Short Ejaculatory Abstinence in Normozoospermic Men is Associated with Higher Clinical Pregnancy Rates in Sub-fertile Couples Undergoing Intra-Cytoplasmic Sperm Injection in Assisted Reproductive Technology: A Retrospective Analysis of 1691 Cycles.

BACKGROUND: The current WHO abstinence recommendations are ideal only for clinical diagnosis, as in recent years a negative correlation of abstinence duration with good embryo development and clinical pregnancy rate has been seen. AIM: The aim of the study was to evaluate the impact of variation in abstinence period on fertilization, embryo development potential, pregnancy, and miscarriage rate in sub-fertile couples undergoing assisted reproductive technology (ART) treatment. SETTING AND DESIGN: A prospective analysis was conducted at a tertiary (level 3) infertility care clinic. MATERIALS AND METHODS: The study included analysis of 1691 cycles for the patient undergoing ART procedures between September 2017 and August 2019. The influence of ejaculatory abstinence (EA) was investigated based on variation in abstinence length with four groups: Group I - 1 day; Group II - 2-5 days; Group III - 6-7 days; and Group IV - EA length of ≥8 days. STATISTICAL ANALYSIS: Analysis of variance and Chi-square test were used to calculate P value. RESULTS: In our primary outcome, we have seen a strong positive correlation of abstinence duration with semen volume, total sperm count, total motile count, and difference between each group was significant. Secondary outcomes showed a significantly higher implantation rate, biochemical pregnancy rate was observed in Group I (1 day) per embryo transfer as compared to longer abstinence groups. This resulted in significantly higher clinical pregnancy rates in Group I 30.0% vs. 25.4% in comparison to longer abstinence groups. CONCLUSIONS: Our study has shown duration of abstinence is negatively correlated with positive ß-human chorionic gonadotropin rate, clinical pregnancy rate, and implantation rate. Lower miscarriage rate was also observed with shorter abstinence duration.

Impact of Vitamin D Supplementation on Semen Quality in Vitamin D-Deficient Infertile Males with Oligoasthenozoospermia.

INTRODUCTION: Male factor contributes to almost 50% cases of infertility. There is evidence of the effect of serum vitamin D on male fertility by helping in spermatozoa maturation and initiating acrosomal reaction. In India, vitamin D deficiency has been documented to be in the range of 50-90% among all the age groups. OBJECTIVE: To study the levels of vitamin D and the impact of vitamin D supplementation in vitamin D-insufficient/deficient infertile males with oligoasthenozoospermia. MATERIAL AND METHODS: This study is a longitudinal observation study. Infertile men with either oligozoospermia or asthenozoospermia with vitamin D levels < 30 ng/ml were enrolled. Vitamin D and calcium supplementation was given, and semen and hormone parameters were assessed at the end of 3 and 6 months. RESULT: There is significant improvement in the mean sperm concentration and progressive sperm motility in infertile males with oligoasthenozoospermia after 6 months of vitamin D supplementation (p value < 0.001). The overall clinical pregnancy rate in the study was 8.33% after vitamin D supplementation (p value 0.24). CONCLUSION: Vitamin D supplementation plays a crucial role in regulating male fertility.

A Preliminary Experience of Integration of an Electronic Witness System, its Validation, Efficacy on Lab Performance, and Staff Satisfaction Assessment in a Busy Indian in vitro Fertilization Laboratory.

BACKGROUND: Electronic witness system (EWS) is one of the recent advancements in the field of in vitro fertilization (IVF) that uses radiofrequency identification (RFID) technology to monitor all critical work carried out in each stage of IVF procedures cycle. OBJECTIVE: The main objective of the study was validation and integration of electronic witnessing system, assessment of its efficacy on lab performance, and staff satisfaction in a busy tertiary IVF center. MATERIALS AND METHODS: The study data included analysis of 187 consecutive cycles for installation and validation of EWS. The laboratory outcomes were analyzed for development of good-quality embryos followed up for the pregnancy outcome. RESULTS: A total of 751 RFIG tags were involved with 77 patient-assigned barcodes for the all the analyzed cycles. During validation of EWS, a total of 02 (0.46%) red flags were highlighted by EWS from pre-allocated tags within the frequency range of the reader. The maturation rate (83.1%), fertilization rate (74.3%), cleavage rate (93.5%), day 3 grade-A embryo development rate (64.6%), good grade blastocyst development rate (26.4%) were observed in EWS group that was comparable to other groups with no significant difference (P > 0.005). Frozen embryo transfer of EWS cases observed a clinical pregnancy rate (50.0%) that was higher than other groups though statistically not significant as sample size was small. CONCLUSIONS: Our preliminary study suggests that EWS does not affect the gametes, embryos, and pregnancy rate, however a larger randomized clinical trials should be undertaken to evaluate the safety and efficacy of EWS.

