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ABSTRACT: Calcium/calmodulin-dependent protein kinase IV (CAMK4) is a multifunctional serine/threonine protein kinase, which plays an important role in the spermatogenesis by phosphorylating protamines. It has been shown to be involved in the regulation of human sperm motility. Moreover, the Camk4 knockout mice were infertile because of severely reduced sperm count and morphological abnormalities. As no study is available on the association of this gene with male infertility, we analysed all the exons of CAMK4 gene in ethnically matched 283 infertile and 268 fertile Indian men. We identified twenty nucleotide substitutions, of which twelve were novel. Of these novel variants, eight were exclusively detected in infertile men. Moreover, two infertile men-specific mutations were non-synonymous replacing amino acids at the highly conserved region. In silico analysis predicted both of these mutations as 'deleterious'. In addition to nucleotide substitutions, we identified five novel insertion-deletion mutations; of these, g.150264_66delGCG was exclusively found in two oligoasthenoteratozoospermic men. In silico analysis of infertile men exclusive mutations predicted that they can alter/diminish the potential binding sites of splicing factors, which may affect the mRNA splicing and protein translation. Our study suggests that the mutations in CAMK4 may lead to abnormal semen parameters.
International Journal of Andrology 08/2012; · 3.59 Impact Factor
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ABSTRACT: The ideal method for sperm selection during Intracytoplasmic sperm injection (ICSI) is still ill-defined. Identification of a viable spermatozoon amongst immotile spermatozoa for ICSI often becomes difficult. Ninety-six ICSI cycles were selected and divided into Group A (azoospermic, n = 58) and Group B (complete asthenozoospermic, n = 38). Oocytes having birefringent meiotic spindle and zona pellucida thickness <20 μm were selected for ICSI. Groups A and B were further divided into A1, A2 and B1, B2, respectively, based on the type of ICSI performed. In Group A1, a motile spermatozoon with normal morphology was injected into a metaphase-II (M-II) oocyte. In Group B1, spermatozoon showing coiling of tail following modified hypo-osmotic swelling test was injected into M-II oocytes. In Groups A2 and B2, ICSI was performed by injecting a spermatozoan with birefringent head. Pronuclear morphology, fertilisation rate, embryo grading and pregnancy rate were assessed. ICSI outcome measures were better in Group A2 than in Group A1 but were statistically insignificant. However, significantly higher percentage of Z1 and Z2 zygotes, Grade I and Grade II embryos and pregnancy rate were observed in Group B2 as compared to Group B1. Selection of birefringent spermatozoa shows promising results in asthenozoospermic men and men undergoing testicular sperm aspiration or extraction before ICSI.
Andrologia 02/2012; 44 Suppl 1:734-8. · 1.55 Impact Factor
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ABSTRACT: Transition nuclear proteins (TNP1 and TNP2) are the major nuclear proteins that replace somatic histones during spermatogenesis. TNPs are required for the maintenance of normal spermatogenesis. Moreover, spermatogenesis was found to be compromised in both Tnp1 and Tnp2 null mice. As no study is available on the role of these genes in Indian infertile men, we have sequenced the entire TNP1 and TNP2 genes in 320 infertile men and 280 control fertile men drawn from two states in India. We identified 18 variants, including 8 previously known and 10 novel. Of the 10 novel variants, 3 were found only in azoospermic men, of which 2 (g.-688A>T in TNP1 and g.1030G>A in TNP2) were predicted to affect the transcription factor binding sites and therefore can cause deregulation of gene expression. Haplotype association analysis showed a significant omnibus association (omnibus χ(2) = 7.87, p = 0.0195) for the single nucleotide polymorphisms (SNPs) in the TNP1 gene with azoospermia. The frequency of the haplotype GCG (H3) was increased in azoospermic men (53.1%) compared with fertile men (43%; χ(2) = 7.964, p = 0.005). However, similar analysis of the TNP2 gene did not show any association with infertility. Furthermore, expression analysis of the TNP1 gene in obstructive azoospermic men showed that haplotypes of the TNP1 gene do not affect its expression level. Our results suggest that the individual SNPs of the TNP1 and TNP2 genes are not associated with infertility; however, the haplotype GCG of the TNP1 gene is a risk factor for azoospermia.
