Tamoxifen is Better than Low-Dose Clomiphene or Gonadotropins in Women with Thin Endometrium (<7 mm) after Clomiphene in Intrauterine Insemination Cycles: A Prospective Study.

AIM: Gonadotropin stimulation is used as the second line of treatment in patients with thin endometrium following clomiphene citrate (CC) administration, which is associated with higher cost, multiple births, and ovarian hyperstimulation syndrome. Tamoxifen (TMX), a selective estrogen receptor modulator, acts as an agonist on the endometrium. The objective of the present study was to compare the efficacy of low-dose CC, TMX, and gonadotropins in women with thin endometrium (<7 mm) following Clomiphene in intrauterine insemination (IUI) cycles. SETTINGS AND DESIGN: A prospective observational study between December 2011 and June 2013 was carried out in a tertiary infertility center. METHODS: Women (n = 502) undergoing IUI with endometrium <7 mm after 100 mg CC were included in the study and divided into three treatment groups. Women in Group A (n = 182, cycles = 364) received clomiphene (50 mg/day from day 3 to 7), Group B (n = 179, cycles = 342) received TMX (40 mg/day from day 3 to 7), and Group C (n = 141, cycles = 226) received continuous urine-derived follicle-stimulating hormone 75-150 IU from day 3 onward until human chorionic gonadotropin injection. Endometrial thickness (ET), pregnancy rate, and live birth rate were considered as main outcome measures. STATISTICAL ANALYSIS: Multiple comparisons using one-way ANOVA and Schiff's test were performed. RESULTS: Pregnancy and live birth rate were significantly higher (P < 0.004) in TMX and gonadotropin groups compared to clomiphene. A number of follicles in the TMX group were found to be lower (P < 0.001) compared to other two groups. In polycystic ovary syndrome patients, ovulation induction with TMX resulted in inadequate response in more than half of the cycles. CONCLUSIONS: TMX can improve ET and live birth rate in patients with thin endometrium after clomiphene.

NMR-based metabonomics for understanding the influence of dormant female genital tuberculosis on metabolism of the human endometrium.

STUDY QUESTION: Does investigation of metabolic perturbations in endometrial tissue of women with dormant genital tuberculosis (GTB) during the window of implantation (WOI) assist in improving the understanding of endometrial receptivity? SUMMARY ANSWER: In dormant GTB cases significant alterations in endometrial tissue metabolites occur, largely related to energy metabolism and amino acid biosynthesis in dormant GTB cases. WHAT IS KNOWN ALREADY: As an intracellular pathogen, Mycobacterium tuberculosis strongly influences the metabolism of host cells causing metabolic dysregulation. It is also accepted that dormant GTB impairs the receptive status of the endometrium. Global metabolic profiling is useful for an understanding of disease progression and distinguishing between diseased and non-diseased groups. STUDY DESIGN, SIZE, DURATION: Endometrial tissue samples were collected from patients reporting at the tertiary infertility care center during the period September 2011-March 2013. Women having tested positive for GTB were considered as the study group (n = 24). Normal healthy women undergoing sterilization (n = 26) and unexplained infertile women with repeated IVF failure (n = 21) volunteered to participate as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial tissue samples were collected 6-10 days after confirmation of ovulation. PCR and BACTEC-460 culture were used for diagnosing GTB. Proton nuclear magnetic resonance (1H NMR) spectra of tissue were recorded using a 700 MHz Bruker Avance AV III spectrometer. Following phase and baseline correction of all NMR spectra by Bruker Topspin 2.1 software, spectral peak alignment of the data was performed. Multivariate analysis was applied to all spectra and individual metabolites identified and multiple correlation analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE: Leucine, isoleucine, acetate, lactate, glutamate, glutamine, methionine, lysine, creatine, glycogen, glycine, proline and choline were found to be significantly increased (P < 0.05) in endometrial tissue of women with dormant GTB compared with unexplained infertile women with repeated implantation failure. Valine, citrate, succinate and aspartate were also observed to be significantly up-regulated (P < 0.01). Furthermore, a significant decrease in glucose (P < 0.05), threonine (P < 0.05), tyrosine (P < 0.01) and phenylalanine (P < 0.0001) was observed in women with dormant GTB. Pearson's correlation analysis between the expression of various endometrial receptivity markers and metabolites showed a significant negative correlation (-0.236 to -0.545, P < 0.05). Also, the metabolites were positively correlated with endometrial receptivity markers (0.207 to 0.618, P < 0.05). LIMITATIONS, REASONS FOR CAUTION: It is often difficult to diagnose dormant GTB because it tends to exist without any clinical signs or symptoms. In addition, the diagnosis of GTB by culture remains a challenge due to low detection rates and its paucibacillary nature. Testing for prostate-specific antigen or the Y chromosome in order to account for the possible influences of recent exposure to semen on endometrial metabolism would be important. WIDER IMPLICATIONS OF THE FINDINGS: The metabolic changes associated with the dormant tubercle infection are of potential relevance to clinicians for the treatment of dormant GTB-related infertility. STUDY FUNDING/COMPETING INTERESTS: Government of India, Indian Council of Medical Research. There are no conflicts of interest.

Congenital malformations among babies born following letrozole or clomiphene for infertility treatment.