An Intrauterine Insemination Audit at Tertiary Care Hospital: A 4½ Years' Retrospective Analysis of 800 Intrauterine Insemination Cycles.

BACKGROUND: There are many variables that may influence the success rates of intrauterine insemination (IUI) treatment. Therefore, a regular audit program is needed for planning effective infertility treatment and improving pregnancy outcomes. AIMS AND OBJECTIVES: The main objective of this study was to identify the crucial predicting factors that can influence the IUI success. MATERIALS AND METHODS: A retrospective analysis of 800 IUI cycles done from January, 2013 to August, 2017 in 651 couples with various etiologies of infertility. The common etiologies included female factor of ovulatory dysfunction, tubal, endocrinal, male factor, male and female factors combined, and unexplained factors. Ovulation induction was done either by clomiphene citrate (CC) alone or in combination of CC with gonadotropins or pure gonadotropins only. Human chorionic gonadotropin trigger was given when at least one dominant follicle measuring ≥18 mm with an endometrial thickness of >7 mm was obtained. IUI was done post 36 h of trigger. The double-density gradient method was the preferred method of sperm preparation. RESULTS: In 800 cycles in corresponding 651 couples, the total outcome was 113 pregnancies (14.1%) per cycle with overall pregnancy rate (PR) per couple of 17.3%. The highest PR was observed in the patient with ovulatory dysfunction (21.2%), followed by patients with combined factor (15.1%) and male factor (14.7%). In the study, a higher PR was achieved in the female ≤25 years (18.9%) P < 0.04 with significant findings with duration of infertility ≤5 years (15.1%) having primary infertility (14.5%) with low body mass index <25 (14.1%). IUI success rate was highest in the first cycle (14.6%) followed by second cycle (14.0%) and third cycle (3.5%). CONCLUSION: IUI audit enables the characterization of prognostic factors to achieve improved PR. This study identifies the factors that can predict improved pregnancy outcome in women age ≤25 years and endometrium thickness between 9 and 11 mm. We also recommend IUI as a first line of infertility treatment for couples in low-income setting provided the women age and duration of infertility are acceptably low.

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects.

BACKGROUND AND OBJECTIVE: Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario. MATERIALS AND METHODS: A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup. RESULTS: Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals. CONCLUSION: Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.

Octaploidy in idiopathic thrombocytopenic purpura.

We report a case of an elderly 68-year-old male who presented in our hospital with chief complaints of petechial rashes and ecchymosis over extremities and bleeding from the oral cavity since 3-4 days prior to hospitalization. He saw a physician before coming to our hospital and received one dose of IV methylprednisolone and oral wysolone. He had come to our hospital for further management. Bone marrow karyotyping was done and chromosomal analysis revealed two cell lines. Eighty percent of the cells analyzed revealed apparently normal male karyotype. However, 20% cells analyzed revealed a total of 184 chromosomes, suggesting octaploidy.

Effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism & sex ratio in mouse preimplantation embryos.

BACKGROUND & OBJECTIVE: There are potential risks of major birth defect in IVF (in vitro fertilization) pregnancy as well as IVF-ICSI (intra cytoplasmic sperm injection) pregnancies in comparison with naturally conceived human pregnancies. This increase risk could be due to either gonadotropins used for ovarian stimulation or in vitro culture conditions or multiple pregnancy or combinations of all the factors. The effects of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio on mouse preimplantation embryos were evaluated through the use of fluorescence in situ hybridization (FISH). METHODS: The study material consisted of 111 preimplantation mouse embryos (2-16 cell stage) in control group and 405 preimplantation mouse embryos in gonadotropin stimulated group from genetically identical Swiss Albino young (6-8 wk) mouse kept in a similar environmental conditions. The study was designed to investigate effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio through the use of FISH technique using chromosome X, Y and 19 probes. All blastomeres of embryos in both groups were assessed. RESULTS: Interpretable FISH results were obtained in 66 embryos in control group and 128 embryos in gonadotropin stimulated group. There was no excess of chromosome aneuploidy (only one case of sex chromosome trisomy in study group; 19, 19, X, Y, Y) or chromosome mosaicism or deviations in sex ratio between the two groups. However, deviation (1.36 M: 1 F in control group & 1.25 M : 1 F in study group) was seen from expected sex ratio (1 M : 1 F) i.e., skewed sex ratio in both the groups. INTERPRETATION & CONCLUSION: Our results showed that gonadotropins used for ovarian stimulation had no effects in causing increase in chromosome X, Y, 19 aneuploidy and mosaicism and skewing of sex ratio in mouse model. A large scale study with more FISH probes on a larger sample size need to be done to confirm the findings.