International Journal of Andrology 04/2011; 34(2):173-82. · 3.59 Impact Factor
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ABSTRACT: A large prospective clinical trial was conducted to compare the efficacy of single dose uFSH and clomiphene citrate combination with clomiphene citrate alone for ovulation induction to improve the pregnancy rate.
The study was a randomized, prospective clinical trial. Totally, 1527 infertile women (4381 cycles) with polycystic ovarian syndrome (PCOS) (n=911/2573 cycles) and unexplained infertility (n=616/1808 cycles) were randomized into two groups. Group A received single dose of uFSH on D(3) of menstrual cycle along with clomiphene. Group B received clomiphene only for ovulation induction. We compared the pregnancy rate and miscarriage rate between two groups.
Group A had a pregnancy rate of 17% compared to 8.3% of Group B which was significantly higher (P=0.0001). The miscarriage rate was 11% in Group A and 10% in Group B which was not significant (P=0.99). Pregnancy rates in PCOS women were 22% in Group A and 9.3% in Group B which shows significantly higher pregnancy rate (P=0.0001) in anovulatory infertility. But in unexplained infertility, there was no significant difference in pregnancy rate between Group A (11%) and Group B(6.3%). Miscarriage rates were 8.8% and 9.5% in Group A and Group B, respectively, in PCOS women and 14% and 13% in women with unexplained infertility.
Addition of single dose of uFSH improves pregnancy outcome particularly in anovulatory infertility (WHO II). Correction of unexplained infertility may need more than simple correction of possible subtle ovulatory effect.
Journal of human reproductive sciences. 05/2010; 3(2):80-4.
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ABSTRACT: The effect of follicular fluid (FF) oxidative stress (OS) on meiotic spindle (MS) formation in oocytes and subsequent outcome in women with polycystic ovarian syndrome (PCOS) are evaluated in this study.
326 oocytes from 35 PCOS women (group A) and 208 oocytes from 32 women with tubal infertility (group B) were visualized for MS using PolScope. FF was analyzed for OS markers including reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity (TAC). Group A was further classified into groups A1 and A2, and group B into groups B1 and B2 depending upon the presence or absence of MS, respectively.
MS formation was absent in a significantly higher number of oocytes in group A compared to group B (p <or= 0.05). OS markers were significantly higher in group A compared to group B (p <or= 0.05). Fertilization rate, number of good quality embryos and clinical pregnancy rates were higher in group B compared to group A, though not statistically significant. FF ROS was significantly higher and TAC significantly lower in groups A2 and B2 compared to groups A1 and B1 (p < 0.001).
The presence of MS and oocyte maturation in PCOS women showed a positive correlation with low levels of OS.
Gynecologic and Obstetric Investigation 01/2010; 69(3):197-202. · 1.28 Impact Factor
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ABSTRACT: The autosomal DAZL (Deleted-in-Azoospermic-Like) gene, mapped to the short arm of the human chromosome 3, is the precursor for the Y-chromosomal DAZ cluster, which encodes for putative RNA-binding proteins. Mutations in the DAZL have been reported to be associated with spermatogenic failure in Taiwanese population but not in Caucasians. As there was no study on Indian populations, we have analysed the entire coding sequences of exons 2 and 3 of DAZL in a total of 1010 men from Indian subcontinent, including 660 infertile men with 598 non-obstructive azoospermia, 62 severe oligozoospermia and 350 normozoospermic fertile control men, to investigate whether mutation(s) in the DAZL is associated with male infertility. Interestingly, none of our samples (1010) showed A386G (T54A) mutation, which was found to be associated with spermatogenic failure in Taiwanese population. In contrast, A260G (T12A) mutation was observed in both infertile and normozoospermic fertile control men, without any significant association with infertile groups (chi2= 0.342; p = 0.556). Similarly, we have found a novel A437G (I71V) mutation, which is also present in both infertile and normozoospermic fertile control men without any significant difference (chi2 = 0.476; p = 0.490). Our study clearly demonstrates the complete absence of the A386G (T54A) mutation in Indian subcontinent and the other two mutations --A260G (T12A) and A437G (I71V)--observed are polymorpic. Therefore, we conclude that these mutations in the DAZL gene are not associated with male infertility in Indian subcontinent.
