Infertility has been recognized as a disease that
requires timely diagnosis, recognition and treatment
in terms of assisted reproduction. The prevalence
of infertility ranges from 7 to 20% in populations
across the globe (
Recently, the role of serum kisspeptin (KP) in regulating HPO axis and maturity of oocytes has been aptly investigated (
A longitudinal cohort study was carried out from August 2014 to May 2015, after Ethical approval was obtained from the Australian Concept Infertility Medical Center. One hundred and twenty-four female patients were recruited after receiving their written informed consent to participate in the study. Females were between 20 and 40 years of age (mean of 32.16 ± 4.8 years), had a mean body mass index (BMI) of 24.19 ± 2.3 kg/m2, were segregated into three groups on the basis of their infertility and all were recommended for ICSI. Cause of infertility due to male, female and unexplained factors was at a frequency of 32 (26%), 33 (27%) and 59 (47%) respectively. The first group consisted of those with a male factor infertility (e.g varicocele, prior surgeries and semen abnormalities). The second group comprised females with diagnoses of uterine fibroids (n=2), endometriosis (n=20) and tubal blockade (n=11). The third group comprised 59 patients with unexplained infertility. Females with metabolic disorder (polycystic ovaries) and endocrine disorders such as thyroid dysfunction and abnormal prolactin levels were excluded from the study. Couples diagnosed with both male and female infertility factors were excluded from the study.
Serum samples were collected on the second day of the menstrual cycle before commencement of down-regulation of ovaries. KP was measured using the KISS-1-ELISA Kit (Shanghai, China). The analytical sensitivity of the KP kit was 10.16 ng/L and intra- and inter-assay coefficients of variation was less than 10 and 12% respectively.
Down-regulation of ovaries by 1 mg of subcutaneous buserelin acetate (Suprefact, Sanofi) on day 21 of previous menstrual cycle was followed by controlled ovarian stimulation with Gonal-f (Merck Sereno) from the second day of periods. Confirmation of maturity of follicles to ≥18 mm in diameter was assessed by transvaginal scan (TVS) and cycles were cancelled when follicles failed to develop in response to gonadotropin stimulation. Endometrial thickness was gauged on the day of ovulation induction in the mid sagittal plane by two-dimensional ultrasound with a 7.5-MHz vaginal probe (Hitachi EUB 525, Hitachi, Japan). Oocytes were retrieved from mature follicles (20 mm in diameter), 36 ± 1 hours after injection of human chorionic gonadotropin (hCG, Ovitrelle 250), on the 14th, 15th or 16th day of stimulation. Semen samples were retrieved by masturbation and sperms were then immobilized by 7% polyvinyl pyrrolidone after which microinjection was performed (Leica DMIRB, Leica Microsystems, Wetzlar, Germany). Finally, the microinjected oocytes were incubated for 16-18 hours at 37°C, 6% CO2 and 5% O2.
Patients were categorized on the basis of β-hCG
concentrations on the 14th day after egg collection
and on the basis of sonographic evidence of an
intrauterine gestational sac 14 days after β-hCG
measurement. Females with beta hCG<25 mIU/ml
were declared as non-pregnant, pre-clinical abortion
was labelled on a beta hCG>25 m IU/ml but
with no cardiac activity on ultrasound while clinical
pregnancy was confirmed based on higher levels
of beta hCG along with intrauterine gestational
sac and cardiac activity on TVS. The implantation
rate (IR) was calculated as the number of pregnancies
per embryo transferred (
Data were analyzed by SPSS version 21 (IBM statistics, Chicago, IL) and was expressed as mean ± SD/standard error of mean wherever appropriate. Analyses were undertaken for the whole group and independently in sub-groups according to the infertility factor. Variation in KP levels in females with different types of infertility and different outcomes after ICSI was compared by Analysis of variance (ANOVA) and Tukey’s Post-hoc test. Pearson’s correlation coefficient was used to test correlation between KP levels and the study variables. In all cases a P<0.05 was considered significant.
Table 1 summarises the characteristics of the study population with respect to type of infertility. Significant difference in KP levels amongst these groups was observed (P<0.001). Post-hoc analyses of KP levels revealed significantly reduced levels in female and unexplained infertility factor sub-groups when compared with those with a male factor infertility (P=0.035 and P<0.001, respectively).
