like signal on human spermatozoa.

There are growing concerns about how electromagnetic waves (EMW) emitted from mobile phones affect human spermatozoa. Several experiments have suggested harmful effects of EMW on human sperm quality, motility, velocity, or the deoxyribonucleic acid (DNA) of spermatozoa. In this study, we analyzed the effects on human spermatozoa (sperm motility and kinetic variables) induced by 1 h of exposure to 1950 MHz Wideband Code Division Multiple Access (W-CDMA)-like EMW with specific absorption rates of either 2.0 or 6.0 W/kg, using a computer-assisted sperm analyzer system. We also measured the percentage of 8-hydroxy-2'-deoxyguanosine (8-OHdG) positive spermatozoa with flow cytometry to evaluate damage to DNA. No significant differences were observed between the EMW exposure and the sham exposure in sperm motility, kinetic variables, or 8-OHdG levels. We conclude that W-CDMA-like exposure for 1 h under temperature-controlled conditions has no detectable effect on normal human spermatozoa. Differences in exposure conditions, humidity, temperature control, baseline sperm characteristics, and age of donors may explain inconsistency of our results with several previous studies. Bioelectromagnetics. 37:373-381, 2016. © 2016 Wiley Periodicals, Inc.

Effects of metformin on endocrine, metabolic milieus and endometrial expression of androgen receptor in patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate effects of metformin on endocrine, metabolic parameters and endometrial androgen receptor (AR) expression in polycystic ovary syndrome (PCOS). DESIGN: Prospective observational study. METHODS: We analyzed blood samples from 10 PCOS patients, of whom endometrium was also analyzed in five patients. Before and after three months of metformin treatment, endocrine, glucose metabolic parameters and the endometrial AR expressions were assessed. The relative expression levels of AR were examined by immunohistochemistry. MAIN OUTCOME MEASURES: The group post-treatment mean of homeostasis model assessment insulin resistance (HOMA-IR) for all patients was significantly lower than the pretreatment. Ovulation was restored in five out of 10 patients. The group post-treatment means of luteinizing hormone (LH) and HOMA-IR for the five ovulatory patients were significantly lower. The relative expression levels of endometrial AR were decreased in four out of the five patients as compared with pretreatment. In the four patients, one or more of the parameters, HOMA-IR, LH or free testosterone, decreased compared with pretreatment. CONCLUSION: Metformin was effective in decreasing HOMA-IR, LH, free testosterone and the relative expression levels of AR. Furthermore, metformin might improve the systemic and local environment of the uterus caused by hyperandrogenemia in PCOS patients with insulin resistance.

Regulation and effects of modulation of telomerase reverse transcriptase expression in primordial germ cells during development.

Telomere length maintenance in the germ line from generation to generation is essential for the perpetuation of eukaryotic organisms. This task is performed by a specialized reverse transcriptase called telomerase. While this critical function of telomerase has been well established, the mechanisms that regulate telomerase in the germ line are still poorly understood. We now show, using a Pou5f1-GFP transgenic mouse model, that telomerase suppression in quiescent male primordial germ cells (PGCs) is accompanied by a decrease in expression of murine telomerase reverse transcriptase (TERT). To further assess the role of TERT in quiescent PGCs, we developed a chicken Actb gene promoter/cytomegalovirus enhancer (CAG)-Tert transgenic mouse strain that constitutively expresses murine TERT. Telomerase activity was detected in quiescent PGCs from CAG-Tert transgenic embryos, demonstrating that re-activation of TERT expression is sufficient to restore telomerase activity in these cells and implying that TERT expression is an important mechanism of telomerase regulation in PGCs. Fluorescence-activated cell-sorting (FACS) analysis of PGC frequency and cell cycle status revealed no effect of either overexpression or deficiency of TERT in CAG-Tert transgenic mice or Tert knock-out mice respectively. These results demonstrate that TERT per se does not affect proliferation or development of PGCs, in contrast with recent studies that suggest that TERT has a telomere-independent effect in certain stem cells. It is possible that the direct effect of TERT on cell behavior may be dependent on cell type.

Tn5 transposase-mediated mouse transgenesis.

We have developed a novel method for mouse transgenesis. The procedure relies on a hyperactive Tn5 transposase to insert a transgene into mouse chromosomes during intracytoplasmic sperm injection. This procedure integrates foreign DNA into the mouse genome with dramatically increased effectiveness as compared to conventional methods such as pronuclear microinjection and traditional sperm injection-mediated transgenesis. Our data indicate that with this method, transgenic mice, both hybrids and inbreds, can be produced more consistently and with lower numbers of manipulated oocytes required for traditional microinjection methods. The transposase-mediated transgenesis technique is also effective with round spermatids, offering the potential for rescuing the fertility of azoospermic animals using sperm precursor cells.

Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI.