International Journal of Andrology 11/2006; 29(5):510-14. · 3.59 Impact Factor
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ABSTRACT: The impact of oxidative stress in female reproduction is not clear. Contradictory reports on the effect of various oxidative stress markers on follicular fluid, oocytes and embryo quality and fertilization potential exist. The objectives of this study were to examine reactive oxygen species (ROS) levels in follicular fluid of women undergoing IVF and to relate these levels to embryo formation and quality.
A total of 208 follicular fluid samples were obtained from 78 women undergoing controlled ovarian stimulation and analysed for ROS and lipid peroxidation (LPO). These samples were divided into groups I and II which represented follicular fluid containing grade III and grade II oocytes, respectively. These groups were further subdivided into groups IA, IB, IIA and IIB according to embryo quality. Subgroups IA and IIA consisted of follicular fluid samples corresponding to grade I/II embryo formation. Subgroups IB and IIB represented fertilization failure/pro-nucleolus (PN) arrest/grade III embryos. No significant correlation was observed in ROS levels on comparing groups I and II (P > 0.05). However, ROS levels were observed to be significantly different on comparing groups IA and IB (P < or = 0.01) and groups IIA and IIB (P < or = 0.05). LPO levels further supported our results.
ROS levels in follicular fluid appear to play a significant role in embryo formation and quality.
Human Reproduction 09/2006; 21(9):2403-7. · 4.47 Impact Factor
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ABSTRACT: Use of letrozole, a selective inhibitor of aromatase, reduces the gonadotrophin dose required to induce follicular maturation. We evaluated whether incorporation of letrozole could be an effective low-cost IVF protocol for poor responders.
A randomized, controlled, single-blind trial was conducted in the Assisted Reproduction Unit, Institute of Reproductive Medicine, Kolkata, India. Thirty-eight women with a history of poor ovarian response to gonadotrophins were recruited. Thirteen women (Let-FSH group) received letrozole 2.5 mg daily from day 3-7, and recombinant FSH (rFSH) 75 IU/day on days 3 and 8; and 25 women (GnRH-ag-FSH group) underwent long GnRH agonist protocol and stimulated with rFSH (300-450 IU/day). Ovulation was triggered by 10,000 IU of HCG followed by IVF-embryo transfer. The main outcome measures were total dose of rFSH (IU/cycle), terminal estradiol (E2) (pg/ml), numbers of follicles, oocytes retrieved and transferable embryo, endometrial thickness (mm), and pregnancy rate.
Compared with the GnRH-ag-FSH group (2865 +/- 228 IU), the Let-FSH group (150 +/- 0 IU) received a significantly (P < 0.001) lower total dose of FSH. Except for terminal E2, which was significantly higher (P < 0.001) in the GnRH-ag-FSH group (380 +/- 46 pg/ml) than the Let-FSH group (227 +/- 45 pg/ml), the treatment outcomes in all other respects, including pregnancy rate, were statistically comparable.
Adjunctive use of letrozole may form an effective means of low-cost IVF protocol in poorly responding women.
Human Reproduction 09/2004; 19(9):2031-5. · 4.47 Impact Factor
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ABSTRACT: The pathophysiological mechanisms underlying premature ovarian failure (POF) are largely unknown. Our objective was to develop a working animal model to explore the pathogenesis of POF. Since galactosaemic women eventually develop POF, we evaluated the potential of experimental galactose toxicity as the proposed model.
Pregnant rats were fed pellets supplemented with or without 35% galactose from day 3 of conception continuing through weaning of the litters. Female offspring were evaluated for serum levels of galactose and galactose-1-phosphate, growth rate, onset of puberty, reproductive cyclicity, ovarian complement of follicles, hypothalamo-pituitary-ovarian function and follicular response to gonadotrophins.