CONTEXT: Clomiphene citrate (CC) is the first line drug for ovulation induction but because of its peripheral antiestrogenic effect, letrozole was introduced as the 2nd line drug. It lacks the peripheral antiestrogenic effect and is associated with similar or even higher pregnancy rates. Since letrozole is a drug for breast cancer, its use for the purpose of ovulation induction became controversial in the light of studies indicating an increased incidence of congenital malformations. AIMS: To evaluate and compare the incidence of congenital malformations among offsprings of infertile couples who conceived naturally or with clomiphene citrate or letrozole treatment. SETTINGS AND DESIGN: A retrospective cohort study done at a tertiary infertility centre. METHODS AND MATERIAL: A total of 623 children born to infertile women who conceived naturally or following clomiphene citrate or letrozole treatment were included in this study. Subjects were sorted out from medical files of both mother and newborn and follow up study was done based on the information provided by parents through telephonic conversations. Babies with suspected anomaly were called and examined by specialists for the presence of major and minor congenital malformations. Other outcomes like multiple pregnancy rate and birth weight were also studied. RESULTS: Overall, congenital malformations, chromosomal abnormalities were found in 5 out of 171 (2.9%) babies in natural conception group and 5 out of 201 babies in the letrozole group (2.5%) and in 10 of 251 babies in the CC group (3.9%). CONCLUSIONS: There was no significant difference in the overall rate of congenital malformations among children born to mothers who conceived naturally or after letrozole or CC treatment. KEY MESSAGES: Congenital malformations have been found to be comparable following natural conception, letrozole and clomiphene citrate. Thus, the undue fear against letrozole may be uncalled for.

Novel mutations in calcium/calmodulin-dependent protein kinase IV (CAMK4) gene in infertile men.

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.

Selection of birefringent spermatozoa under Polscope: effect on intracytoplasmic sperm injection outcome.

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.

The TNP1 haplotype - GCG is associated with azoospermia.

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.

Effect of follicular fluid oxidative stress on meiotic spindle formation in infertile women with polycystic ovarian syndrome.

BACKGROUND: 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. METHODS: 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. RESULTS: MS formation was absent in a significantly higher number of oocytes in group A compared to group B (p

Comparative evaluation of pregnancy outcome in gonadotrophin-clomiphene combination vs clomiphene alone in polycystic ovarian syndrome and unexplained infertility-A prospective clinical trial.

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Hyaluronan binding protein-1: a modulator of sperm-oocyte interaction.

Hyaluronan (HA), a complex glycosaminoglycan, is an important component in reproductive fluids and regulates several reproductive processes. It is thought that the multifaceted biological function of HA is mediated through hyaladherin family protein that binds with HA. We have reported a novel glycoprotein from human that has specific affinity towards hyaluronan, referred to as Hyaluronan Binding Protein-1 (HABP1, Ac. No. NP-001203) and is localized on human chromosome 17 p13.3. Sequence analysis of this gene has further revealed that HABP1 is synthesized as a precursor protein of 282 amino acids which undergoes a post-translational modification to give rise to the mature form of 209 amino acids by proteolytic cleavage of 73 amino acids at the N-terminal. The localization of mature HABP1 in several organs including the sperm surface and its involvement in fertilization have already been demonstrated. Enhanced phosphorylation of HABP1 in motile spermatozoa suggests its involvement in cellular signalling. Though only the mature form of HABP1 is detected in somatic tissues, the precursor form of HABP1 was detected in testicular tubules in a stage-specific manner in pachytene and round spermatids. To study the role of HABP1 in the fertilization process, we have shown the absence of mature HABP1 in cryptorchidic rats testes and an accumulation of the precursor form of HABP1 in giant cells, generated in infertile cryptorchidic rats. The loss of HABP1 from the sperm surface of a patient with very low sperm motility and the absence of the proprotein form of HABP1 in pachytene and round spermatids from testicular biopsy material with spermatogenic arrest, suggests that male infertility may be associated with the level of HABP1 on spermatozoa. In order to examine the role of HABP1 in sperm-oocyte interaction, we found that the number of spermatozoa bound to an oocyte was reduced significantly in the presence of D-mannosylated albumin, the universal blocker of sperm-oocyte interaction, and that this effect could be reversed by the addition of purified recombinant HABP1. In continuation, we have used spermatozoa of a patient, who had failed in IVF: the spermatozoa were incubated with HABP1 containing IVF medium for 2 hrs, then washed and allowed them to interact with the oocyte. The fertilized egg thus devloped up to the 16 cell stage, suggesting that HABP1 can modulate the sperm-oocyte interaction, even when sub-fertile spermatozoa are used.

Reactive oxygen species level in follicular fluid--embryo quality marker in IVF?

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: ROS levels in follicular fluid appear to play a significant role in embryo formation and quality.

A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian population.

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.

A randomized single-blind controlled trial of letrozole as a low-cost IVF protocol in women with poor ovarian response: a preliminary report.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Adjunctive use of letrozole may form an effective means of low-cost IVF protocol in poorly responding women.

Galactose toxicity in the rat as a model for premature ovarian failure: an experimental approach readdressed.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Experimental galactose toxicity may serve as a model for exploring some of the basic tenets of POF.

Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.

BACKGROUND: 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. METHODS: 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. RESULTS: The numbers of PGC at the day-specific sites on all days of examination were significantly lower (P

Significance of an abnormal response during pituitary desensitization in an in vitro fertilization and embryo transfer program.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.