Skewed sex ratio and low aneuploidy in recurrent early missed abortion.

BACKGROUND AND OBJECTIVES: Conventional cytogenetic studies have revealed more number of females in spontaneous abortion and it has been assumed that a large proportion of those were resulted from maternal contamination and overgrowth of maternal decidua in long term culture. In this study we have attempted to overcome difficulties of conventional cytogenetics by using meticulous tissue dissection and molecular methods onto uncultured chorionic villous tissue thus bypassing long term culture to find out true sex ratio and frequency as well as type of common aneuploidy in early missed abortions. METHODS: Early missed abortion products (n=58) were collected from recurrent aborter in and around Lucknow, India, over a period of three years. All the cases were selected on the basis of ultrasonography diagnosis. Chorionic villous tissue was cleaned from maternal tissue and processed for conventional as well as molecular cytogenetic analysis. RESULTS: Conventional cytogenetics was successful in 15, of which 12 were females and 3 males. There were 3 cases of chromosomal abnormality, including one false. Interphase FISH with X, Y, 1, 9, 12, 16, 18 and 13/21 probes was carried out in all 58 cases. There were 43 females and 15 males. Four cases of chromosomal abnormality were detected by interphase FISH (6.9%). Comparative genomic hybridization was successful in 8 cases (6 females and 2 males). There was no aneuploidy; however, suspected gain and losses were seen in 4 cases. INTERPRETATION AND CONCLUSION: Our results suggested skewing of sex ratio (M : F, 1 : 2.9 ) and low aneuploidy rate, indicating that in early missed abortion from recurrent spontaneous abortion female outnumbers male. The various possibilities with literature support are presented that may serve as a template for future work.

A preliminary investigation of genomic screening in cervical carcinoma by comparative genomic hybridization.

BACKGROUND & OBJECTIVE: Available clinical, radiological and histopathologic risk factors are not adequate for accurate prognosis in uterine cervix carcinoma. Hence there is a need to identify indicators to select high risk cases. Most cancers occur and progress through step-wise somatic genetic mutations. Thus screening of whole genome for specific genomic alterations and its outcome following treatment may predict prognosis. The present study was carried out to investigate genomic alterations associated with cervical carcinoma and any association of genomic alterations with clinico-pathologic parameters. METHODS: Cervical carcinoma cases (n = 4) were subjected to protocol based clinical evaluation, treatment and follow up as a double blind procedure. Tumour samples were collected before radiotherapy and 3 months after completion of radiotherapy. All the samples were stored at -80 degrees C. Comparative genomic hybridization (CGH) was carried out to screen genomic alterations in all tumour samples obtained before treatment. Conventional fluorescent in situ hybridization (FISH) was carried out to confirm the CGH findings and to follow up post-treatment samples. Patients were followed up for a minimum of one year or until death. RESULTS: The CGH analysis identified genomic losses and gains. The gains were observed mainly in chromosomes 1q 25.1, 3q 26.1, 6q 13-16, 9p 22 and X, and losses in chromosome 10 and 11q21-24. CGH and FISH results were complementary to each other. Of the four patients, two were alive and two were dead at the end of follow up. INTERPRETATION & CONCLUSION: Initial results indicated that persistence of genomic alterations and appearance of giant nucleus was associated with poor prognosis and the same may be used to follow up patient. Similar studies on large sample with longer period of follow up are warranted to validate our result.

Detection of 22 q11.2 hemizygous deletion by interphase FISH in a patient with features of CATCH 22 syndrome.

We report an eight years female child with clinical and molecular cytogenetic findings consistent with CATCH 22 syndrome characterized by cardiac defect, typical facial dysmorphism, mental deficiency and chromosome 22 q11.2 deletion. Interphase FISH with 22q 11.2 probe demonstrated hemizygous deletion in 98.5% nuclei. Interphase FISH for diagnosis of CATCH 22 syndrome has not been reported previously from India to our knowledge.

Absence of sperm meiotic segregation error of chromosomes 1, 9, 12, 13, 16, 18, 21, X and Y in a case of 100% necrozoospermia.

Varying degrees of necrozoospermia are common findings in cases of male sub-fertility; however, it is rare to find persistent and 100 % necrozoospermia. A case of persistent 100 % necrozoospermia was presented in this paper, where aneuploidy analysis was carried out on sperm. No known associations like thyrotoxicosis, genital infection, spinal injury and diabetes were found. Sperm fluorescent in situ hybridization (FISH) was carried out to evaluate sperm aneuploidy for chromosome 1, 9, 12, 13, 16, 18, 21, X and Y and did not show any excess of aneuploidy over controls. To the best of our knowledge, this is the first attempt on meiotic segregation analysis on 100 % necrozoospermic patients.