BACKGROUND: Recent studies showed that ICSI with cauda epididymal or ejaculated sperm of infertile mice or men, respectively, was less effective in fertilization and normal embryo development than ICSI using sperm from the testes. These studies suggested that sperm nuclear quality declined after release from the testis, but the site where this loss of fertility occurs has not been localized. METHODS: We performed ICSI with testicular, caput, and cauda epididymal sperm from infertile Tnp1-/-Tnp2+/- mutant mice, which have a minimal level of transition nuclear proteins and are sterile by natural mating. RESULTS: When the heads of motile sperm from the testis or caput epididymis of Tnp1-/-Tnp2+/- males were injected into enucleated mouse oocytes, sperm chromosomes showed no difference from those of wild-type mice, but the chromosomes from sperm taken from the cauda epididymis of mutant males showed increased abnormalities. Injection of testicular or caput epididymal sperm from Tnp1-/-Tnp2+/- males into intact oocytes resulted in normal embryonic and fetal development and yields of liveborn equivalent to wild-type, but cauda sperm from Tnp1-/-Tnp2-/- mice produced lower implantation rates and yields of liveborn than did those from wild-type mice. CONCLUSIONS: These results demonstrate that in mice with sperm chromatin abnormalities, the decline in fertility of sperm with ICSI occurs after the caput epididymis. The advantage of using caput epididymal sperm for ICSI in certain situations may be considered as an approach to be tested in human assisted reproduction.

Sperm tail abnormalities in mutant mice with neo(r) gene insertion into an intron of the keratin 9 gene.

Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out. While both heterozygous (K9(+/neo)) and homozygous (K9(neo/neo)) mutant mice expressed the wild-type form of K9 protein, the expression profile of the wild-type K9 in K9(neo/neo) mutants was modified. In addition, the open reading frame of the aberrant mRNA terminated at the exon 6/intron 6 splice site, resulting in a truncated K9 protein. Both K9(+neo) and K9(neo/neo) male mice displayed spermatids with ectopic manchette. Coiled tails were seen in maturing spermatids and epididymal sperm of mutant mice and sperm with deformed tails displayed forward motility. A predominant sperm anomaly was residual cytoplasm at the end of the mitochondria-containing middle piece tail segment. The residual cytoplasm displayed vesicles with random in situ motion, suggesting a transport impediment toward the distal end of the sperm tail. All mutant mice were fertile. Surprisingly, in oocyte nuclear injection experiments using K9(neo/neo) sperm donor, 76% of the resulting animals displayed a deletion of the neo(r) gene from the intron 6 of the mutated K9 allele. Results of this study support the view that intron 6 influences the transcriptional efficiency of the K9 gene by decreasing production of wild-type K9 and changing the expression of K9 proteins.

Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection.

The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.

Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities.

Reversible infertility can be induced in male mice by oral administration of the alkylated imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ). Spermatozoa of these mice have grossly misshapen heads and reduced motility. Because NB-DNJ and related compounds may hold promise as nonhormonal male contraceptives, a comprehensive examination of their effects on male reproduction is necessary. To this end, we further examined reproductive properties of the dysmorphic spermatozoa that are produced after short-term imino sugar administration at the minimal dose that completely abolishes the ability of male C57BL/6 mice to produce offspring by natural mating. Here, we report that, in vitro, the abnormal spermatozoa from the NB-DNJ- and NB-DGJ-treated mice were unable to fertilize oocytes. In addition, we investigated whether the imino sugars damage the genetic integrity of spermatozoa. To test this, we microsurgically injected deformed spermatozoa from imino sugar-treated males into oocytes. The deformed spermatozoa from the testis were able to activate oocytes very efficiently, but those from the cauda epididymis often failed to do so. This problem was overcome when the sperm-injected oocytes were treated with a parthenogenetic agent, Sr(2+). Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr(2+) treatment) into live offspring that grew normally and were normally fertile. This indicates that during short-term administration, alkylated imino sugars alter sperm morphology and physiology but do not diminish the genetic potential of spermatozoa.

Transition nuclear proteins are required for normal chromatin condensation and functional sperm development.

The histone-to-protamine transition is important in the formation of spermatozoa. In mammals this involves two steps: replacement of histones by transition nuclear proteins (TPs) and replacement of TPs by protamines. To determine the functions of the TPs and their importance for sperm development, we generated mice lacking both TPs, since mice lacking only TP1 or TP2 were fertile. Our results indicated that TP1 and TP2 had partially complemented each other. In mice lacking both TPs, nuclear shaping, transcriptional repression, histone displacement, and protamine deposition proceeded relatively normally, but chromatin condensation was irregular in all spermatids, many late spermatids showed DNA breaks, and protamine 2 was not posttranslationally processed. Nevertheless, genomic integrity was maintained in mature spermatids, since efficient fertilization and production of offspring were achieved by intracytoplasmic sperm injection. However, many mature spermatids were retained in the testis, epididymal spermatozoa were drastically reduced in number and were highly abnormal, and the mice were sterile. Most epididymal spermatozoa were incapable of fertilization even using intracytoplasmic sperm injection. Thus, in mammals TPs are required for normal chromatin condensation, for reducing the number of DNA breaks, and for preventing the formation of secondary defects in spermatozoa, eventual loss of genomic integrity, and sterility.