Galactose toxicity delayed the onset of puberty and developed a state of hypergonadotrophic hypoestrogenism. The characteristic low FSH levels at weaning followed by pubertal spurts of gonadotrophins and estradiol (E(2)) secretion of the controls was replaced by a sustained high level of FSH and a low level of E(2) under galactose toxicity. The ovary developed with apparently normal or deficient complement of follicles. Ovarian response to exogenous gonadotrophin stimulation was blunted, but the response improved significantly when the stimulation was preceded by pituitary desensitization.
Experimental galactose toxicity may serve as a model for exploring some of the basic tenets of POF.
Human Reproduction 11/2003; 18(10):2031-8. · 4.47 Impact Factor
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ABSTRACT: In rats, prenatal exposure to high concentrations of galactose may contribute to a condition that is equivalent to the premature ovarian failure (POF) component of human galactosaemia. We investigated if development of POF under experimental galactosaemia-like conditions was attributed to impaired germ cell migration.
Pregnant rats were fed pellets supplemented with, or without, 35% galactose from day 3 of conception continuing through parturition. Between days 12-15, embryos from one uterine horn were dissected out. Primordial germ cells (PGC) were histochemically localized and counted on the basis of binding of Dolichos biflorus agglutinin, a lectin specific for terminal N-acetylgalactosamine (GalNAc), to the surface glycoconjugate of the germ cells. The embryos from the other uterine horn were maintained until parturition. Liver activity of uridine diphosphate galactose 4-epimerase, the enzyme involved at multiple steps in the process of synthesis of GalNAc, was assayed in 1-2 day old female pups.
The numbers of PGC at the day-specific sites on all days of examination were significantly lower (P </= 0.0003), and liver epimerase activity was significantly (P = 0.000001) reduced in the galactose-exposed group.
Impaired germ cell migration leading to the development of gonads with deficient initial pools of germ cells may form the causal link between galactosaemia and POF.
Human Reproduction 02/2003; 18(2):276-82. · 4.47 Impact Factor
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B N Chakravarty
Journal of Assisted Reproduction and Genetics 02/2001; 18(1):10-4. · 1.84 Impact Factor
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B N Chakravarty
Journal of the Indian Medical Association 03/1999; 97(2):43-7.
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ABSTRACT: Our purpose was to evaluate the IVF-ET outcome in patients who did not achieve timely pituitary-ovarian suppression following "long"-protocol GnRH agonist (GnRH-a) administration.
A retrospective analysis was done on 96 IVF treatment cycles characterized by a delayed response (DR) to long-protocol GnRH-a treatment. The study included those patients who either achieved ovarian suppression (E2 < or = 110 pM) despite an elevated LH level (group DR-A) or had pituitary desensitization (LH < or = 1.5 IU/L) without ovarian suppression (group DR-B) on day 12 of GnRH-a treatment but needed an extended course of GnRH-a treatment to achieve complete suppression. These patients had gonadotropin stimulation either from day 12, despite an elevated level of LH (subgroup DR-A1; n = 13) or elevated E2 levels (subgroup DR-B1; n = 9), or after achieving a complete hypogonadotropic-hypopgonadal state following an extended course of GnRH-a treatment [subgroups DR-A2 (n = 46) and DR-B2 (n = 28)]. The outcome was compared with that of 88 cycles of normal responders (group NR) who had pituitary-ovarian suppression by day 12 day GnRH-a administration.
Ovarian response and pregnancy rates in subgroups DR-A1 and DR-A2 were statistically not different and comparable to those in the NR group. In subgroups DR-B1 and DR-B2, E2 response and rates of oocyte retrieval and pregnancy were significantly lower than those in the other groups, but fertilization and cleavage rates were similar. The requirement of gonadotropin for ovarian stimulation was comparatively higher in subgroup DR-A2 and both DR-B subgroups.
There was no treatment cancellation in group NR and both DR-A subgroups, but 22% of the cycles in DR-B1 and 14% of the cycles in DR-B2 were canceled due to poor ovarian response. It therefore appears that during long-protocol pituitary desensitization, the post-GnRH-a level of serum E2, rather than LH, better predicts IVF-ET outcome.
Journal of Assisted Reproduction and Genetics 06/1996; 13(5):374-80. · 1.84 Impact Factor
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Journal of the Indian Medical Association 03/1995; 93(2):71-4.