Clinical variables in different types of infertility
|Variable||Male infertility||Female infertility||Unexplained infertility|
|Kisspeptin (ng/L)||397.6 ± 58.2*^||257.11 ± 24.4*||176.69 ± 5.03|
|No of oocytes fertilized||5.8 ± 0.2||6.02 ± 0.22||5.83 ± 0.16|
|Endometrial thickness (mm)||8.3 ± 0.6||9.90 ± 3.2*||6.74 ± 0.36|
|No of transferred embryos||1.5 ± 0.1*||1.37 ± 0.11*||1.83 ± 0.06|
|Number of gestational sacs||0.3 ± 0.1^||0.75 ± 0.13*||0.102 ± 0.05|
|Implantation rate||19.27 ± 6.35||44.9 ± 6.74||5.08 ± 2.88|
Blood collected on the second day of cycle before initiation of stimulation.
Data expressed as mean ± SD, except kisspeptin levels expressed as mean ± SEM.
Results compared by One-Way ANOVA, significant difference (P<0.05) on Post-hoc analysis with unexplained infertility is expressed as * while that between male infertility and female infertility factors is expressed as ^.
Stratification of outcomes based on type of infertility
|Variable||Male infertility||Female infertility||Unexplained infertility|
|Non pregnant n=6||Preclinical abortion n=18||Clinical pregnancy n=8||Non pregnant n=4||Preclinical abortion n=12||Clinical pregnancy n=17||Non pregnant n=56||Preclinical abortion n=0||Clinical pregnancy n=3|
|Serum KP (ng/L)||352.8 ± 15.9||384.2 ± 38.0||461.1 ± 43.0*||163.1 ± 21.5||164.9 ± 2.0||203.3 ± 12.0*||171.2 ± 3.6||0 (0%)||278.9 ± 24.9*|
|Oocyte maturation rate||81.8 ± 11.5||93.4 ± 3.7||89.4 ± 5.4||100 ± 0.0||98.4 ± 1.0||97.7 ± 1.0||75.9 ± 1.7||0 (0%)||85.4 ± 0.6|
|Fertilization rate||63.9 ± 8.7||79.7 ± 2.7||74.1 ± 5.0||82.0 ± 1.6||83.0 ± 1.4||81.3 ± 1.2||59.9 ± 1.4||0 (0%)||76.9 ± 0.5|
|Implantation rate||0||0||77.0 ± 8.8||0||0||87.2 ± 5.0||0||0 (0%)||3|
Data expressed as mean ± SD, except Kisspeptin (KP) levels which are expressed as mean ± SEM. Highest level of KP was observed in clinical pregnancy group in all stratifications (expressed as *).
After ICSI, 66 (53%) patients failed to conceive, 30 (24%) reported with preclinical abortions while 28 (23%) were confirmed as clinically pregnant. Mean ± SEM values of KP were 87.24 ± 14.94 ng/L in non-pregnant females and 215.11 ± 34.14 ng/L in pre-clinical abortion while it was 296.23 ± 12 ng/L in patients with clinical pregnancy (
Mean levels of Kisspeptin (ng/L) in groups segregated on the bases of ICSI outcomes.
ICSI; Intracytoplasmic sperm injection, **; Clinical pregnancy group versus non-pregnant P<0.001, and ^; Clinical pregnancy versus pre-clinical abortion P<0.05. Error bars represent SEM.
Although variation in KP levels between non-pregnant and pre-clinical abortion groups was marginally significant (P=0.044), it was found to be highly significant between non-pregnant and clinically pregnant females (P<0.001). The outcomes of ICSI treatment based on type of infertility are presented in Table 2. Highest number of clinical pregnancies was achieved in the group of infertile females with diagnosed cause of infertility (51%), however, only 5% of patients with unexplained infertility got pregnant. A weak but significant positive correlation were observed between KP levels and number of fertilized oocytes (r=0.18, P=0.04) and thickness of the endometrium (r=0.27, P=0.01).
With the advancement of ART, couples have been able to conceive, nevertheless, the outcome is associated with a limited success rate (
Fayazi et al. (
During the menstrual cycle, secretory changes in the endometrium account for coordinated signal exchange between hormonally primed endometrium and functional embryo for the implantation of embryo. This is made possible by the interplay of hormones and cytokines, and studies have demonstrated that increased endometrial thickness is associated with higher pregnancy rates (
The prevalence of infertile couples with unexplained factors is estimated at 30% (
Low level of KP in females with unexplained infertility before the initiation of the ICSI protocol was seen. One of the factors causing unexplained infertility may thus be low KP levels. The level of KP has an impact on fertilization of oocytes, preparation of endometrial beds for implantation of embryo and hence successful pregnancy after ICSI. This observation urges the need of further studies at genetic and molecular levels to define and explain the role of KP for preservation of conception and continuation of pregnancy in females with unexplained infertility.